James A Field

Interests

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Courses

Scholarly Contributions

Chapters

• Field, J. A., Adrian, L., & , F. E. (2016). Natural Production of Organohalide Compounds in the Environment. In Organohalide-Respiring Bacteria(pp 7-30). Springer-Verlag.
• Olivares, C. I., Abrell, L. M., Chorover, J. D., Simonich, M., Tanguay, R. L., Sierra-Alvarez, R., & Field, J. A. (2016). Identifying toxic biogransformation products of the insensitive munitions compound, 2,4-dinitroanisole (DNAN), using liquid chromatography coupled to quadrupole time-of-flight mass spectromety (LC-QToF-MS),. In Assessing Transformation Products of Chemicals by Non-Target and Suspect-Screening-Strategies and Workflows(pp 133-145). Chapter 9 Volume 1: Drewes, J. and Letzel (Eds). doi:10.1021/bk-2016-1241.ch009

Journals/Publications

• Jog, K. V., Hess, K. Z., Field, J. A., Krzmarzick, M. J., & Sierra-Alvarez, R. .. (2021). Aerobic biodegradation of emerging azole contaminants by return activated sludge and enrichment cultures. Journal of Hazardous Materials, 417.
• Kadoya, W. M., Sierra-Alvarez, R. .., Jagadish, B., Wong, S., Abrell, L., Mash, E. A., & Field, J. A. (2021). Covalent bonding of aromatic amine daughter products of 2,4-dinitroanisole (DNAN) with model quinone compounds representing humus via nucleophilic addition. Environmental Pollution, 268.
• Lakhey, N., Sierra-Alvarez, R. .., Couger, M. B., Krzmarzick, M. J., & Field, J. A. (2021). Anammox enrichment culture has unexpected capabilities to biotransform azole contaminants of emerging concern. Chemosphere, 264.
• Madeira, C. L., Menezes, O., Park, D., Jog, K. V., Hatt, J. K., Gavazza, S., Krzmarzick, M. J., Sierra-Alvarez, R. .., Spain, J. C., Konstantinidis, K. T., & Field, J. A. (2021). Bacteria Make a Living Breathing the Nitroheterocyclic Insensitive Munitions Compound 3-Nitro-1,2,4-triazol-5-one (NTO). Environmental Science and Technology, 55(9), 5806-5814.
• Menezes, O., Kadoya, W. M., Gavazza, S., Sierra-Alvarez, R. .., Mash, E. A., Abrell, L., & Field, J. A. (2021). Covalent binding with model quinone compounds unveils the environmental fate of the insensitive munitions reduced product 2,4-diaminoanisole (DAAN) under anoxic conditions. Journal of Hazardous Materials, 413.
• Menezes, O., Yu, Y., Root, R. A., Gavazza, S., Chorover, J., Sierra-Alvarez, R. .., & Field, J. A. (2021). Iron(II) monosulfide (FeS) minerals reductively transform the insensitive munitions compounds 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one (NTO). Chemosphere, 285.
• Niu, X., Field, J. A., Paniego, R., Pepel, R. D., Chorover, J., Abrell, L., & Sierra-Alvarez, R. .. (2021). Bioconcentration potential and microbial toxicity of onium cations in photoacid generators. Environmental Science and Pollution Research, 28(7), 8915-8921.
• Niu, X., Pepel, R. D., Paniego, R., Field, J. A., Chorover, J., Abrell, L., Sáez, A., & Sierra-Alvarez, R. .. (2021). Photochemical fate of sulfonium photoacid generator cations under photolithography relevant UV irradiation. Journal of Photochemistry and Photobiology A: Chemistry, 416.
• Zeng, C., Nguyen, C., Boitano, S., Field, J. A., Shadman, F., & Sierra-Alvarez, R. .. (2021). Toxicity of abrasive nanoparticles (SiO2, CeO2, and Al2O3) on Aliivibrio fischeri and human bronchial epithelial cells (16HBE14o-). Journal of Nanoparticle Research, 23(2).
• Bueno-López, J. I., Nguyen, C. H., Rangel-Mendeza, J. R., Sierra-Alvarez, R., Field, J. A., & Cervantes, F. J. (2020). Effects of graphene oxide and reduced graphene oxide on acetoclastic, hydrogenotrophic and methylotrophic methanogenesis", has been accepted for publication in Biodegradation. Biodegradation.
• Bueno-López, J., Nguyen, C. H., Rangel-Mendez, J. R., Sierra-Alvarez, R. .., Field, J. A., & Cervantes, F. J. (2020). Effects of graphene oxide and reduced graphene oxide on acetoclastic, hydrogenotrophic and methylotrophic methanogenesis. Biodegradation, 31(1-2), 35-45.
• Jog, K. V., Sierra-Alvarez, R. .., & Field, J. A. (2020). Rapid biotransformation of the insensitive munitions compound, 3-nitro-1,2,4-triazol-5-one (NTO), by wastewater sludge. World Journal of Microbiology and Biotechnology, 36(5).
• Jog, K. V., Sierra-Alvarez, R. .., & Field, J. A. (2020). Rapid biotransformation of the insensitive munitions compound, 3-nitro-1,2,4-triazol-5-one (NTO), by wastewater sludge. World Journal of Microbiology and Biotechnology, [in press].
• Kadoya, W. M., Sierra-Alvarez, R., Jagadish, B., Wong, S., Abrell, L., Mash, E. A., & Field, J. A. (2020). Covalent Bonding of Aromatic Amine Daughter Products of 2,4-Dinitroanisole (DNAN) with Model Quinone Compounds Representing Humus via Nucleophilic Addition. Environmental Pollution, in press.
• Lakhey, N., Li, G., Sierra-Alvarez, R. .., & Field, J. A. (2020). Toxicity of azoles towards the anaerobic ammonium oxidation (anammox) process. J. Chem. Technol. Biotechnol., 95(4), 1057-1063.
• Lakhey, N., Li, G., Sierra-Alvarez, R. .., & Field, J. A. (2020). Toxicity of azoles towards the anaerobic ammonium oxidation (anammox) process. Journal of Chemical Technology and Biotechnology, 95(4), 1057-1063.
• Li, G., Field, J. A., Zeng, C., Madeira, C. L., Nguyen, C. H., Jog, K. V., Speed, D., & Sierra-Alvarez, R. .. (2020). Diazole and triazole inhibition of nitrification process in return activated sludge. Chemosphere, 241.
• Li, G., Field, J. A., Zeng, C., Madeira, C. L., Nguyen, C. H., Jog, K. V., Speed, D., & Sierra-Alvarez, R. .. (2020). Diazole and triazole inhibition of nitrification process in return activated sludge. Chemosphere, in press.
• Moreno-Andrade, I. .., Regidor-Alfageme, E. .., Durazo, A., Field, J. A., Umlauf, K., & Sierra-Alvarez, R. .. (2020). LC-ICP-OES method for antimony speciation analysis in liquid samples. Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and Environmental Engineering, 55(4), 457-463.
• Moreno-Andrade, I., Regidor-Alfageme, E., Durazo, A., Field, J. A., Umlauf, K., & Sierra-Alvarez, R. (2020). LC-ICP-OES method for antimony speciation analysis in liquid samples.. Journal of Environmental Science and Health, Part A., 55(4), 457-463.
• Nguyen, C. H., Field, J. A., & Sierra-Alvarez, R. .. (2020). Microbial toxicity of gallium- and indium-based oxide and arsenide nanoparticles. Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and Environmental Engineering, 55(2), 168-178.
• Nguyen, C. H., Zeng, C., Boitano, S. A., Field, J. A., & Sierra Alvarez, M. R. (2020). Assessment of Gallium- and Indium-Based Nanoparticles Toward Human Bronchial Epithelial Cells Using an Impedance-Based Real-Time Cell Analyzer. International Journal of Toxicology, 39, 218-231.
• Nguyen, C. H., Zeng, C., Boitano, S., Field, J. A., & Sierra-Alvarez, R. (2020). Cytotoxicity assessment of gallium- and indium-based nanoparticles toward human bronchial epithelial cells using an impedance-based real time cell analyzer. International Journal of Toxicology, [in press].
• Nguyen, C. H., Zeng, C., Boitano, S., Field, J. A., & Sierra-Alvarez, R. .. (2020). Cytotoxicity Assessment of Gallium- and Indium-Based Nanoparticles Toward Human Bronchial Epithelial Cells Using an Impedance-Based Real-Time Cell Analyzer. International Journal of Toxicology, 39(3), 218-231.
• Fernandez-Gonzalez, N. .., Sierra-Alvarez, R. .., Field, J. A., Amils, R., & Sanz, J. L. (2019). Adaptation of granular sludge microbial communities to nitrate, sulfide, and/or p-cresol removal. International Microbiology, 22(3), 305-316.
• Gonzalez-Estrella, J. .., Field, J. A., Ober, C. K., & Sierra-Alvarez, R. (2019). Stability and microbial toxicity of HfO2 and ZrO2 nanoparticles for photolithography. Green Materials, 7(3), 109-117.
• Jagadish, B., Field, J. A., Wong, S., Sierra-alvarez, R., Mash, E. A., Kadoya, W. M., & Abrell, L. (2019). Coupling reactions between reduced intermediates of insensitive munitions compound analog 4-nitroanisole.. Chemosphere, 222, 789-796. doi:10.1016/j.chemosphere.2019.01.163
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  Explosives, pesticides, and pharmaceuticals contain toxic nitroaromatic compounds that may form even more toxic azo compounds if they encounter reducing conditions in the environment. We investigated the mechanism by which 4,4'-dimethoxyazobenzene forms in anaerobic sludge incubations of 4-nitroanisole, an analog for the insensitive munitions compound 2,4-dinitroanisole (DNAN). Because studies have reported the mechanism to involve the coupling of reduced nitroaromatic intermediates, specifically aromatic amines and nitrosoaromatics, by nucleophilic processes, we abiotically paired 10 mM 4-aminoanisole with 2 mM 4-nitrosoanisole in nitrogen-flushed microcosms. However, only 7 μM of 4,4'-dimethoxyazobenzene had formed after 24 h. We identified the major product to be 4-methoxy-4'-nitrosodiphenylamine. Repeating this experiment in phosphate buffer at pH 5.1, 7.1, and 8.6 demonstrated that the formation of this unexpected product is acid catalyzed. We found that 4-methoxy-4'-nitrosodiphenylamine is more toxic than 4,4'-dimethoxyazobenzene to the bioluminescent bacterium Aliivibrio fischeri, with IC50 values of 0.1 μM and 0.5 μM, respectively. Both products are several orders of magnitude more toxic than reduced 4-nitroanisole intermediates 4-aminoanisole and 4-nitrosoanisole, as well as DNAN and its aromatic amine metabolites. Six-fold more 4,4'-dimethoxyazobenzene formed when we incubated 4-nitrosoanisole with ascorbic acid, a reducing agent, than when we incubated 4-nitrosoanisole with 4-aminoanisole in the absence of ascorbic acid. We therefore suspect that 4-hydroxylaminoanisole, the first reduction product of 4-nitrosoanisole, is a better nucleophile than 4-aminoanisole and couples more readily with 4-nitrosoanisole. Slightly basic and reducing conditions can prevent the formation and persistence of toxic coupling products on sites contaminated with nitroaromatics, i.e. DNAN-contaminated firing ranges.
• Kadoya, W. M., Sierra-Alvarez, R. .., Jagadish, B., Wong, S., Abrell, L., Mash, E. A., & Field, J. A. (2019). Coupling reactions between reduced intermediates of insensitive munitions compound analog 4-nitroanisole. Chemosphere, 222, 789-796.
• Madeira, C. L., Jog, K. V., Vanover, E. T., Brooks, M. D., Taylor, D. K., Sierra-Alvarez, R. .., Waidner, L. A., Spain, J. C., Krzmarzick, M. J., & Field, J. A. (2019). Microbial Enrichment Culture Responsible for the Complete Oxidative Biodegradation of 3-Amino-1,2,4-triazol-5-one (ATO), the Reduced Daughter Product of the Insensitive Munitions Compound 3-Nitro-1,2,4-triazol-5-one (NTO). Environmental Science and Technology, 53(21), 12648-12656.
• Madeira, C. L., Kadoya, W. M., Li, G., Wong, S., Sierra-Alvarez, R. .., & Field, J. A. (2019). Reductive biotransformation as a pretreatment to enhance in situ chemical oxidation of nitroaromatic and nitroheterocyclic explosives. Chemosphere, 222, 1025-1032.
• Nguyen, C. H., Field, J. A., & Sierra-Alvarez, R. (2019). Microbial toxicity of gallium- and indium-based oxide and arsenide nanoparticles. Journal of Environmental Science and Health, Part A, in press.
• Simon-Pascual, A. .., Sierra-Alvarez, R. .., & Field, J. A. (2019). Platinum(II) reduction to platinum nanoparticles in anaerobic sludge. Journal of Chemical Technology and Biotechnology, 94(2), 468-474.
• Ayala-Parra, P. .., Liu, Y., Sierra-Alvarez, R. .., & Field, J. A. (2018). Pretreatments to Enhance the Anaerobic Biodegradability of Chlorella protothecoides Algal Biomass. Environmental Progress, 37, 418-424.
• Kadoya, W. M., Sierra-Alvarez, R. .., Wong, S., Abrell, L. M., Mash Jr., ,. E., & Field, J. A. (2018). Evidence of Anaerobic Coupling Reactions between Reduced Intermediates of 4-Nitroanisole. Chemosphere, 195, 372-380.
• Khatiwada, R., Abrell, L., Guangbin, L., Root, R. A., Sierra-Alvarez, R. .., Field, J. A., & Chorover, J. (2018). Adsorption and oxidation of 3-nitro-1,2,4-triazole-5-one (NTO) and its transformation product (3-amino-1,2,4-triazole-5-one, ATO) at ferrihydrite and birnessite surfaces. Environmental Pollution, 240, 200-208.
• Khatiwada, R., Olivares, C., Abrell, L., Root, R. A., Sierra-Alvarez, R. .., Field, J. A., & Chorover, J. (2018). Oxidation of reduced daughter products from 2,4-dinitroanisole (DNAN) by Mn(IV) and Fe(III) oxides. Chemosphere, 201, 790-798.
• Khatiwada, R., Root, R. A., Abrell, L., Sierra-Alvarez, R. .., Field, J. A., & Chorover, J. (2018). Abiotic reduction of insensitive munition compounds by sulfate green rust.. Environmental Chemistry, 15, 259–266.
• Madeira, C. L., Field, J. A., Simonich, M. T., Tanguay, R. L., Chorover, J., & Sierra-Alvarez, R. (2018). Ecotoxicity of the insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) and its reduced metabolite 3-amino-1,2,4-triazol-5-one (ATO). J. Haz. Materials, 343, 340-346.
• Ramos-Ruiz, A., Field, J. A., Sun, W., & Sierra-Alvarez, R. (2018). Gallium arsenide (GaAs) Leaching Behavior and Surface Chemistry Changes in Response to pH and O2 Waste Management. Waste Management, 77, 1-9.
• Simon-Pascual, A., Sierra-Alvarez, R., Ramos-Ruiz, A., & Field, J. A. (2018). Reduction of platinum(IV) ions to elemental platinum nanoparticles by anaerobic sludge. Journal of Chemical Technology & Biotechnology, 93, 1611-1617.
• Zeng, C., Nguyen, C., Boitano, S., Field, J. A., Shadman, F., & Sierra-Alvarez, R. .. (2018). Cerium dioxide (CeO2) nanoparticles decrease arsenite (As(III)) cytotoxicity to 16HBE14o- human bronchial epithelial cells. Environmental Research, 164, 452-458.
• Ayala-Parra, P. .., Liu, Y., Sierra-Alvarez, R. .., & Field, J. A. (2017). Pretreatments to Enhance the Anaerobic Biodegradability of Chlorella protothecoides Algal Biomass. Environmental Progress, in press.
• Ayala-Parra, P., Liu, Y., Field, J. A., & Sierra-Alvarez, R. (2017). Nutrient recovery and biogas generation from the anaerobic digestion of waste biomass from algal biofuel production. Renewable Energy, 108, 410-416.
• Gonzalez-Estrella, J. .., Li, G., Neely, S. E., Puyol, D., Sierra-Alvarez, R. .., & Field, J. A. (2017). Elemental copper nanoparticle toxicity to anaerobic ammonium oxidation and the influence of ethylene diamine-tetra acetic acid (EDTA) on copper toxicity. Chemosphere, 184, 730-737.
• Li, G., Carvajal-Arroyo, J. M., Sierra-Alvarez, R. .., & Field, J. A. (2017). Mechanisms and control of NO2- inhibition of anaerobic ammonium oxidation (anammox). Water Environment Research, 89, 330-336.
• Madeira, C. L., Speet, S. A., Nieto, C. A., Abrell, L., Chorover, J., Sierra-Alvarez, R., & Field, J. A. (2017). Sequential anaerobic-aerobic biodegradation of emerging insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO). Chemosphere, 167, 478-484.
• Moreno-Ramírez, D., Vea, L., Field, J. A., Baker, P. B., Gandolfi, A. J., & Maier, R. M. (2017). Transferable Training Modules: Building Environmental Education Opportunities with and for Mexican Community Health Workers (Promotores de Salud). Family & Community Health, 40(4), 306-315.
• Olivares, C., Madeira, C., Sierra-Alvarez, R. .., Kadoya, W., Leif, A., Chorover, J., & Field, J. (2017). Environmental fate of 14C radiolabeled 2,4-dinitroanisole in soil microcosms. Environ. Sci. Technol., 51(22), 13327-€“13334.
• Ramos-Ruiz, A., Sesma-Martin, J., Sierra-Alvarez, R., & Field, J. A. (2017). Continuous reduction of tellurite to recoverable tellurium nanoparticles using an upflow anaerobic sludge bed (UASB) reactor. Water Research, 108, 189-196.
• Ramos-Ruiz, A., Wilkening, J. V., Field, J. A., & Sierra-Alvarez, R. (2017). Leaching of cadmium and tellurium from cadmium telluride (CdTe) thin-film solar panels under simulated landfill conditions. Journal of Hazardous Materials, 336, 57-64.
• Simon-Pascual, A., Sierra-Alvarez, R., Ramos-Ruiz, A., & Field, J. A. (2017). Reduction of platinum(IV) ions to elemental platinum nanoparticles by anaerobic sludge. Journal of Chemical Technology & Biotechnology, in press.
• Zeng, C., Gonzalez-Alvarez, A., Orenstein, E., Field, J. A., Shadman, F., & Sierra-Alvarez, R. (2017). Ecotoxicity assessment of ionic As(III), As(V), In(III) and Ga(III) species potentially released from novel III-V semiconductor materials. Ecotoxicology and Environmental Safety, 140, 30-36.
• Ayala-Parra, P., Sierra Alvarez, R., & Field, J. A. (2016). Algae as an electron donor promoting sulfate reduction for the bioremediation of acid rock drainage. Journal of Hazradous Materials, 317, 335–343.
• Ayala-Parra, P., Sierra-Alvarez, R., & Field, J. A. (2016). Treatment of acid rock drainage using a sulfate-reducing bioreactor with zero-valent iron. J. Haz. Materials, 308, 97-105.
• Gonzalez-Estrella, J., Gallagher, S., Sierra-Alvarez, R., & Field, J. A. (2016). Iron Sulfide Attenuates the Methanogenic Toxicity of Elemental Copper and Zinc Oxide Nanoparticles and their Soluble Metal Ion Analogs. Sci. Tot. Environ., 548-549, 380-389.
• Li, G., Carvajal-Arroyo, J. M., Sierra-Alvarez, R., & Field, J. A. (2016). Mechanisms and control of NO2- inhibition of anaerobic ammonium oxidation (anammox). Water Environment Research, in press.
• Li, G., Sierra-Alvarez, R., Vilcherrez, D., Weiss, S., Gill, C., Krzmarzick, M., Abrell, L., & Field, J. (2016). Nitrate reverses severe nitrite inhibition of anaerobic ammonium oxidation (Anammox) activity in continuously-fed bioreactors. Environ. Sci. Technol., 50(19), 10518-10526.
• Li, G., Vilcherrez, D., Carvajal-Arroyo, J. M., Sierra-Alvarez, R., & Field, J. A. (2016). Exogenous nitrate attenuates nitrite toxicity to anaerobic ammonium oxidizing (anammox) bacteria. Chemosphere, 144, 2360-2367.
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  E-Pub date 2015. Print date 2016
• Madeira, C. L., Speet, S. A., Nieto, C. A., Abrell, L. M., Chorover, J. D., Sierra-Alvarez, R., & Field, J. A. (2017). Sequential anaerobic-aerobic biodegradatin of emerging insenstive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO). Chemosphere, 167, 478-484.
• Moreno-Ramírez, D., Vea, L., Field, J. A., Baker, P. B., Gandolfi, A. J., & Maier, R. M. (2016). Transferable Training Modules: Building Environmental Education Opportunities with and for Mexican Community Health Workers (Promotores de Salud). Family & Community Health, in press.
• Ochoa-Herrera, V., Field, J. A., Luna-Velasco, A., & Sierra-Alvarez, R. (2016). Microbial toxicity and biodegradability of perfluorooctane sulfonate (PFOS) and shorter chain perfluoroalkyl and polyfluoroalkyl substances (PFASs). Environmental Science: Processes & Impacts, 18(9), 1236-1246.
• Olivares, C. I., Abrell, L. M., Khatiwada, R., Chorover, J. D., Sierra-Alvarez, R., & Field, J. A. (2016). (Bio)transformation of 2,4-dinitroanisole (DNAN) in soils. J Haz Mater, 304, 214-221.
• Olivares, C. I., Abrell, L., Khatiwada, R., Chorover, J., Sierra-Alvarez, R., & Field, J. A. (2016). (Bio)transformation of 2,4-dinitroanisole (DNAN) in Soils. J. Haz. Materials, 304, 214-221.
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  E-Pub date 2015. Print date 2016
• Olivares, C. I., Field, J. A., Simonich, M., Tanguay, R. L., & Sierra-Alvarez, R. (2016). Arsenic (III, V), indium (III), and gallium (III) toxicity to zebrafish embryos using a high-throughput multi-endpoint in vivo developmental and behavioral assay. Chemosphere, 148, 361-368.
• Olivares, C. I., Sierra-Alvarez, R., Abrell, L. M., Chorover, J. D., Simonich, M., Tanguay, R. L., & Field, J. A. (2016). Zebrafish embryo toxicity of anaerobic biotransformation products from the insensitive munitions compout 2,4-dintroanisole. Environ. Toxicol. Chem., 35, 2774-2781.
• Olivares, C. I., Sierra-Alvarez, R., Alvarez-Nieto, C., Abrell, L. M., Chorover, J. D., & Field, J. A. (2016). Microbial toxicity and characterization of DNAN (bio)transformation product mixtures. Chemosphere, 154, 499-506.
• Olivares, C. I., Sierra-Alvarez, R., Alvarez-Nieto, C., Abrell, L., Chorover, J., & Field, J. A. (2016). Microbial toxicity and characterization of DNAN (bio)transformation product mixtures. Chemosphere, 154, 499-506.
• Olivares, C. I., Wang, J., Silva Luna, C. D., Field, J. A., Abrell, L., & Sierra-Alvarez, R. (2016). Continuous treatment of the insensitive munitions compound N-methyl-p-nitro aniline (MNA) in an upflow anaerobic sludge blanket (UASB) bioreactor. Chemosphere, 144, 1116-1122.
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  E-Pub date 2015. Print date 2016
• Olivares, C., Sierra-Alvarez, R., Abrell, L., Chorover, J., Simonich, M., Tanguay, R., & Field, J. (2016). Zebrafish embryo toxicity of anaerobic biotransformation products from the insensitive munitions compound 2,4-dinitroanisole (DNAN). Environmental Toxicology and Chemistry, 35:(11), 2774-2781.
• Pat-Espadas, A. M., Field, J. A., Otero-Gonzalez, L., Razo-Flores, E., Cervantes, F. J., & Sierra-Alvarez, R. (2016). Recovery of palladium(II) by methanogenic granular sludge. Chemosphere, 144, 745–753.
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  E-Pub date 2015. Print date 2016
• Pat-Espadas, A. M., Field, J. A., Razo-Flores, E., Cervantes, F. J., & Sierra-Alvarez, R. (2016). Continuous removal and recovery of palladium in an upflow anaerobic granular sludge bed (UASB) reactor. Journal of Chemical Technology & Biotechnology, 91, 1183-1189.
• Ramos-Ruiz, A., Field, J. A., Wilkening, J. V., & Sierra-Alvarez, R. (2016). Recovery of Elemental Tellurium Nanoparticles by the Reduction of Tellurium Oxyanions in a Methanogenic Microbial Consortium. Environmental Science & Technology, 50(3), 1492-1500.
• Ramos-Ruiz, A., Zeng, C., Sierra-Alvarez, R., Teixeira, L. H., & Field, J. A. (2016). Microbial toxicity of ionic species leached from the II-VI semiconductor materials, cadmium telluride (CdTe) and cadmium selenide (CdSe). Chemosphere, 162, 131-138.
• Rodriguez-Freire, L., Moore, S. E., Sierra-Alvarez, R., Root, R. A., Chorover, J. D., & Field, J. A. (2016). Arsenic remediation by formation of arsenic sulfide minerals in a continous anaerobic bioreactor. Biotechnol. Bioengineer., 113, 522-530.
• Rodriguez-Freire, L., Moore, S. E., Sierra-Alvarez, R., Root, R. A., Chorover, J., & Field, J. A. (2016). Arsenic remediation by formation of arsenic sulfide minerals in a continuous anaerobic bioreactor. Biotechnology and Bioengineering, 113(3), 522-530.
• Gonzalez-Estrella, J., Puyol, D., Gallagher, R., & Field, J. A. (2015). Elemental copper nanoparticle toxicity to different trophic groups involved in anaerobic and anoxic wastewater treatment processes. Sci. Tot. Environ., 512-513, 308–315.
• Gonzalez-Estrella, J., Puyol, D., Sierra-Alvarez, R., & Field, J. A. (2015). Role of biogenic sulfide in attenuating zinc oxide and copper nanoparticle toxicity to acetoclastic methanogenesis. J. Haz. Materials, 283, 755-763.
• Gonzalez-Estrella, J., Puyol, D., Sierra-Alvarez, R., & Field, J. A. (2015). Role of biogenic sulfide in attenuating zinc oxide and copper nanoparticle toxicity to acetoclastic methanogenesis. JOURNAL OF HAZARDOUS MATERIALS, 283, 755-763.
• Kzmarzick, M. J., Khatiwada, J. R., Olivares, C. I., Abrell, L., Sierra Alvarez, M. R., Chorover, J. D., & Field, J. A. (2015). Biotransformation and degradation of the insensitive munitions compound, 3-nitro-1,2,4-triazol-5-one, by soil bacterial communities.. Environ. Sci. Technol., 49, 5681-5688.
• Linker, B. R., Khatiwada, R., Perdrial, N., Abrell, L., Sierra-Alvarez, R., Field, J. A., & Chorover, J. (2015). Adsorption of novel insensitive munitions compounds at clay mineral and metal oxide surfaces. ENVIRONMENTAL CHEMISTRY, 12(1), 74-84.
• Otero-Gonzalez, L., Field, J. A., Calderon, I. A., Aspinwall, C. A., Shadman, F., C., Z., & Sierra-Alvarez, R. (2015). Fate of fluorescent core-shell silica nanoparticles during simulated secondary wastewater treatment. Water Research, 77, 170-178.
• Rodriguez-Freire, L., Moore, S. E., Sierra-Alvarez, R., & Field, J. A. (2015). Adaptation of a methanogenic consortium to arsenite inhibition. Water, Air, & Soil Pollution, 226, in press.
• Zeng, C. C., Huynguyen, C., Boitano, S. A., Field, J. A., Shadman, F., & Sierra Alvarez, M. R. (2017). Cerium dioxide (CeO2) nanoparticles decrease arsenite (As(III)) cytotoxicity to 16HBE14o- human bronchial epithleial cells. Environ Res, 164, 452-458. doi:10.1016/j.envres.2018.03.007
• Zeng, C., Ramos-Ruiz, A., Field, J. A., & Sierra-Alvarez, R. (2015). Cadmium telluride (CdTe) and cadmium selenide (CdSe) leaching behavior and surface chemistry in response to pH and O2. Journal of Environmental Management, 154, 78-85.
• Zeng, C., Ramos-Ruiz, A., Field, J. A., & Sierra-Alvarez, R. (2015). Response to the comments on "Cadmium telluride leaching behavior: Discussion of Zeng et al. (2015)". Journal of Environmental Management, 164, 65-66.
• Abrell, L. M., Mash, E. A., Field, J. A., Chorover, J. D., Sierra Alvarez, M. R., & Bhumasamudram, J. (2014). Synthesis of 13C and 15N labeled 2,4-dinitroanisole. Journal of Labelled Compounds and Radiopharmaceuticals.
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  Abstract Syntheses of [13C6]-2,4-dinitroanisole (ring-13C6) from [13C6]-anisole (ring-13C6) and[15N2]-2,4-dinitroanisole from anisole using in situ generated acetyl nitrate and [15N]-acetylnitrate, respectively, are described. Treatment of [13C6]-anisole (ring-13C6) with acetyl nitrategenerated in 100% HNO3 gave [13C6]-2,4-dinitroanisole (ring-13C6) in 83% yield. Treatment ofanisole with [15N]-acetyl nitrate generated in 10N [15N]-HNO3 gave [15N2]-2,4-dinitroanisole in44% yield after two cycles of nitration. Byproducts in the latter reaction included [15N]-2-nitroanisole and [15N]-4-nitroanisole.
• Billy, L., Raju, K., Nicholas, P., Field, J. A., Abrell, L. M., & Chorover, J. D. (2014). Adsorption of Novel Insensitive Munitions Compounds at Clay Mineral and Metal Oxide Surfaces. Environmental Chemistry.
• Carvajal-Arroyo, J. M., Puyol, D., Li, G., Lucero-Acu\~na, A., Sierra-\'Alvarez, R., & Field, J. A. (2014). Pre-exposure to nitrite in the absence of ammonium strongly inhibits anammox. Water research, 48, 52--60.
• Carvajal-Arroyo, J. M., Puyol, D., Li, G., Sierra-\'Alvarez, R., & Field, J. A. (2014). The intracellular proton gradient enables anaerobic ammonia oxidizing (anammox) bacteria to tolerate NO 2- inhibition. Journal of biotechnology, 192, 265--267.
• Carvajal-Arroyo, J. M., Puyol, D., Li, G., Sierra-\'Alvarez, R., & Field, J. A. (2014). The role of pH on the resistance of resting-and active anammox bacteria to NO2- inhibition. Biotechnology and bioengineering, 111(10), 1949--1956.
• Carvajal-Arroyo, J. M., Puyol, D., Li, G., Swartwout, A., Sierra-\'Alvarez, R., & Field, J. A. (2014). Starved anammox cells are less resistant to inhibition. Water research, 65, 170--176.
• Chorover, J. D., Abrell, L. M., Field, J. A., Nicholas, P., Raju, K., & Billy, L. (2015). Adsorption of Novel Insensitive Munitions Compounds at Clay Mineral and Metal Oxide Surfaces. Environmental Chemistry, 12, 74-84.
• Jagadish, B., Field, J. A., Chorover, J., Sierra-Alvarez, R., Abrell, L., & Mash, E. A. (2014). Synthesis of 13C and 15N labeled 2, 4-dinitroanisole. Journal of Labelled Compounds and Radiopharmaceuticals, 57(6), 434--436.
• Li, G., Puyol, D., Carvajal-Arroyo, J. M., Sierra-Alvarez, R., & Field, J. A. (2014). Inhibition of anaerobic ammonium oxidation by heavy metals. Journal of Chemical Technology and Biotechnology.
• Maier, R. M., Diaz-Barriga, F., Field, J. A., Hopkins, J. C., Klein, B., & Poulton, M. M. (2014). Socially responsible mining: the relationship between mining and poverty, human health and the environment. Reviews on Environmental Health, 29, 83-89.
• Otero-Gonz\'alez, L., Barbero, I., Field, J. A., Shadman, F., & Sierra-Alvarez, R. (2014). Stability of alumina, ceria, and silica nanoparticles in municipal wastewater. Water Science \& Technology, 70(9), 1533--1539.
• Otero-Gonz\'alez, L., Field, J. A., & Sierra-Alvarez, R. (2014). Fate and long-term inhibitory impact of ZnO nanoparticles during high-rate anaerobic wastewater treatment. Journal of environmental management, 135, 110--117.
• Otero-Gonz\'alez, L., Field, J. A., & Sierra-Alvarez, R. (2014). Inhibition of anaerobic wastewater treatment after long-term exposure to low levels of CuO nanoparticles. Water research, 58, 160--168.
• Puyol, D., Carvajal-Arroyo, J., Garcia, B., Sierra-Alvarez, R., & Field, J. (2014). Kinetics and thermodynamics of anaerobic ammonium oxidation process using Brocadia spp. dominated mixed cultures. Water Science \& Technology, 69(8), 1682--1688.
• Puyol, D., Carvajal-Arroyo, J., Li, G., Dougless, A., Fuentes-Velasco, M., Sierra-Alvarez, R., & Field, J. (2014). High pH (and not free ammonia) is responsible for Anammox inhibition in mildly alkaline solutions with excess of ammonium. Biotechnology letters, 36(10), 1981--1986.
• Puyol, D., Carvajal-Arroyo, J., Sierra-Alvarez, R., & Field, J. (2014). Nitrite (not free nitrous acid) is the main inhibitor of the anammox process at common pH conditions. Biotechnology letters, 36(3), 547--551.
• Rodriguez-Freire, L., Sierra Alvarez, M. R., Root, R., Chorover, J. D., & Field, J. A. (2014). Biomineralization of arsenate to arsenic sulfides is greatly enhanced at mildly acidic conditions. Wat. Res., 66, 242-253.
• Rodriguez-Freire, L., Sierra-Alvarez, R., Root, R., Chorover, J., & Field, J. A. (2014). Biomineralization of arsenate to arsenic sulfides is greatly enhanced at mildly acidic conditions. Water Research, 66, 242-253.
• Banihani, Q. H., & Field, J. A. (2013). Treatment of high-strength synthetic sewage in a laboratory-scale upflow anaerobic sludge bed (UASB) with aerobic activated sludge (AS) post-treatment. Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and Environmental Engineering, 48(3), 338-347.
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  PMID: 23245309;Abstract: Performance of a combined system up-flow anaerobic sludge blanket (UASB) followed by aerobic treatment activated sludge (AS) for removal of carbonaceous and nitrogenous contaminants at an average temperature of 25C was investigated. The combined system was fed with high strength synthetic sewage having chemical oxygen demand (COD) of 2500mg L1. The organic loading rate (OLR) of the UASB reactor was increased gradually from 1.1 to 3.8 gCOD Lr1 d1. At steady state condition, the UASB reactor achieved removal efficiency up to 83.5% of total COD (CODtot), 74.0% of volatile fatty acid (VFA) and 94.0% of protein. The combined system performed an excellent organic removal pushing the overall removal efficiency of COD tot, VFA and protein to 91.0%, 99.9% and 98.2%, respectively. When the OLR of the UASB increased to 4.4g COD Lr1 d 1, the UASB was overloaded and; thus, its effluent quality deteriorated. In respect to nitrogen removal, both partial nitrification and complete nitrification took place in aerobic post-treatment. When the dissolved oxygen (DO) concentration was >2.0mg L1, complete nitrification (period B) occurred with an average nitrification efficiency of 96.2%. The partial nitrification occurred due to high OLR to AS during the overloading event (period A) and when DO concentration was
• Field, J., Banihani, Q. H., & Field, J. A. (2013). Treatment of high-strength synthetic sewage in a laboratory-scale upflow anaerobic sludge bed (UASB) with aerobic activated sludge (AS) post-treatment. Journal of environmental science and health. Part A, Toxic/hazardous substances & environmental engineering, 48(3).
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  Performance of a combined system up-flow anaerobic sludge blanket (UASB) followed by aerobic treatment activated sludge (AS) for removal of carbonaceous and nitrogenous contaminants at an average temperature of 25°C was investigated. The combined system was fed with high strength synthetic sewage having chemical oxygen demand (COD) of 2500 mg L(-1). The organic loading rate (OLR) of the UASB reactor was increased gradually from 1.1 to 3.8 gCOD L(r) (-1) d(-1). At steady state condition, the UASB reactor achieved removal efficiency up to 83.5% of total COD (COD(tot)), 74.0% of volatile fatty acid (VFA) and 94.0% of protein. The combined system performed an excellent organic removal pushing the overall removal efficiency of COD(tot), VFA and protein to 91.0%, 99.9% and 98.2%, respectively. When the OLR of the UASB increased to 4.4 g COD L(r) (-1) d(-1), the UASB was overloaded and; thus, its effluent quality deteriorated. In respect to nitrogen removal, both partial nitrification and complete nitrification took place in aerobic post-treatment. When the dissolved oxygen (DO) concentration was >2.0 mg L(-1), complete nitrification (period B) occurred with an average nitrification efficiency of 96.2%. The partial nitrification occurred due to high OLR to AS during the overloading event (period A) and when DO concentration was
• Field, J., Carvajal-Arroyo, J. M., Puyol, D., Li, G., Lucero-Acuña, A., Sierra-Álvarez, R., & Field, J. A. (2013). Pre-exposure to nitrite in the absence of ammonium strongly inhibits anammox. Water research.
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  Anaerobic ammonium oxidizing bacteria (Anammox) are known to be inhibited by their substrate, nitrite. However, the mechanism of inhibition and the physiological conditions under which nitrite impacts the performance of anammox bioreactors are still unknown. This study investigates the role of pre-exposing anammox bacteria to nitrite alone on their subsequent activity and metabolism after ammonium has been added. Batch experiments were carried out with anammox granular biofilm pre-exposed to nitrite over a range of concentrations and durations in the absence of ammonium. The effect of pre-exposure to nitrite alone compared to nitrite simultaneously fed with ammonium was evaluated by measuring the anammox activity and the accumulation of the intermediate, nitric oxide. The results show that the inhibitory effect was more dramatic when bacteria were pre-exposed to nitrite in absence of ammonium, as revealed by the lower activity and the higher accumulation of nitric oxide. The nitrite concentration causing 50% inhibition was 53 and 384 mg N L(-1) in the absence or the presence of ammonium, respectively. The nitrite inhibition was thus 7.2-fold more severe in the absence of ammonium. Biomass exposure to nitrite (25 mg N L(-1)), in absence of ammonium, led to accumulation of nitric oxide. On the other hand when the biomass was exposed to nitrite in presence of ammonium, accumulation of nitric oxide was only observed at much higher nitrite concentrations (500 mg N L(-1)). The inhibitory effect of nitrite in the absence of ammonium was very rapid. The rate of decay of the anammox activity was equivalent to the diffusion rate of nitrite up to 46% of activity loss. The results taken as a whole suggest that nitrite inhibition is more acute when anammox cells are not actively metabolizing. Accumulation of nitric oxide in the headspace most likely indicates disruption of the anammox biochemistry by nitrite inhibition, caused by an interruption of the hydrazine synthesis step.
• Field, J., Carvajal-Arroyo, J. M., Sun, W., Sierra-Alvarez, R., & Field, J. A. (2013). Inhibition of anaerobic ammonium oxidizing (anammox) enrichment cultures by substrates, metabolites and common wastewater constituents. Chemosphere, 91(1).
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  Anaerobic ammonium oxidation (anammox) is an emerging technology for nitrogen removal that provides a more environmentally sustainable and cost effective alternative compared to conventional biological treatment methods. The objective of this study was to investigate the inhibitory impact of anammox substrates, metabolites and common wastewater constituents on the microbial activity of two different anammox enrichment cultures (suspended and granular), both dominated by bacteria from the genus Brocadia. Inhibition was evaluated in batch assays by comparing the N(2) production rates in the absence or presence of each compound supplied in a range of concentrations. The optimal pH was 7.5 and 7.3 for the suspended and granular enrichment cultures, respectively. Among the substrates or products, ammonium and nitrate caused low to moderate inhibition, whereas nitrite caused almost complete inhibition at concentrations higher than 15 mM. The intermediate, hydrazine, either stimulated or caused low inhibition of anammox activity up to 3mM. Of the common constituents in wastewater, hydrogen sulfide was the most severe inhibitor, with 50% inhibitory concentrations (IC(50)) as low as 0.03 mM undissociated H(2)S. Dissolved O(2) showed moderate inhibition (IC(50)=2.3-3.8 mg L(-1)). In contrast, phosphate and salinity (NaCl) posed very low inhibition. The suspended- and granular anammox enrichment cultures had similar patterns of response to the various inhibitory stresses with the exception of phosphate. The findings of this study provide comprehensive insights on the tolerance of the anammox process to a wide variety of potential inhibiting compounds.
• Field, J., Gonzalez-Estrella, J., Sierra-Alvarez, R., & Field, J. A. (2013). Toxicity assessment of inorganic nanoparticles to acetoclastic and hydrogenotrophic methanogenic activity in anaerobic granular sludge. Journal of hazardous materials, 260.
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  Release of engineered nanoparticles (NPs) to municipal wastewater from industrial and residential sources could impact biological systems in wastewater treatment plants. Methanogenic inhibition can cause failure of anaerobic waste(water) treatment. This study investigated the inhibitory effect of a wide array of inorganic NPs (Ag(0), Al₂O₃, CeO₂, Cu(0), CuO, Fe(0), Fe₂O₃, Mn₂O₃, SiO₂, TiO₂, and ZnO supplied up to 1500 mgL(-1)) to acetoclastic and hydrogenotrophic methanogenic activity of anaerobic granular sludge. Of all the NPs tested, only Cu(0) and ZnO caused severe methanogenic inhibition. The 50% inhibiting concentrations determined towards acetoclastic and hydrogenotrophic methanogens were 62 and 68 mgL(-1) for Cu(0) NP; and 87 and 250 mgL(-1) for ZnO NP, respectively. CuO NPs also caused inhibition of acetoclastic methanogens. Cu(2+) and Zn(2+) salts caused similar levels of inhibition as Cu(0) and ZnO NPs based on equilibrium soluble metal concentrations measured during the assays, suggesting that the toxicity was due to the release of metal ions by NP-corrosion. A commercial dispersant, Dispex, intended to increase NP stability did not affect the inhibitory impact of the NPs. The results taken as a whole suggest that Zn- and Cu-containing NPs can release metal ions that are inhibitory for methanogenesis.
• Field, J., Olivares, C., Liang, J., Abrell, L., Sierra-Alvarez, R., & Field, J. A. (2013). Pathways of reductive 2,4-dinitroanisole (DNAN) biotransformation in sludge. Biotechnology and bioengineering, 110(6).
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  As the use of the insensitive munition compound 2,4-dinitroanisole (DNAN) increases, releases to the environment may pose a threat to local ecosystems. Little is known about the environmental fate of DNAN and the conversions caused by microbial activity. We studied DNAN biotransformation rates in sludge under aerobic, microaerophilic, and anaerobic conditions, detected biotransformation products, and elucidated their chemical structures. The biotransformation of DNAN was most rapid under anaerobic conditions with H2 as a cosubstrate. The results showed that the ortho nitro group in DNAN is regioselectively reduced to yield 2-methoxy-5-nitroaniline (MENA), and then the para nitro group is reduced to give 2,4-diaminoanisole (DAAN). Both MENA and DAAN were identified as important metabolites in all redox conditions. Azo and hydrazine dimer derivatives formed from the coupling of DNAN reduction products in anaerobic conditions. Secondary pathways included acetylation and methylation of amine moieties, as well as the stepwise O-demethylation and dehydroxylation of methoxy groups. Seven unique metabolites were identified which enabled elucidation of biotransformation pathways. The results taken as a whole suggest that reductive biotransformation is an important fate of DNAN leading to the formation of aromatic amines as well as azo and hydrazine dimeric metabolites.
• Field, J., Sun, W., Luna-Velasco, A., Sierra-Alvarez, R., & Field, J. A. (2013). Assessing protein oxidation by inorganic nanoparticles with enzyme-linked immunosorbent assay (ELISA). Biotechnology and bioengineering, 110(3).
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  Growth in the nanotechnology industry is leading to increased production of engineered nanoparticles (NPs). This has given rise to concerns about the potential adverse and toxic effects to biological system and the environment. An important mechanism of NP toxicity is oxidative stress caused by the formation of reactive oxygen species (ROS) or via direct oxidation of biomolecules. In this study, a protein oxidation assay was developed as an indicator of biomolecule oxidation by NPs. The oxidation of the protein, bovine serum albumin (BSA) was evaluated with an enzyme-linked immunosorbent assay (ELISA) to measure the protein carbonyl derivatives formed from protein oxidation. The results showed that some NPs such as Cu(0), CuO, Mn(2)O(3), and Fe(0) caused oxidation of BSA; whereas, many of the other NPs tested were not reactive or very slowly reactive with BSA. The mechanisms involved in the oxidation of BSA protein by the reactive NPs could be attributed to the combined effects of ROS-dependent and direct protein oxidation mechanisms. The ELISA assay is a promising method for the assessment of protein oxidation by NPs, which can provide insights on NP toxicity mechanisms.
• Puyol, D., Carvajal-Arroyo, J., Garcia, B., Sierra-Alvarez, R., & Field, J. A. (2013). Kinetic characterization of Brocadia spp.-dominated anammox cultures. Bioresource Technology, 139, 94-100.
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  PMID: 23648757;Abstract: In this study, kinetic analyses were conducted for two Brocadia-dominated enrichment cultures, granular and flocculent, retrieved from lab-scale anaerobic ammonium oxidation (anammox) reactors. Substrate KS ranged from 0.35 to 0.69mMN and VSmax ranged from 0.67 to 0.88mmolNg-1VSSh-1. The model respected the experimentally measured stoichiometry of N-compounds, serving as an independent validation. Growth kinetics of the flocculent sludge was also studied, which indicates a μmax of 0.0984d-1 and a YX/S of 0.105mol C-biomass mol-1 NH4+. The flocculent enrichment culture was used to determine the stoichiometric equation. The kinetic parameters of the Brocadia spp. cultures measured here can be used for optimizing applications of the anammox process. © 2013 Elsevier Ltd.
• Sierra-alvarez, R., Otero-gonzalez, L., Garcia-saucedo, C., & Field, J. A. (2013). Toxicity of TiO₂, ZrO₂, Fe⁰, Fe₂O₃, and Mn₂O₃ nanoparticles to the yeast, Saccharomyces cerevisiae.. Chemosphere, 93(6), 1201-6. doi:10.1016/j.chemosphere.2013.06.075
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  The growing application of engineered nanomaterials is leading to an increased occurrence of nanoparticles (NPs) in the environment. Thus, there is a need to better understand their potential impact on the environment. This study evaluated the toxicity of nanosized TiO₂, ZrO₂, Fe(0), Fe₂O₃, and Mn₂O₃ towards the yeast Saccharomyces cerevisiae based on O₂ consumption and cell membrane integrity. In addition, the state of dispersion of the nanoparticles in the bioassay medium was characterized. All the nanomaterials showed high tendency to aggregate in the bioassay medium. A non-toxic polyacrylate dispersant was used to improve the NP dispersion stability and test the influence of the aggregation state in their toxicity. Mn₂O₃ NPs showed the highest inhibition of O₂ consumption (50% at 170 mg L(-1)) and cell membrane damage (approximately 30% of cells with compromised membrane at 1000 mg L(-1)), while the other NPs caused low (Fe(0)) or no toxicity (TiO₂, ZrO₂, and Fe₂O₃) to the yeast. Dispersant supplementation decreased the inhibition caused by Mn₂O₃ NPs at low concentrations, which could indicate that dispersant association with the particles may have an impact on the interaction between the NPs and the cells.
• Field, J., Otero-González, L., Sierra-Alvarez, R., Boitano, S., & Field, J. A. (2012). Application and validation of an impedance-based real time cell analyzer to measure the toxicity of nanoparticles impacting human bronchial epithelial cells. Environmental science & technology, 46(18).
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  Nanomaterials are increasingly used in a variety of industrial processes and consumer products. There are growing concerns about the potential impacts for public health and environment of engineered nanoparticles. The aim of this work was to evaluate a novel impedance-based real time cell analyzer (RTCA) as a high-throughput method for screening the cytotoxicity of nanoparticles and to validate the RTCA results using a conventional cytotoxicity test (MTT). A collection of 11 inorganic nanomaterials (Ag(0), Al(2)O(3), CeO(2), Fe(0), Fe(2)O(3), HfO(2), Mn(2)O(3), SiO(2), TiO(2), ZnO, and ZrO(2)) were tested for potential cytotoxicity to a human bronchial epithelial cell, 16HBE14o-. The data collected by the RTCA system was compared to results obtained using a more traditional methyl tetrazolium (MTT) cytotoxicity assay at selected time points following application of nanomaterials. The most toxic nanoparticles were ZnO, Mn(2)O(3) and Ag(0), with 50% response at concentrations lower than 75 mg/L. There was a good correlation in cytotoxicity measurements between the two methods; however, the RTCA method maintained a distinct advantage in continually following cytotoxicity over time. The results demonstrate the potential and validity of the impedance-based RTCA technique to rapidly screen for nanoparticle toxicity.
• Field, J., Rodríguez-Freire, L., Sun, W., Sierra-Alvarez, R., & Field, J. A. (2012). Flexible bacterial strains that oxidize arsenite in anoxic or aerobic conditions and utilize hydrogen or acetate as alternative electron donors. Biodegradation, 23(1).
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  Arsenic is a carcinogenic compound widely distributed in the groundwater around the world. The fate of arsenic in groundwater depends on the activity of microorganisms either by oxidizing arsenite (As(III)), or by reducing arsenate (As(V)). Because of the higher toxicity and mobility of As(III) compared to As(V), microbial-catalyzed oxidation of As(III) to As(V) can lower the environmental impact of arsenic. Although aerobic As(III)-oxidizing bacteria are well known, anoxic oxidation of As(III) with nitrate as electron acceptor has also been shown to occur. In this study, three As(III)-oxidizing bacterial strains, Azoarcus sp. strain EC1-pb1, Azoarcus sp. strain EC3-pb1 and Diaphorobacter sp. strain MC-pb1, have been characterized. Each strain was tested for its ability to oxidize As(III) with four different electron acceptors, nitrate, nitrite, chlorate and oxygen. Complete As(III) oxidation was achieved with both nitrate and oxygen, demonstrating the novel ability of these bacterial strains to oxidize As(III) in either anoxic or aerobic conditions. Nitrate was only reduced to nitrite. Different electron donors were used to study their suitability in supporting nitrate reduction. Hydrogen and acetate were readily utilized by all the cultures. The flexibility of these As(III)-oxidizing bacteria to use oxygen and nitrate to oxidize As(III) as well as organic and inorganic substrates as alternative electron donors explains their presence in non-arsenic-contaminated environments. The findings suggest that at least some As(III)-oxidizing bacteria are flexible with respect to electron-acceptors and electron-donors and that they are potentially widespread in low arsenic concentration environments.
• Field, J., Sun, W., Banihani, Q., Sierra-Alvarez, R., & Field, J. A. (2011). Stoichiometric and molecular evidence for the enrichment of anaerobic ammonium oxidizing bacteria from wastewater treatment plant sludge samples. Chemosphere, 84(9).
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  Anammox enrichments were readily developed from seven municipal wastewater treatment plants (WWTPs) sludge, but not with methanogenic granular sludge from two agro-industrial WWTPs. Only 50d was required for the first evidence of anammox activity from a return activated sludge obtained from a WWTP operated for nutrient removal. The molar ratios of nitrite and ammonium consumption of approximately 1.32 as well as nitrate and dinitrogen gas product ratios of approximately 0.095 provided evidence of the anammox reaction. The presence of anammox was confirmed by polymerase chain reaction (PCR) using primer sets (PLA46F and AMX820R) specific for anammox bacteria. The 16S rRNA gene fragment of anammox bacteria was detected in seven enrichment cultures (ECs) with demonstrated anammox activity but not in the original inocula from which the ECs were derived and also not in the two methanogenic sludge samples, which indicates the PCR predicted the anammox activity. Two genera, Brocadia and Kuenenia, were successfully identified as the Planctomycetes occurring in the clone libraries of successful anammox enrichments. Brocadia dominated in cultures that were respiked extensively; whereas Kuenenia predominated in cultures that were less aggressively respiked. These findings indicate that respiking management may play an important role on selecting the genus of anammox bacteria. The batch enrichment results clearly illustrate that anammox can be readily enriched from municipal sludge from a wide variety of process operations at WWTPs.
• Field, J., Sun, W., Sierra-Alvarez, R., & Field, J. A. (2011). Long term performance of an arsenite-oxidizing-chlorate-reducing microbial consortium in an upflow anaerobic sludge bed (UASB) bioreactor. Bioresource technology, 102(8).
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  A chlorate (ClO(3)(-)) reducing microbial consortium oxidized arsenite (As(III)) to arsenate (As(V)) in an upflow anaerobic sludge-bed bioreactor over 550 days operation. As(III) was converted with high conversion efficiencies (>98%) at volumetric loadings ranging from 0.45 to 1.92 mmol As/(L(reactor)d). The oxidation of As(III) was linked to the complete reduction of ClO(3)(-) to Cl(-) and H(2)O, as demonstrated by a molar ratio of approximately 3.0 mol As(III) oxidized per mole of Cl(-) formed and by the greatly lowered ClO(3)(-)-reducing capacity without As(III) feeding. An autotrophic enrichment culture was established from the bioreactor biofilm. A 16S rRNA gene clone library indicated that the culture was dominated by Dechloromonas, and Stenotrophomonas as well as genera within the family Comamonadaceae. The results indicate that the oxidation of As(III) to less mobile As(V) utilizing ClO(3)(-) as a terminal electron acceptor provides a sustainable bioremediation strategy for arsenic contamination in anaerobic environments.
• Halalsheh, M., Kassab, G., Yazajeen, H., Qumsieh, S., & Field, J. (2011). Effect of increasing the surface area of primary sludge on anaerobic digestion at low temperature. Bioresource Technology, 102(2), 748-752.
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  PMID: 20863692;Abstract: Two sets of reactors were operated at 15°C and at sludge retention times (SRTs) of 65days and 30days, respectively. Each set was operated at six different mixing velocities. Shear forces provided by mixing affected particle size distribution and resulted in different sludge surface areas. The aim was to investigate the effect of increasing primary sludge surface area on anaerobic digestion at low temperature. The maximum surface areas at the applied mixing velocities were 5926cm2/cm3of sludge and 4672cm2/cm3 of sludge at SRTs of 65days and 30days, respectively. The corresponding calculated methanogenesis were 49% and 15% at SRTs of 65days and 30days, respectively. Maximum total solids (TS) reductions were 26% and 11% at 65days and 30days SRTs, respectively. Lipase activity increased with increasing SRT and sludge surface area. Results clearly showed that increasing sludge surface area improved sludge digestion at 15°C. © 2010 Elsevier Ltd.
• Tordable-martinez, V., Tapia-rodriguez, A., Sun, W., Sierra-alvarez, R., & Field, J. A. (2011). Uranium bioremediation in continuously fed upflow sand columns inoculated with anaerobic granules.. Biotechnology and bioengineering, 108(11), 2583-91. doi:10.1002/bit.23225
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  Reductive precipitation of soluble hexavalent uranium (U(VI)) to insoluble tetravalent uranium (U(IV)) containing minerals is one of the more promising approaches to uranium remediation. The objective of this study was to evaluate the long-term performance of methanogenic granules for the continuous treatment of U(VI). For this purpose, three sand-packed columns inoculated with anaerobic biofilm were operated with or without ethanol and one column was exposed to nitrate co-contamination. The columns were operated for 373 days and efficiently removed U (24 mg L(-1)) in excess of 99.8%. No long-term benefit of ethanol addition was observed, suggesting that endogenous substrates in the biofilm were sufficient to drive the reduction reactions. Nitrate addition was found to inhibit U(VI) reduction and cause re-oxidation of some U(IV) deposited in the column. Taken as a whole, the results indicate that methanogenic biofilms can be reliably applied in bioreactor technology for sustained U removal from groundwater.
• Field, J., Luna-Velasco, A., Sierra-Alvarez, R., Castro, B., & Field, J. A. (2010). Removal of nitrate and hexavalent uranium from groundwater by sequential treatment in bioreactors packed with elemental sulfur and zero-valent iron. Biotechnology and bioengineering, 107(6).
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  The bioreduction of soluble hexavalent uranium (U(VI)) to insoluble tetravalent uranium (U(IV)) is an attractive bioremediation strategy for the clean-up of contaminated groundwater. High levels of the common occurring co-contaminant, nitrate (NO3(-)), can potentially interfere with uranium bioremediation. In this study, treatment of a synthetic groundwater containing a mixture of NO3(-) and U(VI) was investigated in a sulfur-limestone autotrophic denitrifying (SLAD) bioreactor that was coupled in series with a bioreactor packed with zero-valent iron (Fe(0), ZVI) and sand. An additional aim of the study was to explore the possible role of biological activity in enhancing the reduction of U(VI) by Fe(0). The SLAD reactor removed NO3(-) efficiently (99.8%) at loadings of up to 20 mmol NO3(-) L(r)(-1) d(-1), with near stoichiometric conversion to benign dinitrogen gas (N(2)). The ZVI bioreactor subsequently removed uranium (99.8%) at high (0.22 mM) and low (0.02 mM) influent concentrations of the radionuclide. Aqueous uranium was reliably eliminated to below the maximum contaminant level of 30 µg L(-1) (0.13 µM) when the ZVI reactor was operated at average empty bed hydraulic retention times as low as 2.3 h, demonstrating the feasibility of the sequential treatment strategy in packed bed bioreactors. Sequential extraction of the ZVI reactor packing confirmed that uranium was immobilized as U(IV). Uranium removal was enhanced by microbial activity as confirmed by the increased rate of uranium removal in batch assays inoculated with effluent from the ZVI bioreactor and spiked with Fe(0) compared to abiotic controls.
• Field, J., Sierra-Alvarez, R., Cortinas, I., & Field, J. A. (2010). Methanogenic inhibition by roxarsone (4-hydroxy-3-nitrophenylarsonic acid) and related aromatic arsenic compounds. Journal of hazardous materials, 175(1-3).
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  Roxarsone (4-hydroxy-3-nitro-phenylarsonic acid) and p-arsanilic acid (4-aminophenylarsonic acid) are feed additives widely used in the broiler and swine industry. This study evaluated the inhibitory effect of roxarsone, p-arsanilic, and other phenylarsonic compounds on the activity of acetate- and H(2)-utilizing methanogenic microorganisms. Roxarsone, p-arsanilic, and 4-hydroxy-3-aminophenylarsonic acid (HAPA) inhibited acetoclastic and hydrogenotrophic methanogens when supplemented at concentrations of 1mM, and their inhibitory effect increased sharply with incubation time. Phenylarsonic acid (1mM) inhibited acetoclastic but not H(2)-utilizing methanogens. HAPA, a metabolite from the anaerobic biodegradation of roxarsone, was found to be sensitive to autooxidation by oxygen. The compound (2.6mM) caused low methanogenic inhibition (only 14.2%) in short-term assays of 12h when autooxidation was prevented by supplementing HAPA solutions with ascorbate. However, ascorbate-free HAPA solutions underwent spontaneous autooxidation in the presence of oxygen, leading to the formation of highly inhibitory compounds. These results confirm the microbial toxicity of organoarsenic compounds, and they indicate that biotic as well as abiotic transformations can potentially impact the fate and microbial toxicity of these contaminants in the environment.
• Field, J., Sun, W., Sierra-Alvarez, R., & Field, J. A. (2010). The role of denitrification on arsenite oxidation and arsenic mobility in an anoxic sediment column model with activated alumina. Biotechnology and bioengineering, 107(5).
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  Arsenite (As(III)) is the predominant arsenic (As) species in reducing environments. As(III) is less strongly adsorbed than As(V) at circumneutral pH conditions by common non-iron metal oxides in sediments such as those of aluminum. Therefore, oxidation of As(III) to As(V) could contribute to an improved immobilization of As and thus help mitigate As contamination in groundwater. Microbial oxidation of As(III) is known to readily under aerobic conditions, however, the dissolved oxygen (O₂) concentration in groundwater may be limited due to the poor solubility of O₂ and its high chemical reactivity with reduced compounds. Nitrate (NO₃⁻), can be considered as an alternative electron acceptor, which can support oxidation of As(III) to As(V) by denitrifying bacteria. In this study, two up-flow sediment columns packed with activated alumina (AA) were utilized to demonstrate the role of denitrification on the oxidation of As(III) to As(V) and its contribution to improved As adsorption onto AA. One column was supplied with NO₃⁻(C1) and its performance was compared with a control column lacking NO₃⁻(C2). During most of the operation when the pH was in the circumneutral range (days 50-250), the release of arsenic was greater from C2 compared to C1. The effluent As concentrations started increasing on days 60 and 100 in C2 and C1, respectively. Complete breakthrough started on day 200 in C2; whereas in C1, complete breakthrough was never achieved. The effluent and solid phase As speciation was dominated by As(V) in C1, indicating the occurrence of As(III) oxidation due to NO₃⁻; whereas in C2, only As(III) was dominant. This study illustrates a bioremediation or natural attenuation process based on anoxic microbial NO₃⁻-dependent oxidation of As(III) to more readily adsorbed As(V) as a means to enhance the immobilization of As on alumina oxide particles in subsurface environments.
• Field, J., Sun, W., Sierra-Alvarez, R., Hsu, I., Rowlette, P., & Field, J. A. (2010). Anoxic oxidation of arsenite linked to chemolithotrophic denitrification in continuous bioreactors. Biotechnology and bioengineering, 105(5).
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  In this study, the anoxic oxidation of arsenite (As(III)) linked to chemolithotrophic denitrification was shown to be feasible in continuous bioreactors. Biological oxidation of As(III) was stable over prolonged periods of operation ranging up to 3 years in continuous denitrifying bioreactors with granular biofilms. As(III) was removed with a high conversion efficiency (>92%) to arsenate (As(V)) in periods with high volumetric loadings (e.g., 3.5-5.1 mmol As L(reactor) (-1) day(-1)). The maximum specific activity of sampled granular sludge from the bioreactors was 0.98 +/- 0.04 mmol As(V) formed g(-1) VSS day(-1) when determined at an initial concentration of 0.5 mM As(III). The microbial population adapted to high influent concentrations of As(III) up to 5.2 mM. However, the As(III) oxidation process was severely inhibited when 7.6-8.1 mM As(III) was fed. Activity was restored upon lowering the As(III) concentration to 3.8 mM. Several experimental strategies were utilized to demonstrate a dependence of the nitrate removal on As(III) oxidation as well as a dependence of the As(III) removal on nitrate reduction. The molar stoichiometric ratio of As(V) formed to nitrate removed (corrected for endogenous denitrification) in the bioreactors approximated 2.5, indicating complete denitrification was occurring. As(III) oxidation was also shown to be linked to the complete denitrification of NO(3) (-) to N(2) gas by demonstrating a significantly enhanced production of N(2) beyond the background endogenous production in a batch bioassay spiked with 3.5 mM As(III). The N(2) production also corresponded closely to the expected stoichiometry of 2.5 mol As(III) mol(-1) N(2)-N for complete denitrification.
• Field, J., Sun, W., Sierra-Alvarez, R., Milner, L., & Field, J. A. (2010). Anaerobic oxidation of arsenite linked to chlorate reduction. Applied and environmental microbiology, 76(20).
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  Microorganisms play a significant role in the speciation and mobility of arsenic in the environment. In this study, the oxidation of arsenite [As(III)] to arsenate [As(V)] linked to chlorate (ClO₃⁻) reduction was shown to be catalyzed by sludge samples, enrichment cultures (ECs), and pure cultures incubated under anaerobic conditions. No activity was observed in treatments lacking inoculum or with heat-killed sludge, or in controls lacking ClO₃⁻. The As(III) oxidation was linked to the complete reduction of ClO₃⁻ to Cl⁻, and the molar ratio of As(V) formed to ClO₃⁻ consumed approached the theoretical value of 3:1 assuming the e⁻ equivalents from As(III) were used to completely reduce ClO₃⁻. In keeping with O₂ as a putative intermediate of ClO₃⁻ reduction, the ECs could also oxidize As(III) to As(V) with O₂ at low concentrations. Low levels of organic carbon were essential in heterotrophic ECs but not in autotrophic ECs. 16S rRNA gene clone libraries indicated that the ECs were dominated by clones of Rhodocyclaceae (including Dechloromonas, Azospira, and Azonexus phylotypes) and Stenotrophomonas under autotrophic conditions. Additional phylotypes (Alicycliphilus, Agrobacterium, and Pseudoxanthomonas) were identified in heterotrophic ECs. Two isolated autotrophic pure cultures, Dechloromonas sp. strain ECC1-pb1 and Azospira sp. strain ECC1-pb2, were able to grow by linking the oxidation of As(III) to As(V) with the reduction of ClO₃⁻. The presence of the arsenite oxidase subunit A (aroA) gene was demonstrated with PCR in the ECs and pure cultures. This study demonstrates that ClO₃⁻ is an alternative electron acceptor to support the microbial oxidation of As(III).
• Halalsheh, M. M., M., Z., & Field, J. A. (2010). Anaerobic wastewater treatment of concentrated sewage using a two-stage upflow anaerobic sludge blanket- anaerobic filter system. Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and Environmental Engineering, 45(3), 383-388.
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  PMID: 20390881;Abstract: A two-stage pilot-scale upflow anaerobic sludge blanket - anaerobic filter (UASB-AF) reactors system treating concentrated domestic sewage was operated at 23°C and at hydraulic retention times (HRT) of 15 and 4 h, respectively. Excess sludge from the downstream AF stage was returned to the upstream UASB reactor. The aim was to obtain higher sludge retention time (SRT) in the UASB reactor for better methanization of suspended COD. The UASB-AF system removed 55% and 65% of the total COD (CODtot) and suspended COD (COD ss), respectively. The calculated SRT in the UASB reactor ranged from 20-35 days. The AF reactor removed the washed out sludge from the first stage reactor with average CODss removal efficiency of 55%. The volatile fatty acids concentration in the effluent of the AF was 39 mg COD/L compared with 78 mg COD/L measured for the influent. The slightly higher CODtot removal efficiency obtained in this study compared with a single stage UASB reactor was achieved at 17% reduction in the total volume.
• Halalsheh, M. M., Muhsen, H. H., Shatanawi, K. M., & Field, J. A. (2010). Improving solids retention in upflow anaerobic sludge blanket reactors at low temperatures using lamella settlers. Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and Environmental Engineering, 45(9), 1054-1059.
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  PMID: 20526935;Abstract: Lamella settlers were used to increase sludge concentration in pilot scale UASB reactors treating concentrated sewage at low temperature. The aim was to increase sludge retention time (SRT) and achieve better digestion in UASB reactors without the need for increasing the hydraulic retention time (HRT). Two modified UASB reactors were used for this purpose. In the first reactor, lamella settlers were installed in the settling zone of the UASB reactor and the reactor was named UASB-ESR1. In the second reactor, lamella settlers were installed underneath the gas liquid separator (GLS) and the reactor was named UASB-ESR2. The sludge concentration, sludge profile, and system performance of each reactor were monitored. The obtained sludge concentrations were 50 and 53 g TS/l for UASB-ESR1 and UASB-ESR2, respectively. The measured concentrations were almost double the concentrations reported for conventional UASB reactors ranging 16-26 g TS/l. The calculated SRT in the modified UASB reactors was 103 days in both reactors. The average total COD (CODtot) and suspended COD (CODss) removal efficiencies were 38% and 60%, respectively for the UASB-ESR1. The average CODtot and CODss removal efficiencies for the UASB-ESR2 were 41% and 62%, respectively. The modified reactors were considered at the startup period and the performances of the modified systems are expected to significantly improve when arriving at steady state conditions. Copyright © Taylor & Francis Group, LLC.
• Carreón-Diazconti, C., Santamaría, J., Berkompas, J., Field, J. A., & Brusseau, M. L. (2009). Assessment of in situ reductive dechlorination using compound-specific stable isotopes, functional gene PCR, and geochemical data. Environmental Science and Technology, 43(12), 4301-4307.
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  PMID: 19603638;PMCID: PMC2864078;Abstract: Isotopic analysis and molecular-based bioassay methods were used in conjunction with geochemical data to assess intrinsic reductive dechlorination processes for a chlorinated solvent-contaminated site in Tucson, Arizona. Groundwater samples were obtained from monitoring wells within a contaminant plume comprising tetrachloroethene and its metabolites, trichloroethene, cis-1,2-dichloroethene, vinyl chloride, and ethene, as well as compounds associated with free phase diesel present at the site. Compound-specific isotope analysis was performed to characterize biotransformation processes influencing the transport and fate of the chlorinated contaminants. Polymerase chain reaction (PCR) analysis was used to assess the presence of indigenous reductive dechlorinators. The target regions employed were the 16s rRNA gene sequences of Dehalococcoides sp. and Desulfuromonas sp. and DNA sequences of genes pceA, tceA, bvcA, and vcrA, which encode reductive dehalogenases. The results of the analyses indicate that relevant microbial populations are present and that reductive dechlorination is presently occurring at the site. The results further show that potential degrader populations as well as biotransformation activity is nonuniformly distributed within the site. The results of laboratory microcosm studies conducted using groundwater collected from the field site confirmed the reductive dechlorination of tetrachloroethene to dichloroethene. This study illustrates the use of an integrated, multiple-method approach for assessing natural attenuation at a complex chlorinated solvent-contaminated site. © 2009 American Chemical Society.
• Field, J., Banihani, Q., Sierra-Alvarez, R., & Field, J. A. (2009). Nitrate and nitrite inhibition of methanogenesis during denitrification in granular biofilms and digested domestic sludges. Biodegradation, 20(6).
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  Anaerobic bioreactors that can support simultaneous microbial processes of denitrification and methanogenesis are of interest to nutrient nitrogen removal. However, an important concern is the potential toxicity of nitrate (NO(3) (-)) and nitrite (NO(2) (-)) to methanogenesis. The methanogenic toxicity of the NO (x) (-) compounds to anaerobic granular biofilms and municipal anaerobic digested sludge with two types of substrates, acetate and hydrogen, was studied. The inhibition was the severest when the NO (x) (-) compounds were still present in the media (exposure period). During this period, 95% or greater inhibition of methanogenesis was evident at the lowest concentrations of added NO(2) (-) tested (7.6-10.2 mg NO(2) (-)-N l(-1)) or 8.3-121 mg NO(3) (-)-N l(-1) of added NO(3) (-), depending on substrate and inoculum source. The inhibition imparted by NO(3) (-) was not due directly to NO(3) (-) itself, but instead due to reduced intermediates (e.g., NO(2) (-)) formed during the denitrification process. The toxicity of NO (x) (-) was found to be reversible after the exposure period. The recovery of activity was nearly complete at low added NO (x) (-) concentrations; whereas the recovery was only partial at high added NO (x) (-) concentrations. The recovery is attributed to the metabolism of the NO (x) (-) compounds. The assay substrate had a large impact on the rate of NO(2) (-) metabolism. Hydrogen reduced NO(2) (-) slowly such that NO(2) (-) accumulated more and as a result, the toxicity was greater compared to acetate as a substrate. The final methane yield was inversely proportional to the amount of NO (x) (-) compounds added indicating that they were the preferred electron acceptors compared to methanogenesis.
• Field, J., Gámez, V. M., Sierra-Alvarez, R., Waltz, R. J., & Field, J. A. (2009). Anaerobic degradation of citrate under sulfate reducing and methanogenic conditions. Biodegradation, 20(4).
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  Citrate is an important component of metal processing effluents such as chemical mechanical planarization wastewaters of the semiconductor industry. Citrate can serve as an electron donor for sulfate reduction applied to promote the removal of metals, and it can also potentially be used by methanogens that coexist in anaerobic biofilms. The objective of this study was to evaluate the degradation of citrate with sulfate-reducing and methanogenic biofilms. During batch bioassays, the citrate, acetate, methane and sulfide concentrations were monitored. The results indicate that independent of the biofilm or incubation conditions used, citrate was rapidly fermented with specific rates ranging from 566 to 720 mg chemical oxygen demand (COD) consumed per gram volatile suspended solids per day. Acetate was found to be the main fermentation product of citrate degradation, which was later degraded completely under either methanogenic or sulfate reducing conditions. However, if either sulfate reduction or methanogenesis was infeasible due to specific inhibitors (2-bromoethane sulfonate), absence of sulfate or lack of adequate microorganisms in the biofilm, acetate accumulated to levels accounting for 90-100% of the citrate-COD consumed. Based on carbon balances measured in phosphate buffered bioassays, acetate, CO(2) and hydrogen are the main products of citrate fermentation, with a molar ratio of 2:2:1 per mol of citrate, respectively. In bicarbonate buffered bioassays, acetogenesis of H(2) and CO(2) increased the yield of acetate. The results taken as a whole suggest that in anaerobic biofilm systems, citrate is metabolized via the formation of acetate as the main metabolic intermediate prior to methanogenesis or sulfate reduction. Sulfate reducing consortia must be enriched to utilize acetate as an electron donor in order to utilize the majority of the electron-equivalents in citrate.
• Field, J., Sun, W., Sierra-Alvarez, R., Fernandez, N., Sanz, J. L., Amils, R., Legatzki, A., Maier, R. M., & Field, J. A. (2009). Molecular characterization and in situ quantification of anoxic arsenite-oxidizing denitrifying enrichment cultures. FEMS microbiology ecology, 68(1).
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  To explore the bacteria involved in the oxidation of arsenite (As(III)) under denitrifying conditions, three enrichment cultures (ECs) and one mixed culture (MC) were characterized that originated from anaerobic environmental samples. The oxidation of As(III) (0.5 mM) was dependent on NO(3) (-) addition and N(2) formation was dependent on As(III) addition. The ratio of N(2)-N formed to As(III) fed approximated the expected stoichiometry of 2.5. A 16S rRNA gene clone library analysis revealed three predominant phylotypes. The first, related to the genus Azoarcus from the division Betaproteobacteria, was found in the three ECs. The other two predominant phylotypes were closely related to the genera Acidovorax and Diaphorobacter within the Comamonadaceae family of Betaproteobacteria, and one of these was present in all of the cultures examined. FISH confirmed that Azoarcus accounted for a large fraction of bacteria present in the ECs. The Azoarcus clones had 96% sequence homology with Azoarcus sp. strain DAO1, an isolate previously reported to oxidize As(III) with nitrate. FISH analysis also confirmed that Comamonadaceae were present in all cultures. Pure cultures of Azoarcus and Diaphorobacter were isolated and shown to be responsible for nitrate-dependent As(III) oxidation. These results, taken as a whole, suggest that bacteria within the genus Azoarcus and the family Comamonadaceae are involved in the observed anoxic oxidation of As(III).
• Field, J., Sun, W., Sierra-Alvarez, R., Milner, L., Oremland, R., & Field, J. A. (2009). Arsenite and ferrous iron oxidation linked to chemolithotrophic denitrification for the immobilization of arsenic in anoxic environments. Environmental science & technology, 43(17).
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  The objective of this study was to explore a bioremediation strategy based on injecting NO3- to support the anoxic oxidation of ferrous iron (Fe(II)) and arsenite (As(II)) in the subsurface as a means to immobilize As in the form of arsenate (As(V)) adsorbed onto biogenic ferric (Fe(III)) (hydr)oxides. Continuous flow sand filled columns were used to simulate a natural anaerobic groundwater and sediment system with co-occurring As(III) and Fe(II) in the presence (column SF1) or absence (column SF2) of nitrate, respectively. During operation for 250 days, the average influent arsenic concentration of 567 microg L(-1) was reduced to 10.6 (+/-9.6) microg L(-1) in the effluent of column SF1. The cumulative removal of Fe(II) and As(II) in SF1 was 6.5 to 10-fold higher than that in SF2 Extraction and measurement of the mass of iron and arsenic immobilized on the sand packing of the columns were close to the iron and arsenic removed from the aqueous phase during column operation. The dominant speciation of the immobilized iron and arsenic was Fe(III) and As(V) in SF1, compared with Fe(II) and As(III) in SF2. The speciation was confirmed by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). The results indicate that microbial oxidation of As(III) and Fe(II) linked to denitrification resulted in the enhanced immobilization of aqueous arsenic in anaerobic environments by forming Fe(III) (hydr)oxide coated sands with adsorbed As(V).
• Field, J., Cortinas, I., Sierra-Alvarez, R., & Field, J. A. (2008). Biologically mediated mobilization of arsenic from granular ferric hydroxide in anaerobic columns fed landfill leachate. Biotechnology and bioengineering, 101(6).
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  To gain insight on the fate of arsenic (As) from drinking water treatment residuals in landfills, the mobilization of arsenate adsorbed onto granular ferric hydroxide (GFH) was studied in continuous anaerobic columns fed with a synthetic landfill leachate. The release of As was compared in biologically active and abiotic columns. More than 150 days of incubation were required before noteworthy As release occurred. After 400 days of operation, 19% of the As was mobilized as identified species in the biologically active column, which was 25.5-fold greater than that of the abiotic column. Fine colloids accounted for up to 81% of the As released. Arsenite was the predominant species identified in filtered (0.45 microm) effluent samples. Dimethylarsinic acid and monomethylarsonic acid were also observed as metabolites. During column operation, approximately 30% of the iron (hydr)oxide mass was lost and most of the mass loss was attributed to changes in iron mineralogy that could be demonstrated in a batch bioassay. The results indicate that As-laden GFH residuals from drinking water treatment are subject to mobilization in municipal landfills and that biologically mediated changes in the iron mineralogy may play an important role in the mobilization mechanism.
• Field, J., Ochoa-Herrera, V., Sierra-Alvarez, R., Somogyi, A., Jacobsen, N. E., Wysocki, V. H., & Field, J. A. (2008). Reductive defluorination of perfluorooctane sulfonate. Environmental science & technology, 42(9).
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  Perfluorooctane sulfonate (PFOS) is under increased scrutiny as an environmental pollutant due to recent reports of its worldwide distribution, environmental persistence, and bioaccumulation potential. The susceptibility of technical PFOS and PFOS branched isomers to chemical reductive dehalogenation with vitamin B12 (260 microM) as catalyst and Ti(III)-citrate (36 mM) as bulk reductant in anoxic aqueous solution at 70 degrees C and pH 9 was evaluated in this study. Defluorination was confirmed by fluoride release measurements of 18% in technical PFOS, equivalent to the removal 3 mol F-/mol PFOS, and 71% in PFOS branched isomers equivalent to the removal of 12 mol F-/mol PFOS. Degradation of PFOS was further confirmed by monitoring the disappearance of PFOS compounds with reaction time by suppressed conductivity ion chromatography, LC-MS/MS, and 19F NMR studies. The PFOS compounds differed in their susceptibility to reductive degradation by vitamin B12Ti(III) citrate. Chromatographic peaks corresponding to branched PFOS isomers disappeared whereas the peak corresponding to linear PFOS was stable. To our knowledge this is the first report of reductive dehalogenation of PFOS catalyzed by a biomolecule.
• Field, J., Sun, W., Sierra, R., & Field, J. A. (2008). Anoxic oxidation of arsenite linked to denitrification in sludges and sediments. Water research, 42(17).
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  In this study, denitrification linked to the oxidation of arsenite (As(III)) to arsenate (As(V)) was shown to be a widespread microbial activity in anaerobic sludge and sediment samples that were not previously exposed to arsenic contamination. When incubated with 0.5mM As(III) and 10mM NO(3)(-), the anoxic oxidation of As(III) commenced within a few days, achieving specific activities of up to 1.24mmol As(V) formed g(-1) volatile suspended solids d(-1) due to growth (doubling times of 0.74-1.4d). The anoxic oxidation of As(III) was partially to completely inhibited by 1.5 and 5mM As(III), respectively. Inhibition was minimized by adding As(III) adsorbed onto activated aluminum (AA). The oxidation of As(III) was shown to be linked to the complete denitrification of NO(3)(-) to N(2) by demonstrating a significantly enhanced production of N(2) beyond the background endogenous production as a result of adding As(III)-AA to the cultures. The N(2) production corresponded closely the expected stoichiometry of the reaction, 2.5mol As(III) mol(-1)N(2)-N. The oxidation of As(III) linked to the use of common-occurring nitrate as an electron acceptor may be an important missing link in the biogeochemical cycling of arsenic.
• Cervantes, F. J., Enríquez, J. E., Galindo-Petatán, E., Arvayo, H., Razo-Flores, E., & Field, J. A. (2007). Biogenic sulphide plays a major role on the riboflavin-mediated decolourisation of azo dyes under sulphate-reducing conditions. Chemosphere, 68(6), 1082-1089.
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  PMID: 17350080;Abstract: The effect of high concentrations of sulphate on the reductive decolourisation of different azo dyes by anaerobic sludge was studied in batch cultures. Sludge cultures were pre-incubated under sulphate-reducing conditions prior addition of dyes. Little or no effects of sulphate (5-10 g sulphate l-1) on the rate of decolourisation of Reactive Orange 14 (RO14), Direct Blue 53 (DB53) and Direct Blue 71 (DB71) were observed when no external redox mediator was provided. However, an increase in sulphate concentration, in the presence of riboflavin (20 μM), enhanced the decolourisation of all dyes. The first-rate constant of decolourisation (k) was increased up to 2-, 3.6- and 2-fold for RO14, DB53 and DB71, respectively, by supplying high sulphate concentrations, compared to the controls lacking sulphate, in the presence of the redox mediator. Sulphate reduction did not take place during the course of azo reductions, but was only evident before dye addition and after complete decolourisation, suggesting azo dyes reduction out-competed sulphate reduction for the available reducing equivalents. The experimental data suggest that reduction of azo dyes by riboflavin, which had been reduced by biogenic sulphide, was the major mechanism implicated during decolourisations, which was corroborated by abiotic incubations. Riboflavin greatly accelerated the abiotic reduction of RO14, so that the k value was increased up to 44-fold compared to the control lacking riboflavin. © 2007 Elsevier Ltd. All rights reserved.
• Field, J., Sierra-Alvarez, R., Beristain-Cardoso, R., Salazar, M., Gómez, J., Razo-Flores, E., & Field, J. A. (2007). Chemolithotrophic denitrification with elemental sulfur for groundwater treatment. Water research, 41(6).
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  Denitrification for the treatment of nitrates in wastewater typically relies on organic electron donating substrates. However, for groundwater treatment, inorganic compounds such as elemental sulfur (S0) are being considered as alternative electron donors in order to overcome concerns that residual organics can cause biofouling. In this study, a packed-bed bioreactor supplied with S0:limestone granules (1:1, v/v) was started up utilizing a chemolithotrophic denitrifying enrichment culture in the form of biofilm granules that was pre-cultivated on thiosulfate. The granular enrichment culture enabled a rapid start-up of the bioreactor. A nearly complete removal of nitrate (7.3 mM) was NO3- attained by the bioreactor at nitrate loading rates of up to 21.6 mmol/(L(reactor)d). With lower influent concentrations (1.3 mM nitrate) comparable to those found in contaminated groundwater, high nitrate loads of 18.1 mmol/(L(reactor)d) were achieved with an average nitrate removal efficiency of 95.9%. The recovery of nitrogen as benign N2 gas was nearly stoichiometric. The concentration of undesirable products from S0-based denitrification such as nitrite and sulfide were low. Comparison of bioreactor results with batch kinetic studies revealed that denitrification rates were dependent on the surface area of the added S0. The surface area normalized denitrification rate was determined to be 26.4 mmol /(m2 S0 d).
• Cervantes, F. J., Enriquez, J. E., Mendoza-Hernandez, M., Razo-Flores, E., & Field, J. A. (2006). The role of sulphate reduction on the reductive decolorization of the azo dye reactive orange 14. Water Science and Technology, 54(2), 171-177.
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  PMID: 16939099;Abstract: The aim of this study was to investigate the impact of a broad range of sulphate concentrations (0-10 g SO4-2 L-1) on the reduction of an azo dye (reactive orange 14 (RO14)) by an anaerobic sludge. An increase in the sulphate concentration generally stimulated the reduction of RO14 by sludge incubations supplemented with glucose, acetate or propionate as electron donor. Sulphate and azo dye reductions took place simultaneously in all incubations. However, there was a decrease on the rate of decolorization when sulphate was supplied at 10 g SO4-2 L-1. Abiotic incubations at different sulphide concentrations (0-2.5 g sulphide L-1) promoted very poor reduction of RO14. However, addition of riboflavin (20 μM), as a redox mediator, accelerated the reduction of RO14 up to 44-fold compared to a control lacking the catalyst. Our results indicate that sulphate-reduction may significantly contribute to the reduction of azo dyes both by biological mechanisms and by abiotic reductions implicating sulphide as an electron donor. The contribution of abiotic decolorization by sulphide, however, was only significant when a proper redox mediator was included. Our results also revealed that sulphate-reduction can out-compete with azo reduction at high sulphate concentrations leading to a poor decolorising performance when no sufficient reducing capacity is available. © IWA Publishing 2006.
• Encinas-Yocupicio, A., Razo-Flores, E., Sánchez-Diaz, F., Santos, A. d., Field, J. A., & Cervantes, F. J. (2006). Catalytic effects of different redox mediators on the reductive decolorization of azo dyes. Water Science and Technology, 54(2), 165-170.
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  PMID: 16939098;Abstract: The catalytic effects of redox mediators, with distinct standard redox potentials (E′0), were evaluated on the first-order rate constant of decolorization (Kd) of recalcitrant azo dyes by an anaerobic granular sludge. The dyes studied included mono-azo (Reactive Orange 14, RO14), di-azo (Direct Blue 53, DB53), and tri-azo (Direct Blue 71, DB71) compounds. Toxicity and auto-catalytic aspects seemed to play a role in determining the rate of decolorization. Addition of riboflavin, anthraquinone-2,6-disulphonate (AQDS) or lawsone as a redox mediator, increased the Kd value for all dyes studied, although their impact varied in every case. Kd values were increased from 1.1-fold up to 3.8-fold depending on the redox mediator applied. Moreover, catalysts with moderately similar E′0 value caused distinct stimulation on the rate of decolorization. These results should be considered for selecting the proper redox mediator to be applied during the anaerobic treatment of textile wastewaters and effluents containing electron-withdrawing pollutants, such as nitro-aromatic and polychlorinated compounds. © IWA Publishing 2006.
• Field, J. A. (2006). Vitamin B12 precursors enhance bioremediation. Industrial Bioprocessing, 28(8), 5-6.
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  Abstract: University of Arizona researchers found that porphobilinogen (PB), involved in the formation of the corrin ring, increased carbon tetrachloride (CT) biotransformation rates by 2.7-, 8.8- and 10.9-fold when supplemented at 160, 500 and 900 micromoles. A control using 10 micrograms of B12 resulted in a 5.9-fold increase. PB addition resulted in a 57% molar yield of inorganic chloride from CT. The large impact of PB indicates that corrin ring formation is the rate-limiting step of vitamin B12 in methanogens and acetogens found in the environment.
• Field, J., Cardoso, R. B., Sierra-Alvarez, R., Rowlette, P., Flores, E. R., Gómez, J., & Field, J. A. (2006). Sulfide oxidation under chemolithoautotrophic denitrifying conditions. Biotechnology and bioengineering, 95(6).
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  Chemolithoautotrophic denitrifying microorganisms oxidize reduced inorganic sulfur compounds coupled to the reduction of nitrate as an electron acceptor. These denitrifiers can be applied to the removal of nitrogen and/or sulfur contamination from wastewater, groundwater, and gaseous streams. This study investigated the physiology and kinetics of chemolithotrophic denitrification by an enrichment culture utilizing hydrogen sulfide, elemental sulfur, or thiosulfate as electron donor. Complete oxidation of sulfide to sulfate was observed when nitrate was supplemented at concentrations equal or exceeding the stoichiometric requirement. In contrast, sulfide was only partially oxidized to elemental sulfur when nitrate concentrations were limiting. Sulfide was found to inhibit chemolithotrophic sulfoxidation, decreasing rates by approximately 21-fold when the sulfide concentration increased from 2.5 to 10.0 mM, respectively. Addition of low levels of acetate (0.5 mM) enhanced denitrification and sulfate formation, suggesting that acetate was utilized as a carbon source by chemolithotrophic denitrifiers. The results of this study indicate the potential of chemolithotrophic denitrification for the removal of hydrogen sulfide. The sulfide/nitrate ratio can be used to control the fate of sulfide oxidation to either elemental sulfur or sulfate.
• Field, J., Guerrero-Barajas, C., & Field, J. A. (2006). Enhanced anaerobic biotransformation of carbon tetrachloride with precursors of vitamin B(12) biosynthesis. Biodegradation, 17(4).
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  Relatively low concentrations of Vitamin B(12) are known to accelerate the anaerobic biotransformation of carbon tetrachloride (CT) and chloroform (CF). However, the addition of vitamin B(12) for field-scale bioremediation is expected to be costly. The present study considered a strategy to generate vitamin B(12) by addition of biosynthetic precursors. One of the precursors, porphobilinogen (PB) involved in the formation of the corrin ring, significantly increased the CT biotransformation rates by 2.7-, 8.8- and 10.9-fold when supplemented at 160, 500 and 900 microM, respectively. A positive control with 10 microM of vitamin B(12) resulted in a 5.9-fold increase in the CT-bioconversion rate. PB additions provided high molar yields of inorganic chloride (57% of CT organochlorine), comparable to that obtained with vitamin B(12) supplemented cultures. The primary substrate, methanol, known to induce vitamin B(12) production in methanogens and acetogens, was required for PB to have a significant impact on CT conversion. The observation suggests that PB's role was due to stimulating vitamin B(12) biosynthesis. The present study therefore provides insights on how to achieve vitamin B(12) enhanced rates of CT bioremediation through the use of less complex compounds that are precursors of vitamin B(12). Although PB is a costly chemical, its large impact points to corrin ring formation as the rate-limiting step.
• Field, J., Karri, S., Sierra-Alvarez, R., & Field, J. A. (2006). Toxicity of copper to acetoclastic and hydrogenotrophic activities of methanogens and sulfate reducers in anaerobic sludge. Chemosphere, 62(1).
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  Heavy metals could potentially negatively impact microorganisms in anaerobic sulfate reducing bioreactors. The objective of this is study was to evaluate the inhibitory effect of copper to acetoclastic and hydrogenotrophic activities of methanogens and sulfate reducers in sludge obtained from a full-scale sulfate reducing bioreactor. The 50% inhibiting concentration (50%IC) of Cu(2+) to acetoclastic and hydrogenotrophic methanogens was 20.7 and 8.9 mg l(-1), respectively. The 50%IC of Cu(2+) to acetoclastic sulfate reduction was 32.3 mg l(-1). The hydrogenotrophic sulfate reducers were only inhibited by 27% at the highest concentration of Cu(2+) tested, 200 mg l(-1), indicating a high level of tolerance. The soluble Cu(2+) was observed to decrease rapidly in both the methanogenic and sulfate reducing assays. The highest level of decrease was observed in the hydrogenotrophic sulfate reducing assay which was over 99% in 5h. The results of this study indicate that sulfate reducing biotechnologies would be robust at relatively high inlet concentrations of Cu(2+).
• Guerrero-Barajas, C., & Field, J. A. (2006). Enhanced anaerobic biotransformation of carbon tetrachloride with precursors of vitamin B₁₂ biosynthesis. Biodegradation, 17(4), 317-329.
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  PMID: 16491307;Abstract: Relatively low concentrations of Vitamin B12 are known to accelerate the anaerobic biotransformation of carbon tetrachloride (CT) and chloroform (CF). However, the addition of vitamin B12 for field-scale bioremediation is expected to be costly. The present study considered a strategy to generate vitamin B12 by addition of biosynthetic precursors. One of the precursors, porphobilinogen (PB) involved in the formation of the corrin ring, significantly increased the CT biotransformation rates by 2.7-, 8.8- and 10.9-fold when supplemented at 160, 500 and 900 μM, respectively. A positive control with 10 μM of vitamin B12 resulted in a 5.9-fold increase in the CT-bioconversion rate. PB additions provided high molar yields of inorganic chloride (57% of CT organochlorine), comparable to that obtained with vitamin B12 supplemented cultures. The primary substrate, methanol, known to induce vitamin B12 production in methanogens and acetogens, was required for PB to have a significant impact on CT conversion. The observation suggests that PB's role was due to stimulating vitamin B12 biosynthesis. The present study therefore provides insights on how to achieve vitamin B12 enhanced rates of CT bioremediation through the use of less complex compounds that are precursors of vitamin B12. Although PB is a costly chemical, its large impact points to corrin ring formation as the rate-limiting step. © Springer 2006.
• C., N., Leeuwen, A. V., M., E., Slenders, P., Prenafeta-Boldú, F. X., Temmink, H., Lettinga, G., & Field, J. A. (2005). Fate and biodegradability of sulfonated aromatic amines. Biodegradation, 16(6), 527-537.
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  PMID: 15865345;Abstract: Ten sulfonated aromatic amines were tested for their aerobic and anaerobic biodegradability and toxicity potential in a variety of environmental inocula. Of all the compounds tested, only two aminobenzenesulfonic acid (ABS) isomers, 2- and 4-ABS, were degraded. The observed degradation occurred only under aerobic conditions with inocula sources that were historically polluted with sulfonated aromatic amines. Bioreactor experiments, with non-sterile synthetic wastewater, confirmed the results from the aerobic batch degradation experiments. Both ABS isomers were degraded in long-term continuous experiment by a bioaugmented enrichment culture. The maximum degradation rate in the aerobic bioreactor was 1.6-1.8 gl-1 d-1 for 2-ABS and a somewhat lower value for 4-ABS at hydraulic retention times (HRT) of 2.8-3.3h. Evidence for extensive mineralization of 2- and 4-ABS was based on oxygen uptake and carbon dioxide production during the batch experiments and the high levels of chemical oxygen demand (COD) removal in the bioreactor. Furthermore, mineralization of the sulfonate group was demonstrated by high recovery of sulfate. The sulfonated aromatic amines did not show any toxic effects on the aerobic and anaerobic bacterial populations tested. The poor biodegradability of sulfonated aromatic amines indicated under the laboratory conditions of this study suggests that these compounds may not be adequately removed during biological wastewater treatment. © Springer 2005.
• Field, J. A., & Guerrero, C. (2005). Vitamin B12 precursors stimulate the anaerobic bioremediation of chlorinated solvents. Proceedings of the 8th International In Situ and On-Site Bioremediation Symposium, 4, 1940-.
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  Abstract: Methods to stimulate vitamin B12 production in natural anaerobic consortia which in turn will stimulate degradation of halomethanes were explored. Two approaches were considered, one based on supplementing cultures with precursors of vitamin B12 biosynthesis and the second approach was based on supplying primary substrates which are known to induce vitamin B12 biosynthesis. Initial experiments evaluating vitamin B12 precursors were conducted in anaerobic batch assays with 0.5 g volatile suspended solids of methanogenic sludge, a mixture of volatile fatty acids, 100 μM carbon tetrachloride, and mineral medium. Only one of the precursors, porphobilinogen, significantly stimulated the reductive dechlorination. A second experiment was conducted evaluating a concentration series ranging from 40 to 900 μM porphobilinogen. As little as 40 μM of porphobilinogen had a significant stimulating effect on CT-dechlorination; however several hundred μM of porphobilinogen was required to achieve a similar effect as that obtained with 20 μM vitamin B12. Other assays were conducted evaluating the use of primary substrates which are known to stimulate vitamin B12 production such as 1,2-propanediol. Both propanediol and ethylene glycol were stimulatory to CT degradation compared to other substrates such as volatile fatty acid and methanol. This is an abstract of a paper presented at the proceedings of the 8th International In Situ and On-Site Bioremediation Symposium (Baltimore, MD 6/6-9/2005).
• Field, J., Guerrero-Barajas, C., & Field, J. A. (2005). Enhancement of anaerobic carbon tetrachloride biotransformation in methanogenic sludge with redox active vitamins. Biodegradation, 16(3).
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  Carbon tetrachloride (CT) is an important groundwater pollutant which is only subject to biotransformation in the absence of oxygen. The anaerobic biotransformation of CT is influenced by electron shuttling compounds. The purpose of this study was to evaluate the impact of redox active vitamins on CT (100 microM) metabolism in a methanogenic sludge consortium (0.5 g VSS l(-1)) supplied with volatile fatty acids as electron donor (0.2 g COD l(-1)). The redox active vitamins, tested at concentrations ranging from 0.5 to 20 microM, were riboflavin (RF) and two forms of vitamin B12, cyanocobalamin (CNB12) and hydroxycobalamin (HOB12), and these were compared with a redox mediating quinone, anthraquinone-2,6-disulfonate (AQDS). Substoichiometric concentrations of RF, CNB12, HOB12 at molar ratios of vitamin: CT as low as 0.005 significantly increased rates of CT-bioconversion. These are the lowest molar ratios of vitamin B12 reported having an impact on dechlorination. Additionally, this study constitutes the first report of RF having a role in reductive dechlorination. At molar ratios of 0.1 vitamin: CT, RF, CNB12, HOB12 increased the first order rate constant of CT bioconversion by 4.0-, 13.3-and 13.6-fold, respectively. The redox active vitamins also enhanced the rates of abiotic CT conversion in heat killed sludge treatments, but the rates were approximately 4- to 5-fold lower than the corresponding vitamin enhanced rates of biological CT conversion. The addition of CNB12 or HOB12 to the live methanogenic sludge consortium increased the yield of inorganic chloride (Cl-) from CT-converted. Chloroform was a transient intermediate in CNB12 or HOB12 supplemented cultures. In contrast, the addition of RF increased the yield of chloroform from CT-converted. Taken as a whole the results clearly demonstrate that very low concentrations of redox active vitamins could potentially play an important role in accelerating the anaerobic the bioremediation of CT as well as influencing the proportions of biotransformation products formed.
• Field, J., Guerrero-Barajas, C., & Field, J. A. (2005). Riboflavin- and cobalamin-mediated biodegradation of chloroform in a methanogenic consortium. Biotechnology and bioengineering, 89(5).
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  Chloroform (CF) is an important priority pollutant contaminating groundwater. Reductive dechlorination by anaerobic microorganisms is a promising strategy towards the remediation of CF. The objective of this study was to evaluate the use of redox active vitamins as electron shuttles to enhance the anaerobic biodegradation of CF in an unadapted methanogenic consortium not previously exposed to chlorinated compounds. Only negligible degradation of CF was observed in control cultures lacking redox active vitamins. The addition of riboflavin (RF), cyanocobalamin (CNB12), and hydroxycobalamin (HOB12) enabled biodegradation of CF. The reactions were predominantly catalyzed biologically as evidenced by the lack of any CF conversion in heat-killed controls amended with the cobalamins or minor conversion with RF. In live cultures, significant increases in the rate of CF conversion was observed at substoichiometric molar ratios as low as 0.1 to 0.01 vitamin:CF for RF and CNB12, respectively. At the highest molar vitamin:CF ratios tested of 0.2, the first-order rate constant of CF degradation was 5.3- and 91-fold higher in RF and CNB12 amended cultures, respectively, compared to the unamended control culture. The distribution of biotransformation products was highly impacted by the type of redox active vitamin utilized. Cultures supplemented with RF provided high yields of dichloromethane (DCM). On the other hand, cobalamins promoted the near complete mineralization of organochlorine in CF to inorganic chloride and lowered the yield of DCM. In cultures where no or little CF bioconversion occurred, prolonged exposure to CF resulted in cell lysis, as evidenced by the release of intracellular chloride. The results taken as a whole suggest that the anaerobic bioremediation of CF-contaminated sites can greatly be improved with strategies aimed at increasing the concentration of redox active vitamins.
• Field, J., Hollingsworth, J., Sierra-Alvarez, R., Zhou, M., Ogden, K. L., & Field, J. A. (2005). Anaerobic biodegradability and methanogenic toxicity of key constituents in copper chemical mechanical planarization effluents of the semiconductor industry. Chemosphere, 59(9).
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  Copper chemical mechanical planarization (CMP) effluents can account for 30-40% of the water discharge in semiconductor manufacturing. CMP effluents contain high concentrations of soluble copper and a complex mixture of organic constituents. The aim of this study is to perform a preliminary assessment of the treatability of CMP effluents in anaerobic sulfidogenic bioreactors inoculated with anaerobic granular sludge by testing individual compounds expected in the CMP effluents. Of all the compounds tested (copper (II), benzotriazoles, polyethylene glycol (M(n) 300), polyethylene glycol (M(n) 860) monooleate, perfluoro-1-octane sulfonate, citric acid, oxalic acid and isopropanol) only copper was found to be inhibitory to methanogenic activity at the concentrations tested. Most of the organic compounds tested were biodegradable with the exception of perfluoro-1-octane sulfonate and benzotriazoles under sulfate reducing conditions and with the exception of the same compounds as well as Triton X-100 under methanogenic conditions. The susceptibility of key components in CMP effluents to anaerobic biodegradation combined with their low microbial inhibition suggest that CMP effluents should be amenable to biological treatment in sulfate reducing bioreactors.
• Field, J., Karri, S., Sierra-Alvarez, R., & Field, J. A. (2005). Zero valent iron as an electron-donor for methanogenesis and sulfate reduction in anaerobic sludge. Biotechnology and bioengineering, 92(7).
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  Zero valent iron (ZVI) is a reactive media commonly utilized in permeable reactive barriers (PRBs). Sulfate reducing bacteria are being considered for the immobilization of heavy metals in PRBs. The purpose of this study was to evaluate the potential of ZVI as an electron donor for sulfate reduction in natural mixed anaerobic cultures. The ability of methanogens to utilize ZVI as an electron-donor was also explored since these microorganisms often compete with sulfate reducers for common substrates. Four grades of ZVI of different particle sizes (1.120, 0.149, 0.044, and 0.010 mm diameter) were compared as electron donor in batch bioassays inoculated with anaerobic bioreactor sludge. Methanogenesis was evaluated in mineral media lacking sulfate. Sulfate reduction was evaluated in mineral media containing sulfate and the specific methanogenic inhibitor, 2-bromoethane sulfonate. ZVI contributed to significant increases in methane production and sulfate reduction-compared to endogenous substrate controls. The rates of methane formation or sulfate reduction were positively correlated with the surface area of ZVI. The highest rates of 0.310 mmol CH4 formed/mol Fe0.day and 0.804 mmol SO4(2-) reduced/mol Fe0.day were obtained with the finest grade of ZVI (0.01 mm). The results demonstrate that ZVI is readily utilized as a slow-release electron donor for methanogenesis and sulfate reduction in anaerobic sludge; and therefore, has a promising potential in bioremediation applications.
• Guerrero-Barajas, C., & Field, J. A. (2005). Enhancement of anaerobic carbon tetrachloride biotransformation in methanogenic sludge with redox active vitamins. Biodegradation, 16(3), 215-228.
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  PMID: 15865146;Abstract: Carbon tetrachloride (CT) is an important groundwater pollutant which is only subject to biotransformation in the absence of oxygen. The anaerobic biotransformation of CT is influenced by electron shuttling compounds. The purpose of this study was to evaluate the impact of redox active vitamins on CT (100 μM) metabolism in a methanogenic sludge consortium (0.5 g VSS1 -1) supplied with volatile fatty acids as electron donor (0.2 g COD1-1). The redox active vitamins, tested at concentrations ranging from 0.5 to 20 μM, were riboflavin (RF) and two forms of vitamin B12, cyanocobalamin (CNB12) and hydroxycobalamin (HOB12), and these were compared with a redox mediating quinone, anthraquinone-2,6-disulfonate (AQDS). Substoichiometric concentrations of RF, CNB12, HOB12 at molar ratios of vitamin:CT as low as 0.005 significantly increased rates of CT-bioconversion. These are the lowest molar ratios of vitamin B12 reported having an impact on dechlorination. Additionally, this study constitutes the first report of RF having a role in reductive dechlorination. At molar ratios of 0.1 vitamin:CT, RF, CNB12, HOB12 increased the first order rate constant of CT bioconversion by 4.0-, 13.3-and 13.6-fold, respectively. The redox active vitamins also enhanced the rates of abiotic CT conversion in heat killed sludge treatments, but the rates were approximately 4- to 5-fold lower than the corresponding vitamin enhanced rates of biological CT conversion. The addition of CNB12 or HOB12 to the live methanogenic sludge consortium increased the yield of inorganic chloride (Cl-) from CT-converted. Chloroform was a transient intermediate in CNB12 or HOB12 supplemented cultures. In contrast, the addition of RF increased the yield of chloroform from CT-converted. Taken as a whole the results clearly demonstrate that very low concentrations of redox active vitamins could potentially play an important role in accelerating the anaerobic the bioremediation of CT as well as influencing the proportions of biotransformation products formed.
• Guerrero-Barajas, C., & Field, J. A. (2005). Riboflavin- and cobalamin-mediated biodegradation of chloroform in a methanogenic consortium. Biotechnology and Bioengineering, 89(5), 539-550.
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  PMID: 15669086;Abstract: Chloroform (CF) is an important priority pollutant contaminating groundwater. Reductive dechlorination by anaerobic microorganisms is a promising strategy towards the remediation of CF. The objective of this study was to evaluate the use of redox active vitamins as electron shuttles to enhance the anaerobic biodegradation of CF in an unadapted methanogenic consortium not previously exposed to chlorinated compounds. Only negligible degradation of CF was observed in control cultures lacking redox active vitamins. The addition of riboflavin (RF), cyanocobalamin (CNB12), and hydroxycobalamin (HOB12) enabled biodegradation of CF. The reactions were predominantly catalyzed biologically as evidenced by the lack of any CF conversion in heat-killed controls amended with the cobalamins or minor conversion with RF. In live cultures, significant increases in the rate of CF conversion was observed at substoichiometric molar ratios as low as 0.1 to 0.01 vitamin:CF for RF and CNB12, respectively. At the highest molar vitamin:CF ratios tested of 0.2, the first-order rate constant of CF degradation was 5.3- and 91-fold higher in RF and CNB12 amended cultures, respectively, compared to the unamended control culture. The distribution of biotransformation products was highly impacted by the type of redox active vitamin utilized. Cultures supplemented with RF provided high yields of dichloromethane (DCM). On the other hand, cobalamins promoted the near complete mineralization of organochlorine in CF to inorganic chloride and lowered the yield of DCM. In cultures where no or little CF bioconversion occurred, prolonged exposure to CF resulted in cell lysis, as evidenced by the release of intracellular chloride. The results taken as a whole suggest that the anaerobic bioremediation of CF-contaminated sites can greatly be improved with strategies aimed at increasing the concentration of redox active vitamins. © 2005 Wiley Periodicals, Inc.
• Hollingsworth, J. R., Hollingsworth, J. R., Sierra Alvarez, R., Sierra Alvarez, M. R., Zhou, M., Zhou, M., Ogden, K. L., Ogden, K. L., Field, J. A., & Field, J. A. (2005). Anaerobic biodegradability and methanogenic toxicity of key constituents in copper chemical mechanical planarization effluents of the semiconductor industry. Chemosphere, 59, 1219-1228.
• Cervantes, F. J., Vu-Thi-Thu, L., Lettinga, G., & Field, J. A. (2004). Quinone-respiration improves dechlorination of carbon tetrachloride by anaerobic sludge. Applied Microbiology and Biotechnology, 64(5), 702-711.
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  PMID: 14762702;Abstract: The impact of humic acids and the humic model compound, anthraquinone-2,6-disulfonate (AQDS), on the biodegradation of carbon tetrachloride (CT) by anaerobic granular sludge was studied. Addition of both humic acids and AQDS at sub-stoichiometric levels increased the first-order rate of conversion of CT up to 6-fold, leading to an increased production of inorganic chloride, which accounted for 40-50% of the CT initially added. Considerably less dechlorination occurred in sludge incubations lacking humic substances. By comparison, very limited dechlorination occurred in sterile controls with autoclaved sludge. Accumulation of chloroform (1-10%) and dichloromethane (traces) also accounted for the CT converted. The accumulation of a chlorinated ethene, perchloroethylene (up to 9% of added CT), is also reported for the first time as an end-product of CT degradation. A humus-respiring enrichment culture (composed primarily of a Geobacter sp.) derived from the granular sludge also dechlorinated CT, yielding products similar to the AQDS-supplemented granular sludge consortium. The dechlorination of CT by the Geobacter enrichment was dependent on the presence of AQDS or humic acids, which were reduced during the assays. The reduced form of AQDS, anthrahydroquinone-2,6-disulfonate, was shown to cause the chemical reduction of CT when incubated in sterile medium. The results taken as a whole indicate that the formation of reduced humic substances by quinone-respiring microorganisms can contribute to the reductive dechlorination of CT. © Springer-Verlag 2004.
• Field, J., Sierra-Alvarez, R., Cortinas, I., Yenal, U., & Field, J. A. (2004). Methanogenic inhibition by arsenic compounds. Applied and environmental microbiology, 70(9).
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  The acute acetoclastic methanogenic inhibition of several inorganic and organic arsenicals was assayed. Trivalent species, i.e., methylarsonous acid and arsenite, were highly inhibitory, with 50% inhibitory concentrations of 9.1 and 15.0 microM, respectively, whereas pentavalent species were generally nontoxic. The nitrophenylarsonate derivate, roxarsone, displayed moderate toxicity.
• Cervantes, F. J., Duong-Dac, T., Ivanova, A. E., Roest, K., Akkermans, A. D., Lettinga, G., & Field, J. A. (2003). Selective enrichment of Geobacter sulfurreducens from anaerobic granular sludge with quinones as terminal electron acceptors. Biotechnology Letters, 25(1), 39-45.
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  PMID: 12882304;Abstract: A quinone-respiring, enrichment culture derived from methanogenic granular sludge was phylogenetically characterized by using a combined cloning-denaturing gradient gel electrophoresis (DGGE) method, which revealed that the consortium developed was dominated by a single microorganism: 97% related, in a sequence of 1520 base pairs, to Geobacter sulfurreducens. The enrichment culture could grow with acetate, formate or H2 when humic acids, the humic model compound, anthraquinone-2,6-disulfonate (AQDS), or chelated Fe(III) was provided as a terminal electron acceptor. The occurrence of a humic acid- or quinone-respiring microorganism in the microbial community of a wastewater treatment system suggests that this type of microorganisms may play a potential role in anaerobic bioreactors treating humus-containing wastewaters.
• Cervantes, F. J., Duong-Dac, T., Roest, K., Akkermans, A. D., Lettinga, G., & Field, J. A. (2003). Enrichment and immobilization of quinone-respiring bacteria in anaerobic granular sludge. Water Science and Technology, 48(6), 9-16.
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  PMID: 14640194;Abstract: The capacity of an anaerobic granular sludge for serving as an immobilizing mechanism for quinone-respiring bacteria was evaluated. The inoculum was continuously fed with a basal medium containing the humic model compound, anthraquinone-2,6-disulfonate (AQDS), as a terminal electron acceptor. Complete reduction of AQDS was achieved by the granular sludge for a prolonged period in an anaerobic bioreactor provided with a mixture of volatile fatty acids as a substrate. Phylogenetic analysis revealed the enrichment and immobilization of AQDS-respiring bacteria appearing as dominant organisms in the microbial population of the AQDS-supplemented reactor, compared to a reactor control operated under methanogenic conditions. The consistent quinone-reducing capacity observed in the consortium indicates that it is feasible to apply quinone-reducing microorganisms in continuous bioreactors and this ability can potentially be important in wastewaters rich in humic substances. The quinone reducing activity could also be applied to accelerate the conversion of xenobiotics susceptible to reductive biotransformations such as azo dyes and polychlorinated compounds in continuous bioreactors.
• Field, J. A., & Brady, J. (2003). Riboflavin as a redox mediator accelerating the reduction of the azo dye Mordant Yellow 10 by anaerobic granular sludge. Water Science and Technology, 48(6), 187-193.
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  PMID: 14640217;Abstract: Azo dyes are important persistent pollutants of textile industry effluents. Reduction of these dyes to their corresponding aromatic amines under anaerobic conditions can be used to initiate biodegradation. Since electron transfer is suggested to be rate limiting, redox mediators are being considered to improve dye reduction kinetics. This study evaluates the use of riboflavin, the redox active moiety of common occurring enzyme cofactors, as a redox mediator to accelerate the reduction of the azo dye, mordant yellow 10 (MY10). Dye reduction was found to follow zero order kinetics, the total rate constant (Vtotal) could be separated into two components: the rate of reduction due to direct contact between enzymes in the sludge with the dye (Vdirect); and the rate of reduction mediated by riboflavin (Vmediated). Riboflavin increased the Vtotal by 61 % at extremely sub-stoichiometric concentrations of 9.1 μmol l-1, which corresponded to a molar riboflavin:dye ratio of 1:60. The accelerating effect of riboflavin displayed saturation kinetics at higher concentrations, with a maximum increase of Vtotal of approximately 2-fold. A model is presented which assumes that Vmediated depends on the activity of riboflavin reductase (RR) and thus follows Michaelis-Menton kinetics with respect to the riboflavin concentration. The half-velocity constant (Km) was very low (6.3 μmol l-1), indicating a high affinity of the sludge RR for riboflavin. Both Vdirect and Vmediated were found to be proportional to the assay sludge concentration. The results taken as a whole indicate that vitamin levels of riboflavin can be utilized to improve the kinetics of azo dye reduction during anaerobic treatment.
• P., F., A., I., Lettinga, G., & Field, J. A. (2003). Activated carbon as an electron acceptor and redox mediator during the anaerobic biotransformation of azo dyes. Environmental Science and Technology, 37(2), 402-408.
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  PMID: 12564915;Abstract: Activated carbon (AC) has a long history of applications in environmental technology as an adsorbent of pollutants for the purification of drinking waters and wastewaters. Here we describe novel role of AC as redox mediator in accelerating the reductive transformation of pollutants as well as a terminal electron acceptor in the biological oxidation of an organic substrate. This study explores the use of AC as an immobilized redox mediator for the reduction of a recalcitrant azo dye (hydrolyzed Reactive Red 2) in laboratory-scale anaerobic bioreactors, using volatile fatty acids as electron donor. The incorporation of AC in the sludge bed greatly improved dye removal and formation of aniline, a dye reduction product. These results indicate that AC acts as a redox mediator. In supporting batch experiments, bacteria were shown to oxidize acetate at the expense of reducing AC. Furthermore, AC greatly accelerated the chemical reduction of an azo dye by sulfide. The results taken as a whole clearly suggest that AC accepts electrons from the microbial oxidation of organic acids and transfers the electrons to azo dyes, accelerating their reduction. A possible role of quinone surface groups in the catalysis is discussed.
• P., F., Bisschops, I. A., Blanchard, V. G., Bouwman, R. H., Lettinga, G., & Field, J. A. (2003). The contribution of biotic and abiotic processes during azo dye reduction in anaerobic sludge. Water Research, 37(13), 3098-3109.
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  PMID: 14509696;Abstract: Azo dye reduction results from a combination of biotic and abiotic processes during the anaerobic treatment of dye containing effluents. Biotic processes are due to enzymatic reactions whereas the chemical reaction is due to sulfide. In this research, the relative impact of the different azo dye reduction mechanisms was determined by investigating the reduction of Acid Orange 7 (AO7) and Reactive Red 2 (RR2) under different conditions. Reduction rates of two azo dyes were compared in batch assays over a range of sulphide concentrations in the presence of living or inactivated anaerobic granular sludge. Biological dye reduction followed zero order kinetics and chemical dye reduction followed second-order rate kinetics as a function of sulfide and dye concentration. Chemical reduction of the dyes was greatly stimulated in the presence of autoclaved sludge; whereas chemical dye reduction was not affected by living or γ-irradiated-sludge. Presumably redox-mediating enzyme cofactors released by cell lysis contributed to the stimulatory effect. This hypothesis was confirmed in assays evaluating the chemical reduction of AO7 utilizing riboflavin, representative of the heat stable redox-mediating moieties of common occurring flavin enzyme cofactors. Sulfate influenced dye reduction in accordance to biogenic sulfide formation from sulfate reduction. In assays lacking sulfur compounds, dye reduction only readily occurred in the presence of living granular sludge, demonstrating the importance of enzymatic mechanisms. Both chemical and biological mechanisms of dye reduction were greatly stimulated by the addition of the redox-mediating compound, anthraquinone-disulfonate. Based on an analysis of the kinetics and demonstration in lab-scale upward-flow anaerobic sludge bed reactors, the relative importance of chemical dye reduction mechanisms in high rate anaerobic bioreactors was shown to be small due to the high biomass levels in the reactors. © 2003 Elsevier Science Ltd. All rights reserved.
• Paulo, P. L., Stams, A. J., Field, J. A., Dijkema, C., Lier, J. V., & Lettinga, G. (2003). Pathways of methanol conversion in a thermophilic anaerobic (55°C) sludge consortium. Applied Microbiology and Biotechnology, 63(3), 307-314.
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  PMID: 12856164;Abstract: The pathway of methanol conversion by a thermophilic anaerobic consortium was elucidated by recording the fate of carbon in the presence and absence of bicarbonate and specific inhibitors. Results indicated that about 50% of methanol was directly converted to methane by the methylotrophic methanogens and 50% via the intermediates H2/CO2 and acetate. The deprivation of inorganic carbon species [∑(HCO3-+CO2)] in a phosphate-buffered system reduced the rate of methanol conversion. This suggests that bicarbonate is required as an electron (H2) sink and as a co-substrate for the efficient and complete removal of the chemical oxygen demand. Nuclear magnetic resonance spectroscopy was used to investigate the route of methanol conversion to acetate in bicarbonate-sufficient and bicarbonate-depleted environments. The proportions of [1,2-13C]acetate, [1-13C]acetate and [2-13C]acetate were determined. Methanol was preferentially incorporated into the methyl group of acetate, whereas HCO3- was the preferred source of the carboxyl group. A small amount of the added H13CO3- was reduced to form the methyl group of acetate and a small amount of the added 13CH 3OH was oxidised and found in the carboxyl group of acetate when 13CH3OH was converted. The recovery of [ 13C]carboxyl groups in acetate from 13CH3OH was enhanced in bicarbonate-deprived medium. The small amount of label incorporated in the carboxyl group of acetate when 13CH 3OH was converted in the presence of bromoethanesulfonic acid indicates that methanol can be oxidised to CO2 prior to acetate formation. These results indicate that methanol is converted through a common pathway (acetyl-CoA), being on the one hand reduced to the methyl group of acetate and on the other hand oxidised to CO2, with CO2 being incorporated into the carboxyl group of acetate.
• Razo-Flores, E., Iniestra-González, M., Field, J. A., Olguin-Lora, P., & Puig-Grajales, L. (2003). Biodegradation of mixtures of phenolic compounds in an upward-flow anaerobic sludge blanket reactor. Journal of Environmental Engineering, 129(11), 999-1006.
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  Abstract: The anaerobic biodegradability of mixtures of phenolic compounds was studied under continuous and batch systems. Continuous experiments were carried out in up-flow anaerobic sludge bed (UASB) reactors degrading a mixture of phenol and p-cresol as the main carbon and energy sources. The total chemical oxygen demand (COD) removal above 90% was achieved even at organic loading rates as high as 7 kg COD/m3/day. Batch experiments were conducted with mixtures of phenolic compounds (phenol, p-cresol, and o-cresol) to determine the specific biodegradation rates using unadapted and adapted anaerobic granular sludge. Phenol and p-cresol were mineralized by adapted sludge with rates several orders of magnitude higher than unadapted sludge. Additionally, an UASB reactor was operated with the mixture phenol, p-cresol, and o-cresol. After 54 days of operation, 80% of o-cresol (supplied at 132 mg/L) was eliminated. The phenol biodegradation was not affected by the presence of o-cresol. These results demonstrate that major phenolic components in petrochemical effluents can be biodegraded simultaneously during anaerobic treatment.
• Cervantes, F. J., A., F., Duong-Dac, T., J., A., Lettinga, G., & Field, J. A. (2002). Reduction of humic substances by halorespiring, sulphate-reducing and methanogenic microorganisms. Environmental Microbiology, 4(1), 51-57.
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  PMID: 11966825;Abstract: Physiologically distinct anaerobic microorganisms were explored for their ability to oxidize different substrates with humic acids or the humic analogue, anthraquinone-2,6-disulphonate (AQDS), as a terminal electron acceptor. Most of the microorganisms evaluated including, for example, the halorespiring bacterium, Desulfitobacterium PCE1, the sulphate-reducing bacterium, Desulfovibrio G11 and the methanogenic archaeon, Methanospirillum hungatei JF1, could oxidize hydrogen linked to the reduction of humic acids or AQDS. Desulfitobacterium dehalogenans and Desulfitobacterium PCE1 could also convert lactate to acetate linked to the reduction of humic substances. Humus served as a terminal electron acceptor supporting growth of Desulfitobacterium species, which may explain the recovery of these microorganisms from organic rich environments in which the presence of chlorinated pollutants or sulphite is not expected. The results suggest that the ubiquity of humus reduction found in many different environments may be as a result of the increasing number of anaerobic microorganisms, which are known to be able to reduce humic substances.
• Field, J. A. (2002). Limits of anaerobic biodegradation.. Water Science and Technology, 45(10), 9-18.
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  PMID: 12188583;Abstract: The main factors responsible for anaerobic recalcitrance are reviewed. Anaerobic recalcitrance is associated with hydrocarbons lacking functional groups, branched molecules (gasoline oxygenates), aromatic amines and aromatic sulfonates. The most recalcitrant compounds are high molecular weight nonhydrolyzable polymers such as plastic, lignin and humus, which cannot be taken up by cells. Recently new capabilities of anaerobic microorganisms have been discovered to degrade compounds previously considered to be recalcitrant. For example, anaerobic bacteria initiate the degradation of alkylbenzenes and alkanes with an unusual addition reaction with fumarate, forming a hydrocarbon-succinate adduct. Finally, new evidence indicates that the most recalcitrant compounds (humic substances) are not so inert and can play important roles in aiding the biodegradation of other compounds by serving as an electron acceptor or redox mediator.
• Cervantes, F. J., Dijksma, W., Duong-Dac, T., Ivanova, A., Lettinga, G., & Field, J. A. (2001). Anaerobic Mineralization of Toluene by Enriched Sediments with Quinones and Humus as Terminal Electron Acceptors. Applied and Environmental Microbiology, 67(10), 4471-4478.
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  PMID: 11571145;PMCID: PMC93192;Abstract: The anaerobic microbial oxidation of toluene to CO2 coupled to humus respiration was demonstrated by use of enriched anaerobic sediments from the Amsterdam petroleum harbor (APH) and the Rhine River. Both highly purified soil humic acids (HPSHA) and the humic quinone moiety model compound anthraquinone-2,6-disulfonate (AQDS) were utilized as terminal electron acceptors. After 2 weeks of incubation, 50 and 85% of added uniformly labeled [13C] toluene were recovered as 13CO2 in HPSHA- and AQDS-supplemented APH sediment enrichment cultures, respectively; negligible recovery occurred in unsupplemented cultures. The conversion of [13C] toluene agreed with the high level of recovery of electrons as reduced humus or as anthrahydroquinone-2,6-disulfonate. APH sediment was also able to use nitrate and amorphous manganese dioxide as terminal electron acceptors to support the anaerobic biodegradation of toluene. The addition of substoichiometric amounts of humic acids to bioassay reaction mixtures containing amorphous ferric oxyhydroxide as a terminal electron acceptor led to more than 65% conversion of toluene (1 mM) after 11 weeks of incubation, a result which paralleled the partial recovery of electron equivalents as acid-extractable Fe(II). Negligible conversion of toluene and reduction of Fe(III) occurred in these bioassay reaction mixtures when humic acids were omitted. The present study provides clear quantitative evidence for the mineralization of an aromatic hydrocarbon by humus-respiring microorganisms. The results indicate that humic substances may significantly contribute to the intrinsic bioremediation of anaerobic sites contaminated with priority pollutants by serving as terminal electron acceptors.
• Cervantes, F. J., Van, F., Lettinga, G., & Field, J. A. (2001). Enhanced decolourisation of acid orange 7 in a continuous UASB reactor with quinones as redox mediators. Water Science and Technology, 44(4), 123-128.
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  PMID: 11575075;Abstract: The reductive biotransformation of acid orange 7 (AO7) was explored in a lab-scale upflow anaerobic sludge blanket (UASB) reactor at low hydraulic residence times (HRT). A colour removal of 85% was achieved when the reactor was operated at a HRT of 6 hours, but decreased up to 70% when the HRT was lowered to 2 hours. Addition of the quinone model compound, anthraquinone 2,6-disulfonate (AQDS), as redox mediator, allowed for a considerably higher decolourising efficiency (>90% at all the HRT evaluated). The results indicate that the use of catalytic concentrations of AQDS (AQDS/AO7 molar ratio about 0.01) can accelerate decolourising processes achieving satisfying extent of decolourisation.
• Eekert, M. V., Schröder, T., Rhee, A. v., Stams, A. J., Schraa, G., & Field, J. A. (2001). Constitutive dechlorination of chlorinated ethenes by a methanol degrading methanogenic consortium. Bioresource Technology, 77(2), 163-170.
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  PMID: 11272023;Abstract: The ability of granular methanogenic sludge to dechlorinate chloroethenes was investigated with unadapted sludge from an upflow anaerobic sludge blanket (UASB) reactor fed with methanol. The sludge degraded chlorinated ethenes, but the degradation rates were low. The addition of primary substrate was necessary to sustain dechlorination. The dechlorinating activity seemed to be constitutively present in the anaerobic bacteria. Usually, one chlorine atom was removed via reductive hydrogenolysis. Only trichloroethene (TCE) was converted to substantial amounts of vinylchloride (VC). 1,1-Dichloroethene (1,1DCE) was observed to be an important intermediate in the dechlorination by unadapted granular sludge, although previously this compound had not been commonly observed. Furthermore, the dechlorination of 1,1DCE was faster than the dechlorination of the other chloroethenes. Copyright © 2001 Elsevier Science Ltd.
• Field, J. A. (2001). Recalcitrance as a catalyst for new developments. Water Science and Technology, 44(8), 33-40.
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  PMID: 11730134;Abstract: Humic substances are very recalcitrant in anaerobic environments as substrates, mostly due to their non-hydrolyzable high molecular weight structure, which is incompatible for cell uptake. The recalcitrant nature can be applied towards immobilizing pollutants into non-toxic humic substances. Certain anaerobic bioconversions such as demethylation of lignin and reduction of nitro- or azo groups can active pollutants for humification processes upon subsequent exposure to air. Although humic substances provide no carbon or engery for microorganisms, the evidence presented here indicates that they are very active as terminal electron acceptors and redox mediators promoting the anaerobic degradation and biotransformation of priority pollutants.
• Hage, A., Petra, D. G., Field, J. A., Schipper, D., Wijnberg, J. B., Kamer, P. C., Reek, J. N., W.N.M., P., Wever, R., & Schoemaker, H. E. (2001). Asymmetric reduction of ketones via whole cell bioconversions and transfer hydrogenation: Complementary approaches. Tetrahedron Asymmetry, 12(7), 1025-1034.
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  Abstract: Prochiral aryl and dialkyl ketones were enantioselectively reduced to the corresponding alcohols using whole cells of the white-rot fungus Merulius tremellosus ono991 as a biocatalytic reduction system and ruthenium(II)-amino alcohol and iridium(I)-amino sulfide complexes as metal catalysts in asymmetric transfer hydrogenation. Comparison of the results showed that the corresponding chiral alcohols could be obtained with moderate to high enantioselectivities (e.e.s of up to 98%). The biocatalytic and transfer hydrogenation approaches appear to be complementary. The biocatalytic approach is the most suitable for the enantioselective reduction of chloro-substituted (aryl) ketones, whereas in the reduction of α,β-unsaturated compounds excellent results were obtained using the catalytic hydrogenation protocol. © 2001 Elsevier Science Ltd.
• Hage, A., Schoemaker, H. E., & Field, J. A. (2001). Optimization of stereoselective ketone reduction by the white-rot fungus Merulius tremellosus ono991. Applied Microbiology and Biotechnology, 57(1-2), 79-84.
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  PMID: 11693938;Abstract: A recently isolated white-rot fungal strain, Merulius tremellosus ono991, displays high stereoselectivity during the reduction of arylketones. In order to increase the productivity and specific yield of the optically active alcohols, the culture conditions for the reduction of the model ketone compound 1′-acetonaphtone to α-methyl-1-naphtalenemethanol were optimized with respect to oxygen supply, choice of primary substrate and arylketone concentration. Alternative electron acceptors were also used to elucidate the role of reduction equivalents in the reduction process. The optimal yields of α-methyl-1-naphtalenemethanol were obtained in N2-flushed incubations with glycerol as primary substrate. The specific yield was increased from 57% to 98% compared to incubations under air with glucose. Most of the yield increase was due to N2-flushing and could be attributed to two factors. First, an increased stability of the product, α-methyl-1-naphtalenemethanol, in anaerobic compared to aerobic atmosphere was demonstrated. Second, fermentative metabolism increased reduced enzyme cofactors available for the reduction. Diverting reducing equivalents away from fermentation with alternative electron acceptors correlated with a decreased yield of α-methyl-1-naphtalenemethanol. Furthermore, the dependency of ketone reductase for common occurring metabolic reducing equivalents, NAD(P)H, was demonstrated by the reduction of 1′-acetonaphtone in cell extracts of M. tremellosus ono991.
• Lexmond, M. J., Lier, J. V., Lens, P. N., Field, J. A., Pol, L. H., & Zeeman, G. (2001). Farewell seminar for Prof. Dr. Ir Gatze Lettinga, Wageningen, The Netherlands, 29-30 March 2001: Preface. Water Science and Technology, 44(8), vii-viii.
• P., F., H., R., P., D., Lettinga, G., & Field, J. A. (2001). Application of redox mediators to accelerate the transformation of reactive Azo dyes in anaerobic bioreactors. Biotechnology and Bioengineering, 75(6), 691-701.
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  PMID: 11745147;Abstract: Azo dyes are nonspecifically reduced under anaerobic conditions but the slow rates at which reactive azo dyes are converted presents a serious problem for the application of anaerobic technology as a first stage in the complete biodegradation of these compounds. As quinones have been found to catalyze reductive transfers by acting as redox mediators, the application of anthraquinone-2,6-disulfonic acid (AQDS) during continuous anaerobic treatment of the reactive azo dye, Reactive Red 2 (RR2), was evaluated. A mixture of volatile fatty acids was used as the electron-donating primary substrate. Batch experiments demonstrated that AQDS could increase the first-order rate constant of RR2 reductive cleavage by one order of magnitude. In the continuous experiment, treatment of RR2 containing synthetic wastewater in a lab-scale upflow anaerobic sludge blanket (UASB) reactor yielded low dye removal eciencies (
• P., F., Lettinga, G., & Field, J. A. (2001). Azo dye decolourisation by anaerobic granular sludge. Chemosphere, 44(5), 1169-1176.
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  PMID: 11513405;Abstract: The decolourisation of 20 selected azo dyes by granular sludge from an upward-flow anaerobic sludge bed (UASB) reactor was assayed. Complete reduction was found for all azo dyes tested, generally yielding colourless products. The reactions followed first-order kinetics and reaction rates varied greatly between dyes: half-life times ranged from 1 to about 100 h. The slowest reaction rates were found for reactive dyes with a triazine reactive group. There was no correlation between a dye's half-life time and its molecular weight, indicating that cell penetration was probably not an important factor. Since granular sludge contains sulphide, eight dyes were also monitored for direct chemical decolourisation by sulphide. All these dyes were reduced chemically albeit at slower rates than in the presence of sludge at comparable sulphide levels. Increasing sulphide concentrations, even when present in huge excess, stimulated the azo reduction rate. The results indicate that granular sludge can decolourise a broad spectrum of azo dye structures due to non-specific extracellular reactions. Reducing agents (e.g., sulphide) in sludge play an important role. The presence of anaerobic biomass is probably beneficial for maintaining the pools of these reduced compounds. © 2001 Elsevier Science Ltd. All rights reserved.
• Cervantes, F. J., Van, S., Lettinga, G., & Field, J. A. (2000). Competition between methanogenesis and quinone respiration for ecologically important substrates in anaerobic consortia. FEMS Microbiology Ecology, 34(2), 161-171.
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  Abstract: Anaerobic consortia obtained from a wide variety of environments were tested for oxidizing several ecologically significant substrates with the humic model compound, anthraquinone-2,6-disulfonate (AQDS), as terminal electron acceptor. All the substrates, including hydrogen, acetate, propionate, methanol and lactate, were completely or partially converted to methane when bicarbonate was the only electron acceptor available. Addition of AQDS (20 mM) to the cultures prevented methanogenesis in most cases and AQDS reduction became the preferred pathway. AQDS was shown to be toxic for methanogenesis and this effect played an important role in enabling quinone-respiring bacteria to outcompete methanogens. Furthermore, AQDS respiration is thermodynamically more favorable than methanogenesis. All the consortia evaluated were capable of oxidizing hydrogen linked to the reduction of AQDS. Most inocula tested were also able to oxidize acetate and lactate in the same way. When methanol was provided as an electron donor competition between methanogenesis and acetogenesis occurred. Acetate accumulated from the latter process was responsible for quinone respiration. These results suggest that quinone-respiring bacteria are ubiquitous and that quinones in humus may significantly contribute to carbon cycling process by serving as a terminal electron acceptor for the anaerobic microbial oxidation of a wide variety of ecologically important substrates. Copyright (C) 2000 Federation of European Microbiological Societies.
• Cervantes, F. J., Van, S., Lettinga, G., & Field, J. A. (2000). Quinones as terminal electron acceptors for anaerobic microbial oxidation of phenolic compounds. Biodegradation, 11(5), 313-321.
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  PMID: 11487061;Abstract: The capacity of anaerobic granular sludge for oxidizing phenol and p-cresol under anaerobic conditions was studied. Phenol and p-cresol were completely converted to methane when bicarbonate was the only terminal electron acceptor available. When the humic model compound, anthraquinone-2,6-disulfonate, was included as an alternative electron acceptor in the cultures, the oxidation of the phenolic compounds was coupled to the reduction of the model humic compound to its corresponding hydroquinone, anthrahydroquinone-2,6-disulfonate. These results demonstrate for the first time that the anaerobic degradation of phenolic compounds can be coupled to the reduction of quinones as terminal electron acceptor.
• Field, J. A., Cervantes, F. J., Van, F., & Lettinga, G. (2000). Role of quinones in the biodegradation of priority pollutants: A review. Water Science and Technology, 42(5-6), 215-222.
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  Abstract: Evidence is accumulating that inert humic substances can play important roles in the anaerobic degradation of priority pollutants by shuttling electrons. This paper reviews the roles of humus and quinone analogues as electron acceptors, redox mediators and electron donors for microbial and abiotic degradation processes. An eventual technology based on pumping quinones as terminal electron acceptors into aquifers and sediments to stimulate xenobiotic degradation offers promising potential. Also quinone redox mediators can be considered to accelerate reductive transformations (e.g. dechlorination, azo cleavage) of priority pollutants.
• Have, R. T., C., M., & Field, J. A. (2000). Lignin peroxidase initiates O₂-dependent self-propagating chemical reactions which accelerate the consumption of 1-(3',4'-dimethoxyphenyl)propene. Biochemical Journal, 347(2), 585-591.
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  PMID: 10749690;PMCID: PMC1220993;Abstract: Lignin peroxidase (LiP) has been used to study the C(α)-C(β) cleavage of the propylene side chain in 1-(3',4'-dimethoxyphenyl)propene (DMPP) to 3,4-dimethoxybenzaldehyde (veratraldehyde, VAD). Under an air atmosphere, LIP oxidized DMPP to VAD (27.8%) and 1-(3',4'-dimethoxyphenyl)propan-2-one (DMPA, 8.7%), after 10 min of incubation. Dissolved O2 was rapidly consumed during DMPP conversion, of which one-third was converted into superoxide. The remaining two-thirds of the consumed O2 was involved in C(α)-C(β) cleavage of DMPP to VAD and in self-propagating chemical reactions stimulating the consumption of DMPP. The involvement of peroxyl radicals, in the chemical consumption of DMPP, was confirmed bq using the well-known peroxyl radical reductant Mn2+. This metal ion severely inhibited the DMPP consumption rate under air, but did not affect the lower enzymic DMPP consumption rate under N2. The substoichiometric requirement of LiP for H2O2 during DMPP oxidation could be explained in part by dismutation of superoxide, but more importantly by direct chemical reactions of DMPP-derived peroxyl radicals with fresh DMPP. Another VAD-producing route was found by incubating the DMPP oxidation product, DMPA, with LiP. Under air the molar yield of VAD was 29.7%. In the absence of O2, the C(α)-C(β) cleavage of DMPA to VAD was strongly inhibited and side-chain coupling products (dimers) were formed instead. As a whole, the results suggest two new roles of O2 in LiP-mediated oxidation of aromatic substrates. First, O2 is responsible for the formation of reactive peroxyl intermediates, which can directly react with other substrate molecules and thereby accelerate consumption rates. Secondly, O2 can prevent coupling reactions by lowering the pool of carbon-centred radicals accumulating during LIP catalysis.
• Have, R. T., Thouars, R. D., Swarts, H. J., & Field, J. A. (2000). Erratum: Veratryl alcohol-mediated oxidation of isoeugenyl acetate by lignin peroxidase (European Journal of Biochemistry (1999) 265 (1008-1014)). European Journal of Biochemistry, 267(2), 625-.
• Puig-Grajales, L., Tan, N. G., Van, F., Razo-Flores, E., & Field, J. A. (2000). Anaerobic biodegradability of alkylphenols and fuel oxygenates in the presence of alternative electron acceptors. Applied Microbiology and Biotechnology, 54(5), 692-697.
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  PMID: 11131397;Abstract: Alkylphenols and fuel oxygenates are important environmental pollutants produced by the petrochemical industry. A batch biodegradability test was conducted with selected ortho-substituted alkylphenols (2-cresol, 2,6-dimethylphenol and 2-ethylphenol), fuel oxygenates (methyl tert-butyl ether, ethyl tert-butyl ether and tert-amylmethyl ether) and tert-butyl alcohol (TBA) as model compounds. The ortho-substituted alkylphenols were not biodegraded after 100 days of incubation under methanogenic, sulfate-, or nitratereducing conditions. However, biodegradation of 2-cresol and 2-ethylphenol (150 mg l-1) was observed in the presence of Mn (IV) as electron acceptor. The biodegradation of these two compounds took place in less than 15 days and more than 90% removal was observed for both compounds. Mineralization was indicated since no UV-absorbing metabolites accumulated after 23 days of incubation. These alkylphenols were also slowly chemically oxidized by Mn (IV). No biodegradation of fuel oxygenates or TBA (1 g l-1) was observed after 80 or more days of incubation under methanogenic, Fe (III)-, or Mn (IV)-reducing conditions, suggesting that these compounds are recalcitrant under anaerobic conditions. The fuel oxygenates caused no toxicity towards acetoclastic methanogens activity in anaerobic granular sludge.
• Tan, N. C., Borger, A., Slenders, P., Svitelskaya, A., Lettinga, G., & Field, J. A. (2000). Degradation of azo dye Mordant Yellow 10 in a sequential anaerobic and bioaugmented aerobic bioreactor. Water Science and Technology, 42(5-6), 337-344.
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  Abstract: Complete biodegradation of azo dyes requires an anaerobic and aerobic step, in the anaerobic step sulfonated azo dyes (SADs) are reduced, yielding (sulfonated) aromatic amines ((S)AAs) which can be degraded aerobically. The complete biodegradation of the SAD Mordant Yellow 10 (MY10) was studied in a sequential anaerobic and aerobic bioreactor. Anaerobically, MY10 was reductively cleaved and the resulting aromatic amines, 5-aminosalicylic acid (5-ASA) and sulfanilic acid (SA), were both recovered in high stoichiometric yields. One of the AAs, 5-ASA, was readily degraded under aerobic conditions. However, SA was not degraded aerobically in the continuous experiment because no SA-degrading bacterial activity was present in the system. Therefore, a SA-degrading enrichment culture derived from Rhine sediment was used as an inoculum source. This enrichment culture was bioaugmented into the aerobic reactor by increasing the hydraulic retention time (HRT), thus enabling SA-degrading activity to develop and maintain in the aerobic reactor. After decreasing the HRT, the SA-degrading activity remained in the bioreactor and the stoichiometric recovery of sulfate (a SA biodegradation product) indicated the mineralization of SA after bioaugmentation. Batch experiments with aerobic reactor sludge confirmed the biodegradation of SA and 5-ASA. The sequential anaerobic and aerobic bioreactor was able to completely remove the sulfonated azo dye MY10 at a maximum loading rate of 210 mg MY10 (l(reactor) d)-1 after the appropriate microorganisms for aerobic degradation of SA were bioaugmented into the aerobic bioreactor.
• Van, F., Lettinga, G., & Field, J. A. (2000). The role of (auto)catalysis in the mechanism of an anaerobic azo reduction. Water Science and Technology, 42(5-6), 301-308.
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  Abstract: Azo dyes are non-specifically reduced under anaerobic conditions, but the slow rates at which many dyes react may present a serious problem for the application of anaerobic technology as a first stage in the complete biodegradation of these compounds. Therefore, it is significant to explore the mechanism of anaerobic azo reduction, especially with respect to its kinetics. With that purpose, decolouration of the monoazo dye C.I. Acid Orange 7 (AO7) was studied in batch experiments. Experiments indicated that chemical reduction by sulphide is partially responsible for the anaerobic conversions of AO7. Mathematical evaluation of the experimental results pointed out that autocatalysis played an important role in the chemical reduction of AO7. Further tests made clear that 1 -amino-2-naphthol was the dye's constituent aromatic amines that accelerated the reduction process, possibly by mediating the transfer of reducing equivalents. The impact of redox mediation by quinones was further evaluated by testing the catalysing effects of anthraquinone-2,6-disulphonic acid (AQDS) and of autoclaved sludge. AQDS appeared to be an extremely powerful catalyst, capable of increasing the first-order chemical reduction rate constants by a factor 10 to 100. Also autoclaved sludge, possibly because of mediation by sludge organic matter, accomplished accelerated azo reduction rates. Azo reduction in living sulphidogenic anaerobic sludge environments is 3 times more rapid than the chemically catalysed reaction with sulphide. The exact role of the biological activity remains to be clarified.
• B., J., Veldhuizen, A. V., Swarts, H. J., Frankland, J. C., & Field, J. A. (1999). Novel monochlorinated metabolites with a 1-benzoxepin skeleton from Mycena galopus. Tetrahedron Letters, 40(31), 5767-5770.
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  Abstract: Two novel monochlorinated 2,3-dihydro-1-benzoxepin derivatives were isolated from the ethyl acetate extracts of the stipes of Mycena galopus. The structures were established on the basis of their spectral data.
• Eekert, M. V., Stams, A. J., Field, J. A., & Schraa, G. (1999). Gratuitous dechlorination of chloroethanes by methanogenic granular sludge. Applied Microbiology and Biotechnology, 51(1), 46-52.
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  Abstract: The dechlorinating activity of a methanogenic granular sludge from a methanol-fed upflow anaerobic sludge blanket reactor was investigated with chlorinated ethanes. This unadapted methanogenic consortium degraded all chloroethanes tested. The product formation rates decreased with the number of chlorine substituents. The more highly chlorinated ethanes were also converted, although at a lower rate, in the presence of autoclaved (dead) sludge, indicating the involvement of reduced heat-stable cofactors like vitamin B12 and F430. Direct chemical dechlorination of hexa-, penta- and tetrachloroethanes was also observed in medium without sludge, although at a much lower rate. The results show the importance of cometabolic and abiotic (chemical) conversions for the transformation of chlorinated ethanes by the methanogenic consortium. The types of reaction and the products formed were correlated with the Gibbs free-energy change (ΔG(0')). Reductive hydrogenolysis and dichloroelimination were important dechlorinating mechanisms. Generally, these reactions have a higher ΔG(0')) value than dehydrochlorination reactions, which occurred less frequently during the transformation of chloroethanes by the methanogenic granular sludge.
• Hage, A., Schoemaker, H. E., & Field, J. A. (1999). Reduction of aryl acids by white-rot fungi for the biocatalytic production of aryl aldehydes and alcohols. Applied Microbiology and Biotechnology, 52(6), 834-838.
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  PMID: 10616717;Abstract: Ligninolytic basidiomycetes were screened for their ability to reduce aryl acids to the corresponding aldehydes and alcohols. Seven fungal strains converted p-anisic acid in high molar yields to the reduced products. The white-rot fungus Bjerkandera sp. strain BOS55 was one of the best reducing strains and was highly tolerant towards high concentrations of different aromatic acids. It was tested for the reduction of p-anisic, veratric, 3- chloro-4-methoxybenzoic, 3,5-dichloro-4-methoxybenzoic, 3,4-dichlorobenzoic, 4-fluorobenzoic, and 3-nitrobenzoic acids. All of these compounds were reduced to their corresponding aldehydes and alcohols.
• Have, R. T., G., R., Swarts, H. J., & Field, J. A. (1999). Veratryl alcohol-mediated oxidation of isoeugenyl acetate by lignin peroxidase. European Journal of Biochemistry, 265(3), 1008-1014.
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  PMID: 10518796;Abstract: The mechanism of the veratryl alcohol (VA)-mediated oxidation of isoeugenyl acetate (IEA) by lignin peroxidase, and the subsequent spontaneous C(α)-C(β) cleavage of IEA to vanillyl acetate were studied, IEA oxidation only occurred in the presence of VA. It probably did not bind to lignin peroxidase as evidenced by an unaffected K(m) for VA in the presence of IEA, and by the fact that a 10-fold molar excess of the unreactive IEA counterpart, eugenyl acetate, did not affect the IEA oxidation rate. IEA was very efficient in recycling VA. Up to 34 mol of IEA were oxidized per mol VA. Formation of the predominant VA oxidation product, veratraldehyde, was postponed until IEA was almost completely oxidized. Together these findings suggest that IEA was oxidized by VA·+ rather than directly by lignin peroxidase. Thus, VA functioned as a redox mediator during IEA oxidation which is remarkable considering the high calculated ionization potential of 8.81 eV. Regardless of the presence of O2, ≃ 2 mol of IEA were consumed per mol H2O2, which indicated that IEA was enzymatically oxidized by one electron to the putative radical cation (IEA·+). After formation of IEA·+, a series of O2-dependent chemical reactions were responsible for C(α)-C(β) cleavage to the major oxidation product vanillyl acetate, as evidenced by the observation that an N2 atmosphere did not inhibit IEA oxidation, but almost completely inhibited vanillyl acetate formation. GC-MS analyses revealed that under an air atmosphere 1-(4'-acetoxy-3'- methoxyphenyl)-2-propanone, 1-(4'-acetoxy-3'-methoxyphenyl)-1-hydroxy-2- propanone, and 1-(4'-acetoxy-3'-methoxyphenyl)-2-hydroxy-1-propanone were also formed. Formation of the latter two was diminished under an N2 atmosphere.
• Razo-Flores, E., Lettinga, G., & Field, J. A. (1999). Biotransformation and biodegradation of selected nitroaromatics under anaerobic conditions. Biotechnology Progress, 15(3), 358-365.
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  Abstract: The fate of four nitroaromatic compounds (5-nitrosalicylate, 5NSA; 4-nitrobenzoate, 4NBc; 2,4-dinitrotoluene, 2,4DNT; nitrobenzene, NB) was studied in 160 mL laboratory-scale upward-flow anaerobic sludge bed reactors supplied with a mixture of volatile fatty acids and/or glucose as electron donors. All the nitroaromatics were transformed stoichiometrically to their corresponding aromatic amines. After prolonged reactor operation, 5NSA and 4NBc were completely mineralized to CH4 and CO2, whereas 2,4DNT was partially transformed to a nonidentified and nondegradable metabolite. Batch nitro-reduction experiments indicated that the position of the nitro group in relation to the other substituents in the aromatic ring plays a key role in the rate of the nitro-group reduction. The results obtained indicate that certain nitroaromatic compounds can be completely mineralized and serve as a carbon and energy source for anaerobic bacteria.
• Razo-Flores, E., Smulders, P., Prenafeta-Boldú, F., Lettinga, G., & Field, J. A. (1999). Treatment of anthranilic acid in an anaerobic expanded granular sludge bed reactor at low concentrations. Water Science and Technology, 40(8), 187-194.
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  Abstract: The mineralization of anthranilic acid (2AB) as the only carbon and energy source was studied in batch and continuous conditions using methanogenic granular sludge. Under batch conditions in serum vials, 2AB (300 mg/l) was completely mineralized to methane within 55 days time. The experiment with the anaerobic continuous expanded granular sludge bed (EGSB) reactor was initially conducted at an upflow velocity (V(up)) of 5 m/h, a hydraulic retention time (HRT) of 12 h and an organic loading rate (OLR) of 1.5 g chemical oxygen demand (COD)/l-d. After 102 days, 2AB was not degraded at all and the reactor operation was shifted to batch mode by recycling the effluent. After some days, 2AB was completely mineralized and accumulation of flocculent sludge was observed. Batch biodegradability assays demonstrated that this flocculent biomass had a higher specific biodegradation rate compared to the granular sludge. During EGSB reactor operation, the flocculent biomass which was not attached to the granules probably washed-out under the applied hydrodynamic conditions. When the EGSB reactor was operated at V(up) of 2 m/h to favour the retention of the flocculent biomass, 2AB was mineralized even at influent concentrations as low as 140 mg COD/L. Kinetic calculations indicated that the sludge had an apparent K(s) value for the mineralization of 2AB as low as 24 mg COD/l.
• Tan, N. C., Lettinga, G., & Field, J. A. (1999). Reduction of the azo dye Mordant Orange 1 by methanogenic granular sludge exposed to oxygen. Bioresource Technology, 67(1), 35-42.
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  Abstract: Integration of anaerobic and aerobic conditions in a single bioreactor is a good strategy for the complete mineralization of azo dyes. In order for this strategy to work, azo dye reduction should occur in biofilms exposed to oxygen. Therefore, the effect of oxygen on the azo dye reduction by methanogenic granular sludge was studied using Mordant Orange 1 (MO1) as a model. Azo dye-reduction rates by two different granular sludges were determined in batch assays with various concentrations of oxygen in the headspace. Azo dye reduction occurred in the presence of oxygen if co-substrates, either ethanol or acetate, were added. The rate of dye reduction was highly positively correlated with the oxygen-consuming activity of the sludge. The results suggest that co-substrates stimulate oxygen respiration, which lowers oxygen penetration into the biofilm and thereby creates anaerobic microniches where azo dye reduction can occur.
• Tan, N. C., Prenafeta-Boldú, F., Opsteeg, J. L., Lettinga, G., & Field, J. A. (1999). Biodegradation of azo dyes in cocultures of anaerobic granular sludge with aerobic aromatic amine degrading enrichment cultures. Applied Microbiology and Biotechnology, 51(6), 865-871.
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  PMID: 10422231;Abstract: A prerequisite for the mineralization (complete biodegradation) of many azo dyes is a combination of reductive and oxidative steps. In this study, the biodegradation of two azo dyes, 4-phenylazophenol (4-PAP) and Mordant Yellow 10 (4-sulfophenylazo-salicylic acid; MY10), was evaluated in batch experiments where anaerobic and aerobic conditions were integrated by exposing anaerobic granular sludge to oxygen. Under these conditions, the azo dyes were reduced, resulting in a temporal accumulation of aromatic amines. 4-Aminophenol (4-AP) and aniline were detected from the reduction of 4-PAP. 5-Aminosalicylic acid (5-ASA) and sulfanilic acid (SA) were detected from the reduction of MY10. Subsequently, aniline was degraded further in the presence of oxygen by the facultative aerobic bacteria present in the anaerobic granular sludge. 5-ASA and SA were also degraded, if inocula from aerobic enrichment cultures were added to the batch experiments. Due to rapid autoxidation of 4-AP, no enrichment culture could be established for this compound. The results of this study indicate that aerobic enrichment cultures developed on aromatic amines combined with oxygen-tolerant anaerobic granular sludge can potentially be used to completely biodegrade azo dyes under integrated anaerobic/aerobic conditions.
• Collins, P. J., D., A., & Field, J. A. (1998). Reduction of the 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) cation radical by physiological organic acids in the absence and presence of Manganese. Applied and Environmental Microbiology, 64(6), 2026-2031.
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  Abstract: Laccase is a copper-containing phenoloxidase, involved in lignin degradation by white rot fungi. The laccase substrate range can be extended to include nonphenolic lignin subunits in the presence of a noncatalytic cooxidant such as 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), with ABTS being oxidized to the stable cation radical, ABTS.+, which accumulates. In this report, we demonstrate that the ABTS.+ can be efficiently reduced back to ABTS by physiologically occurring organic acids such as oxalate, glyoxylate, and malonate. The reduction of the radical by oxalate results in the formation of H2O2, indicating the formation of O2.- as an intermediate. O2.- itself was shown to act as an ABTS.+ reductant. ABTS.+ reduction and H2O2 formation are strongly stimulated by the presence of Mn2+, with accumulation of Mn3n+ being observed. Additionally, 4-methyl-O-isoeugenol, an unsaturated lignin monomer model, is capable of directly reducing ABTS.+. These data suggest several mechanisms for the reduction of ABTS.+ which would permit the effective use of ABTS as a laccase cooxidant at catalytic concentrations.
• Grotenhuis, T., Field, J., Wasseveld, R., & Rulkens, W. (1998). Biodegradation of polyaromatic hydrocarbons (PAH) in polluted soil by the white-rot fungus Bjerkandera. Journal of Chemical Technology and Biotechnology, 71(4), 359-360.
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  Abstract: The feasibility of using the White-rot fungi Bjerkandera for bioremediation of polluted soil through polyaromatic hydrocarbon (PAH) biodegradation was investigated. Following laboratory experiments which demonstrated large increase in PAH degradation with increase in bioavailability by the addition of surfactants, bench-scale experiments with real contaminated soil were conducted, achieving PAH degradation in soil with up to 70% PAH removal in real aged soil pollution.
• H., M., Schröder, T. J., J., A., Schraa, G., & Field, J. A. (1998). Degradation and fate of carbon tetrachloride in unadapted methanogenic granular sludge. Applied and Environmental Microbiology, 64(7), 2350-2356.
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  Abstract: The potential of granular sludge from upfiow anaerobic sludge blanket (UASB) reactors for bioremediation of chiorinated pollutants was evaluated by using carbon tetrachloride (CT) as a model compound. Granular sludges cultivated in UASB reactors on methanol, a volatile fatty acid mixture, or sucrose readily degraded CT supplied at a concentration of 1,500 nmol/batch (approximately 10 μM) without any prior exposure to organohalogens. The maximum degradation rate was 1.9 μmol of CT g of volatile suspended solids-1 day-1. The main end products of CT degradation were CO2 and Cl-, and the yields of these end products were 44 and 68%, respectively, of the initial amounts of [14C]CT and CT-Cl. Lower chlorinated methanes accumulated in minor amounts temporarily. Autoclaved (dead) sludges were capable of degrading CT at rates two- to threefold lower than those for living sludges, indicating that abiotic processes (mediated by cofactors or other sludge components) played an important role in the degradation observed. Reduced components in the autoclaved sludge were vital for CT degradation. A major part (51%) of the CT was converted abiotically to CS2. The amount of CO2 produced (23%) was lower and the amount of Cl- produced (86%) was slightly higher with autoclaved sludge than with living sludge. Both living and autoclaved sludges could degrade chloroform. However, only living sludge degraded dichloromethane and methylchloride. These results indicate that reductive dehalogenation, which was mediated better by living sludge than by autoclaved sludge, is only a minor pathway for CT degradation. The main pathway involves substitutive and oxidative dechlorination reactions that lead to the formation of CO2. Granular sludge, therefore, has outstanding potential for gratuitous dechlorination of CT to safe end products.
• Have, R. T., Hartmans, S., Teunissen, P. J., & Field, J. A. (1998). Purification and characterization of two lignin peroxidase isozymes produced by Bjerkandera sp. strain BOS55. FEBS Letters, 422(3), 391-394.
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  PMID: 9498823;Abstract: The white-rot fungus Bjerkandera sp. strain BOS55 excretes at least seven lignin peroxidase (LIP) isozymes. Two of these, LiP-2 and LIP-5 (molecular weight 40-42 kDa), were purified to homogeneity. Both isozymes had the same N-terminal amino acid sequences which showed strong homology with LiP isozymes produced by other white-rot fungi. The kinetics of both isozymes were similar, LIP-5 oxidized veratryl alcohol optimally only in the presence of H2O2 near pH 3.0 (16.7 U/mg) and LiP-2 did this below pH 2.5 (33.8 U/mg). Also at normal physiological pHs for fungal growth (pH 5.0-6.5) both isozymes were still active. Further characterization of LiP-2 and LiP-5 revealed that the K(m) for H2O2 strongly decreased with increasing pH. As a result of this the catalytic efficiency (TN/K(m)) calculated on the basis of the K(m) for H2O2 in the oxidation of veratryl alcohol was constant over wide pH range.
• Have, R. T., Rietjens, I. M., Hartmans, S., Swarts, H. J., & Field, J. A. (1998). Calculated ionisation potentials determine the oxidation of vanillin precursors by lignin peroxidase. FEBS Letters, 430(3), 390-392.
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  PMID: 9688577;Abstract: In view of the biocatalytic production of vanillin, this research focused on the lignin peroxidase (LiP) catalysed oxidation of naturally occurring phenolic derivatives: O-methyl ethers, O-acetyl esters, and O- glucosyl ethers. The ionisation potential (IP) of a series of model compounds was calculated and compared to their experimental conversion by LiP, defining a relative IP threshold of approximately 9.0 eV. Based on this threshold value only the O-acetyl esters and glucosides of isoeugenol and coniferyl alcohol would be potential LiP substrates. Both O-acetyl esters were tested and were shown to be converted to O-acetyl vanillin in molar yields of 51.8 and 2.3%, respectively.
• Hoekstra, E. J., Verhagen, F. J., Field, J. A., W.B., E., & Brinkman, U. A. (1998). Natural production of chloroform by fungi. Phytochemistry, 49(1), 91-97.
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  Abstract: Chloroform production was detected in the headspace of pure cultures of the basidiomycetes Mycena metata and Peniophora pseudopini and the deuteromycete Caldariomyces fumago. The average production rates were in the range of 0.07-70 μg/l culture fluid/day for Caldariomyces fumago and 0.7-40 ng/1 culture fluid/day for the basidiomycetes; they depended on the composition of the medium, pH and the initial concentration of oxygen. In incidental cases, chloroform was identified in the headspace of pure cultures of the basidiomycetes Agaricus arvensis, Bjerkandera sp. BOS55, and Phellinus pini. It is suggested that fungi are important sources of elevated concentrations of chloroform in soil air.
• J., F., Swarts, H. J., B., J., & Field, J. A. (1998). Biotransformation of the major fungal metabolite 3,5-dichloro-p-anisyl alcohol under anaerobic conditions and its role in formation of bis(3,5- dichloro-4-hydroxyphenyl)methane. Applied and Environmental Microbiology, 64(9), 3225-3231.
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  PMID: 9726864;PMCID: PMC106714;Abstract: Higher fungi have a widespread capacity for biosynthesis of organohalogens. Commonly occurring chloroaromatic fungal metabolites can end up in anaerobic microniches at the boundary of fungal colonies and wetland soils. The aim of this study was to investigate the environmental fate of a major fungal metabolite, 3,5-dichloro-p-anisyl alcohol, under anaerobic conditions. This compound was incubated with methanogenic sludge to study its biotransformation reactions. Initially, 3,5-dichloro-p-anisyl alcohol was readily demethylated in stoichiometric quantities to 3,5-dichloro-4- hydroxybenzyl alcohol. The demethylated product was converted further via two routes: a biotic route leading to the formation of 3,5-dichloro-4- hydroxybenzoate and 2,6-dichlorophenol, as well as an abiotic route leading to the formation of bis(3,5-dichloro-4-hydroxyphenyl)methane. In the first route, the benzyl alcohol moiety on the aromatic ring was oxidized, giving 3,5-dichloro-4-hydroxybenzoate as a transient or accumulating product, depending on the type of methanogenic sludge used. In sludge previously adapted to low-molecular-weight lignin from straw, a part of the 3,5- dichloro-4-hydroxybenzoate was decarboxylated, yielding detectable levels of 2,6-dichlorophenol. In the second route, 3,5-dichloro-4-hydroxybenzyl alcohol dimerized, leading to the formation of a tetrachlorinated bisphenolic compound, which was identified as bis(3,5-dichloro-4-hydroxyphenyl)methane. Since formation of this dimer was also observed in incubations with autoclaved sludge spiked with 3,5-dichloro-4-hydroxybenzyl alcohol, it was concluded that its formation was due to an abiotic process. However, demethylation of the fungal metabolite by biological processes was a prerequisite for dimerization. The most probable reaction mechanism leading to the formation of the tetrachlorinated dimer in the absence of oxygen is presented, and the possible environmental implications of its natural occurrence are discussed.
• J., M., Rietberg, H., Hage, A., & Field, J. A. (1998). Polycyclic aromatic hydrocarbon oxidation by the white-rot fungus Bjerkandera sp. strain BOS55 in the presence of nonionic surfactants. Biotechnology and Bioengineering, 57(2), 220-227.
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  PMID: 10099197;Abstract: The effect of nonionic surfactants on the polycyclic aromatic hydrocarbon (PAH) oxidation rates by the extracellular ligninolytic enzyme system of the white-rot fungus Bjerkandera sp. strain BOS55 was investigated. Various surfactants increased the rate of anthracene, pyrene, and benzo[a]pyrene oxidation by two to fivefold. The stimulating effect of surfactants was found to be solely due to the increased bioavailability of PAH, indicating that the oxidation of PAH by the extracellular ligninolytic enzymes is limited by low compound bioavailability. The surfactants were shown to improve PAH dissolution rates by increasing their aqueous solubility and by decreasing the PAH precipitate particle size. The surfactant Tween 80 was mineralized by Bjerkandera sp. strain BOS55; as a result both the PAH solubilizing activity of Tween 80 and its stimulatory effect on anthracene and pyrene oxidation rates were lost within 24 h after addition to 6-day-old cultures. It was observed that the surfactant dispersed anthracene precipitates recrystallized into larger particles after Tween 80 was metabolized. However, benzo[a]pyrene precipitates remained dispersed, accounting for a prolonged enhancement of the benzo[a]pyrene oxidation rates. Because the endogenous production of H2O2 is also known to be rate limiting for PAH oxidation, the combined effect of adding surfactants and glucose oxidase was studied. The combined treatment resulted in anthracene and benzo[a]pyrene oxidation rates as high as 1450 and 450 mg L-1 d-1, respectively, by the extracellular fluid of 6-day-old fungal cultures.
• J., M., Vis, E. H., & Field, J. A. (1998). Successive mineralization and detoxification of benzo[a]pyrene by the white rot fungus Bjerkandera sp. strain BOS55 and indigenous microflora. Applied and Environmental Microbiology, 64(8), 2853-2858.
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  PMID: 9687440;PMCID: PMC106782;Abstract: White rot fungi can oxidize high-molecular-weight polycyclic aromatic hydrocarbons (PAH) rapidly to polar metabolites, but only limited mineralization takes place. The objectives of this study were to determine if the polar metabolites can be readily mineralized by indigenous microflora from several inoculum sources, such as activated sludge, forest soils, and PAH-adapted sediment sludge, and to determine if such metabolites have decreased mutagenicity compared to the mutagenicity of the parent PAH. 14C- radiolabeled benzo [a] pyrene was subjected to oxidation by the white rot fungus Bjerkandera sp. strain BOS55. After 15 days, up to 8.5% of the [14C]benzo[a]pyrene was recovered as 14CO2 in fungal cultures, up to 73% was recovered as water-soluble metabolites, and only 4% remained soluble in dibutyl ether. Thin-layer chromatography analysis revealed that many polar fluorescent metabolites accumulated. Addition of indigenous microflora to fungal cultures with oxidized benzo[a]pyrene on day 15 resulted in an initially rapid increase in the level of 14CO2 recovery to a maximal value of 34% by the end of the experiments (> 150 days), and the level of water- soluble label decreased to 16% of the initial level. In fungal cultures not inoculated with microflora, the level of 14CO2 recovery increased to 13.5%, while the level of recovery of water-soluble metabolites remained as high as 61%. No large differences in 14CO2 production were observed with several inocula, showing that some polar metabolites of fungal benzo[a] pyrene oxidation were readily degraded by indigenous microorganisms, while other metabolites were not. Of the inocula tested, only PAH-adapted sediment sludge was capable of directly mineralizing intact benzo[a] pyrene, albeit at a lower rate and to a lesser extent than the mineralization observed after combined treatment with white rot fungi and indigenous microflora. Fungal oxidation of benzo[a]pyrene resulted in rapid and almost complete elimination of its high mutagenic potential, as observed in the Salmonella typhimurium revertant test performed with strains TA100 and TA98. Moreover, no direct mutagenic metabolite could be detected during fungal oxidation. The remaining weak mutagenic activity of fungal cultures containing benzo [a] pyrene metabolites towards strain TA98 was further decreased by subsequent incubations with indigenous microflora.
• J., P., & Field, J. A. (1998). 2-Chloro-1,4-dimethoxybenzene as a novel catalytic cofactor for oxidation of anisyl alcohol by lignin peroxidase. Applied and Environmental Microbiology, 64(3), 830-835.
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  PMID: 16349526;PMCID: PMC106334;Abstract: 2-Chloro-1,4-dimethoxybenzene (2C1-14DMB) is a natural compound produced de novo by several white rot fungi. This chloroaromatic metabolite was identified as a cofactor superior to veratryl alcohol (VA) in the oxidation of anisyl alcohol (AA) by lignin peroxidase (LIP). Our results reveal that good LiP substrates, such as VA and tryptophan, are comparatively poor cofactors in the oxidation of AA. Furthermore, we show that a good cofactor does not necessarily serve a role in protecting LiP against H2O2 inactivation. 2Cl-14DMB was not a direct mediator of AA oxidation, since increasing AA concentrations did not inhibit the oxidation of 2Cl-14DMB at all. However, the high molar ratio of anisaldehyde formed to 2Cl-14DMB consumed, up to 13:1, indicates that a mechanism which recycles the cofactor is present.
• J., P., Sheng, D., Vijay, G., Moënne-Loccoz, P., Field, J. A., & Gold, M. H. (1998). 2-Chloro-1,4-dimethoxybenzene cation radical: Formation and role in the lignin peroxidase oxidation of anisyl alcohol. Archives of Biochemistry and Biophysics, 360(2), 233-238.
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  PMID: 9851835;Abstract: 2-Chloro-1,4-dimethoxybenzene (2Cl-1,4-DMB) oxidation by lignin peroxidase (LIP) proceeds via the formation of the 2Cl-1,4-DMB cation radical as indicated by ESR and UV/vis spectroscopy. The products of the LiP- catalyzed oxidation of 2Cl-1,4-DMB were identified as 2-chloro-1,4- benzoquinone and the dimers dichlorotetramethoxybiphenyl and chloro(chlorodimethoxyphenyl)benzoquinone. The addition of anisyl alcohol (AA) rapidly quenched the 2Cl-1,4-DMB cation radical optical absorption bands, suggesting that the cation radical directly mediates the oxidation of AA. When LiP reactions are conducted in the presence of 50 μM 2Cl-1,4-DMB, the enzyme is inactivated; however, this inactivation can be prevented by the addition of AA. This also suggests that the 2Cl-1,4-DMB cation radical formed in the reaction, in turn, oxidizes AA.
• Kortekaas, S., Vidal, G., Yan-Ling, H., Lettinga, G., & Field, J. A. (1998). Anaerobic-aerobic treatment of toxic pulping black liquor with upfront effluent recirculation. Journal of Fermentation and Bioengineering, 86(1), 97-110.
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  Abstract: Alkaline pulping liquors are problematic for anaerobic treatment due to their toxicity to methanogens and their relatively large fraction of inert lignin. In previous research, toxicity due to wood extractives was shown to be highly eliminated during aerobic wastewater treatment, but not during anaerobic treatment. These observations have led to the proposal of a detoxification strategy denominated upfront dilution, based on the sequenced anaerobic-aerobic treatment of the pulping liquor, recirculating the aerobic effluent to dilute the incoming influent to sub-toxic concentrations. In this study, the treatment of highly toxic hemp stem wood black liquor (HSWBL) in lab-scale UASB reactors with upfront dilution was compared with direct anaerobic treatment and with direct aerobic treatment. Direct anaerobic treatment of 12 g COD/l HSWBL led to almost complete inhibition of the methanogenic activity within 14 d. However, recirculation of 75% of the aerobic post-treatment effluent for upfront dilution of the toxic HSWBL, enabled anaerobic treatment at loading rates up to 21.5 g COD/l(UASB) · d without significant inhibition of the methanogenic activity. Extensive detoxification was confirmed during anaerobic-aerobic treatment of 20 g COD/l HSWBL recirculating 86% of the aerobic effluent. COD and BOD removal was 72% and 97%, respectively, after anaerobic-aerobic treatment at an overall loading rate of 3.6 g COD/l · d, while 30-35% of the incoming COD was recovered as methane. Batch-assays demonstrated significant detoxification after anaerobic-aerobic treatment of HSWBL. Treatment efficiencies and detoxification during anaerobic-aerobic and aerobic treatment were similar. However, the anaerobic-aerobic treatment system provided 50% lower surplus sludge production, production of 0.16 m2 methane/kg COD(removed) as an energy source, less nutrient dosage and substantial reductions in aeration costs. During anaerobic-aerobic treatment as well as aerobic treatment significant lignin removal was obtained, ranging from 28-58%. Lignin removal could be attributed to biodegradation of low molecular weight lignin (MW < 2.2 kD). The lignin which survived biological treatment was extensively polymerized to MW of > 34 kD.
• Kortekaas, S., Wijngaarde, R. R., Klomp, J., Lettinga, G., & Field, J. A. (1998). Anaerobic treatment of hemp thermomechanical pulping wastewater. Water Research, 32(11), 3362-3370.
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  Abstract: Biological treatment is an indispensable instrument for water management of non-wood pulp mills, either as internal measure or end of pipe. In this paper, anaerobic treatment of wastewaters derived from the thermomechanical pulping (TMP) of hemp (Cannabis sativa L.) is described. Hemp stem wood and hemp bark TMP wastewaters were treated in laboratory scale upflow anaerobic sludge blanket (UASB) reactors. For both types of wastewater, maximal COD removal of 72% were obtained at loading rates of 13-16 g COD·l-1 d-1, providing 59-63% recovery of the influent COD as methane. The reactors continued to provide excellent COD removal efficiencies of 63-66% up to a loading rate of 27 g COD·l-1 d-1 being the highest loading rate tested. Batch toxicity assays revealed the absence of methanogenic inhibition by hemp TMP wastewaters, coinciding with the high acetoclastic activity of the reactor sludge of approximately 1 g COD·g VSS-1 d-1. Due to the relatively low molecular weight of hemp TMP lignin, its removal (measured as UV280) during anaerobic treatment was remarkably high and averaged 45 and 31% for the hemp stem wood and the hemp bark TMP UASB reactors, respectively. Gel permeation chromatography revealed that the lignin removed corresponded to the lowest molecular weight derivatives. Subsequent batch aerobic post-treatment led to a considerable increase of color levels and polymerization of the residual lignin to molecular weights in excess of 34 kD.
• Mester, T., & Field, J. A. (1998). Characterization of a novel manganese peroxidase-lignin peroxidase hybrid isozyme produced by Bjerkandera species strain BOS55 in the absence of manganese. Journal of Biological Chemistry, 273(25), 15412-15417.
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  PMID: 9624124;Abstract: A novel manganese-dependent peroxidase (MnP) isozyme produced in manganese-free cultures of Bjerkandera sp. strain BOS55 was purified and characterized. The production of the enzyme was greatly stimulated by the exogenous addition of various physiological organic acids such as glycolate, glyoxylate, and oxalate. The physical properties of the enzyme are similar to those of MnP isozymes from different white rot fungi (M(r) = 43,000, pI 3.88, and ε407(nm) = 123 mM-1 cm-1). The Bjerkandera MnP was efficient in the oxidation of Mn(II), as indicated by the kinetic constants (low K(m) of 51 μM and turnover number of 59 s-1). Furthermore, the isozyme was able to oxidize various substrates in the absence of manganese, such as 2,6- dimethoxyphenol, guaiacol, ABTS, 3-hydroxyanthranilic acid, and o- and p- anisidine. An interesting characteristic of the isozyme was its ability to oxidize nonphenolic substrates, veratryl alcohol and 1,4-dimethoxybenzene, without manganese addition. The affinity for veratryl alcohol (K(m) = 116 μM) and its turnover number (2.8 s-1) are comparable to those of lignin peroxidase (LIP) isozymes from other white rot fungi. Manganese at concentrations greater than 0.1 mM severely inhibited the oxidation of veratryl alcohol. The results suggest that this single isozyme is a hybrid between MnP and LiP found in other white rot fungi. The N-terminal amino acid sequence showed a very high homology to those of both MnP and LiP isozymes from Trametes versicolor.
• Swarts, H. J., J., F., Field, J. A., & B., J. (1998). Identification and synthesis of novel chlorinated p-anisylpropanoid metabolites from Bjerkandera species. Journal of Natural Products, 61(9), 1110-1114.
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  Abstract: Analysis of the EtOAc extracts from the culture medium of Bjerkandera sp. BOS55 and B. fumosa revealed the presence of two novel chlorinated metabolites. Their structures were unambiguously established as erythro-1- (3',5'-dichloro-4'-methoxyphenyl)-1,2-propanediol (2) and 1-(3'-chloro-4'- methoxyphenyl)-3-hydroxy-1-propanone (3) through synthesis of authentic samples and comparison of retention times and mass spectral data. The production of trametol (1) by Bjerkandera sp, BOS55 was also a new finding.
• Swarts, H. J., J., F., Field, J. A., & B., J. (1998). Trichlorinated phenols from Hypholoma elongatum. Phytochemistry, 49(1), 203-206.
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  Abstract: Three trichlorinated phenols, 2,4,6-trichloro-3-methoxyphenol, 3,5,6- trichloro-2,4-dimethoxy-phenol and 3,4,6-trichloro-2,5-dimethoxyphenol, were detected as novel metabolites in the ethyl acetate extract from the culture medium of the Basidiomycete, Hypholoma elongatum (strain WIJS94-28).
• Terrón, M. C., Verhagen, F. J., Franssen, M. C., & Field, J. A. (1998). Chemical bromination of phenol red by hydrogen peroxide is possible in the absence of haloperoxidases. Chemosphere, 36(6), 1445-1452.
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  Abstract: The dye phenol red (phenolsulphonphthalein) was chemically brominated to bromophenol blue (3',3',5',5'-tetrabromophenolsulphonphthalein) directly by hydrogen peroxide (20 mM or higher) under acidic pH (0.5-4.5) and moderate temperature (30°C). Since this bromination reaction takes place in the absence of any chemical or biological catalyst, the results represent an important warning concerning a commonly used assay for detecting or screening haloperoxidases. Bromophenol red (5',5'-dibromophenolsulphonphthalein) and another unidentified compound were observed as temporary intermediates. Incubation of the reaction mixture with KCl instead of NaBr did not yield any chlorinated products under the same conditions.
• Teunissen, P. J., & Field, J. A. (1998). 2-Chloro-1,4-dimethoxybenzene as a mediator of lignin peroxidase catalyzed oxidations. FEBS Letters, 439(3), 219-223.
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  PMID: 9845326;Abstract: Poly R-478, 4-methoxymandelic acid and oxalic acid were oxidized by lignin peroxidase (LiP) in the presence of the fungal metabolite 2-chloro-1,4-dimethoxybenzene (2Cl-14DMB), whereas no oxidation occurred in the absence of 2Cl-14DMB. These substrates clearly inhibited the consumption of 2Cl-14DMB and the formation of 2-chloro-1,4-benzoquinone from 2Cl-14DMB by LiP. The results suggest that 2Cl-14DMB can replace the function of veratryl alcohol as a redox mediator in lignin peroxidase catalyzed oxidations. Copyright (C) 1998 Federation of European Biochemical Societies.
• Verhagen, F. J., B.J., F., Boekema, B. K., Swarts, H. J., Wijnberg, J. B., & Field, J. A. (1998). Dynamics of organohalogen production by the ecologically important fungus Hypholoma fasciculare. FEMS Microbiology Letters, 158(2), 167-178.
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  Abstract: The ecologically important white rot basidiomycete Hypholoma fasciculare was previously shown to produce large amounts of adsorbable organic halogens (AOX). The purposes of this study were to identify the time period of AOX production in relation to the primary and secondary metabolic phases of the growth cycle of the fungus, to determine the maximal specific AOX production rates and final AOX yields on the different substrates and to account for the measured AOX in identifiable compounds. The AOX production was observed to take place during the transition between the primary and secondary metabolic phases of the growth cycle of the fungus. The maximum AOX production rates ranged from 0.63 to 3.23 mg AOX per gram of dry mycelium per day and the final AOX yields ranged from 0.88 and 1.50 percent of dry weight of mycelium on five different substrates including natural woody substrates. The AOX produced by the fungus was stable in all five substrates, even after prolonged incubation periods. However, the composition of the AOX changed drastically. Initially most of the AOX was accounted for the compound 3,5-dichloro-p-anisyl alcohol; however, after prolonged incubation this compound was largely converted into 3,5-dichloro-p-anisic acid in N-rich medium and into unidentified organohalogens in N-limited medium.
• Verhagen, F. J., Swarts, H. J., Wijnberg, J. B., & Field, J. A. (1998). Organohalogen production is a ubiquitous capacity among basidiomycetes. Chemosphere, 37(9-12), 2091-2104.
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  Abstract: Several species of basidiomycetes are capable of producing de novo high concentrations of chloroaromatic metabolites. However, the extent to which basidiomycetes contribute to the natural pool of adsorbable organic halogen (AOX) found in the environment is unknown yet. The purpose of this study was to determine the ubiquity of organohalogen production among basidiomycetes and to determine maximal specific organohalogen production rates. Finally, the fate of the fungal chloroaromatic compounds in the environment was studied. A total of 191 fungal strains were tested for AOX production when grown on defined liquid medium. Approximately 50% of the strains tested and 55% of the genera tested produced AOX. Organohalogen production seemed to be a ubiquitous capacity among basidiomycetes. Many highly ecologically significant fungal species were identified among the moderate and high producers. Although it was found that the final AOX concentrations produced by Hypholoma fasciculare was strongly influenced by the substrate used, all maximal specific AOX production rates on different substrates were in the same order of magnitude. Seven new species and four new genera of basidiomycetes could be added to the list of known chlorinated anisyl metabolites (CAM) producing basidiomycetes. In degradation studies of the major fungal metabolite 3,5-dichloro-anisyl alcohol, it was found that in forest soils there seems to be ubiquitous mineralizing capacity for this chlorinated aromatic compound. It was found that Burkholderia cepacia was responsible for the fast degradation of the fungal compound in the oak forest soils.
• Collins, P. J., Field, J. A., Teunissen, P., & D., A. (1997). Stabilization of lignin peroxidases in white rot fungi by tryptophan. Applied and Environmental Microbiology, 63(7), 2543-2548.
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  PMID: 9212404;PMCID: PMC168551;Abstract: Supplementation of various cultures of white rot fungi with tryptophan was found so have a large stimulatory effect on lignin peroxidase activity levels. This enhancement was greater than that observed in the presence of the lignin peroxidase recycling agent veratryl alcohol. Using reverse transcription-PCR, we found that tryptophan does not act to induce lignin peroxidase expression at the level of gene transcription. Instead, the activity enhancement observed is likely to result from the protective effect of tryptophan against H2O2 inactivation. In experiments using a partially purified lignin peroxidase preparation, tryptophan and its derivative indole were determined to function in the same way as veratryl alcohol in converting compound II, an oxidized form of lignin peroxidase, to ferric enzyme, thereby completing the catalytic cycle. Furthermore, tryptophan was found to be a better substrate for lignin peroxidase than veratryl alcohol. Inclusion of either tryptophan or indole enhanced the oxidation of the azo dyes methyl orange and Eriochrome blue black. Stimulation of azo dye oxidations by veratryl alcohol has previously been shown to be due to its enzyme recycling function. Our data allow us to propose that tryptophan stabilizes lignin peroxidase by acting as a reductant for the enzyme.
• Donlon, B., Razo-Flores, E., Luijten, M., Swarts, H., Lettinga, G., & Field, J. (1997). Detoxification and partial mineralization of the azo dye mordant orange 1 in a continuous upflow anaerobic sludge-blanket reactor. Applied Microbiology and Biotechnology, 47(1), 83-90.
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  Abstract: In batch toxicity assays, azo dye compounds were found to be many times more toxic than their cleavage products (aromatic amines) towards methanogenic activity in anaerobic granular sludge. Considering the ability of anaerobic microorganisms to reduce azo groups, detoxification of azo compounds towards methanogens can be expected to occur during anaerobic wastewater treatment. In order to test this hypothesis, the anaerobic degradation of one azo dye compound Mordant orange 1 (MO1), by granular sludge was investigated in three separate continuous upflow anaerobic sludge-blanket reactors. One reactor, receiving no cosubstrate, failed after 50 days presumably because of a lack of reducing equivalents. However, the two reactors receiving either glucose or a volatile fatty acids (acetate, propionate, butyrate) mixture, could eliminate the dye during operation for 217 days. The azo dye was reductively cleaved to less toxic aromatic amines (1,4-phenylenediamine and 5-aminosalicylic acid) making the treatment of MO1 feasible at influent concentrations that were over 25 times higher than their 50% inhibitory concentrations. In the reactor receiving glucose as cosubstrate, 5-aminosalicylic acid could only be detected at trace levels in the effluent after day 189 of operation. Batch biodegradability assays with the sludge sampled from this reactor confirmed the mineralization of 5-aminosalicylic acid to methane.
• Florencio, L., Field, J. A., & Lettinga, G. (1997). High-rate anaerobic treatment of alcoholic wastewaters. Brazilian Journal of Chemical Engineering, 14(4), 409-416.
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  Abstract: Modern high-rate anaerobic wastewater treatment processes are rapidly becoming popular for industrial wastewater treatment. However, until recently stable process conditions could not be guaranteed for alcoholic wastewaters containing higher concentrations of methanol. Although methanol can be directly converted into methane by methanogens, under specific conditions it can also be converted into acetate and butyrate by acetogens. The accumulation of volatile fatty acids can lead to reactor instability in a weakly buffered reactor. Since this process was insufficiently understood, the application of high-rate anaerobic reactors was highly questionable. This research investigated the environmental factors that are of importance in the predominance of methylotrophic methanogens over acetogens in a natural mixed culture during anaerobic wastewater treatment in upflow anaerobic sludge bed reactors. Technological and microbiological aspects were investigated. Additionally, the route by which methanol is converted into methane is also presented.
• Have, R. T., Hartmans, S., & Field, J. A. (1997). Interference of peptone and tyrosine with the lignin peroxidase assay. Applied and Environmental Microbiology, 63(8), 3301-3303.
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  PMID: 9251220;PMCID: PMC168631;Abstract: The N-unregulated white rot fungus Bjerkandera sp. strain BOS55 was cultured in 1 liter of peptone-yeast extract medium tO produce lignin peroxidase (LiP). During the LiP assay, the oxidation of veratryl alcohol to veratraldehyde was inhibited due to tyrosine present in the peptone and the yeast extracts.
• Jong, E. D., & Field, J. A. (1997). Sulfur tuft and turkey tail: Biosynthesis and biodegradation of organohalogens by Basidiomycetes. Annual Review of Microbiology, 51, 375-414.
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  PMID: 9343355;Abstract: Chlorinated aliphatic and aromatic compounds are generally considered to be undesirable xenobiotic pollutants. However, the higher fungi, Basidiomycetes, have a widespread capacity for organohalogen biosynthesis. Adsorbable organic halogens (AOX) and/or low-molecular-weight halogenated compounds are produced by Basidiomycetes of 68 genera from 20 different families. Most of the 81 halogenated metabolites identified from Basidiomycetes to date are chlorinated, although brominated and iodated metabolites have also been described. Two broad categories of Basithomycete organohalogen metabolites are the halogenated aromatic compounds and the haloaliphatic compounds. Some of these organohalogen metabolites have demonstrable physiological roles as antibiotics and as metabolites involved in lignin degradation. Basidiomycetes produce large amounts of low-molecular- weight organohalogens or adsorbable organic halogens (AOX) when grown on lignocellulosic substrates. In our view, Basidiomycetes, as decomposers of forest litter, are a major source of natural organohalogens in terrestrial environments. Basidiomycetes are also potent degraders of a wide range of chlorinated pollutants, such as bleachery effluent from kraft mills and pentachlorophenol, polychlorinated dioxins, and polychlorinated biphenyls. The extracellular, lignin-degrading enzymes of the Basidiomycetes are involved in the oxidative degradation of chlorophenols and dioxin and can cause reductive dechlorination of halomethanes. There is no clear-cut separation between 'polluters' and 'clean-uppers' within the Basidiomycetes. Several genera, e.g. Bjerkandera, Hericium, Phlebia, and Trametes, produce significant amounts of chlorinated compounds but are also highly effective in metabolizing or biotransforming chlorinated pollutants.
• Kato, M. T., Field, J. A., & Lettinga, G. (1997). Anaerobe tolerance to oxygen and the potentials of anaerobic and aerobic cocultures for wastewater treatment. Brazilian Journal of Chemical Engineering, 14(4), 395-407.
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  Abstract: The anaerobic treatment processes are considered to be well-established methods for the elimination of easily biodegradable organic matter from wastewaters. Some difficulties concerning certain wastewaters are related to the possible presence of dissolved oxygen. The common belief is that anaerobes are oxygen intolerant. Therefore, the common practice is to use sequencing anaerobic and aerobic steps in separate tanks. Enhanced treatment by polishing off the residual biodegradable oxygen demand from effluents of anaerobic reactors, or the biodegradation of recalcitrant wastewater pollutants, usually requires sequenced anaerobic and aerobic bacteria activities. However, the combined activity of both bacteria can also be obtained in a single reactor. Previous experiments with either pure or mixed cultures showed that anaerobes can tolerate oxygen to a certain extent. The oxygen toxicity to methanogens in anaerobic sludges was quantified in batch experiments, as well as in anaerobic reactors. The results showed that methanogens have a high tolerance to oxygen. In practice, it was confirmed that dissolved oxygen does not constitute any detrimental effect on reactor treatment performance. This means that the coexistence of anaerobic and aerobic bacteria in one single reactor is feasible and increases the potentials of new applications in wastewater treatment.
• Kato, M. T., Field, J. A., & Lettinga, G. (1997). The anaerobic treatment of low strength wastewaters in UASB and EGSB reactors. Water Science and Technology, 36(6-7), 375-382.
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  Abstract: The application of the UASB and EGSB reactors for the treatment of low strength wastewaters was investigated. The effect of dissolved oxygen on the methanogenic activity of granular sludges, the low substrate levels inside reactors and lower temperatures on the treatment performance were evaluated. The results showed that methanogens located in granular sludge have a high tolerance to oxygen. The concentration to cause 50% inhibition to methanogenic activity was between 7% and 41% oxygen in the head space of flasks, corresponding to 0.05 mg/l and 6 mg/l of DO prevailing in the media, respectively. The feasibility of UASB and EGSB reactors at 30°C was demonstrated. In UASB reactors, COD removal efficiencies exceeded 95% at organic loading rates up to 6.8 g COD/l.d and influent COD concentrations ranging from 422 to 722 mg/l, during the treatment of ethanol substrate. In EGSB reactors, efficiencies were above 80% at OLRs up to 12 g COD/l.d with COD as low as 100 to 200 mg/l. The studies confirmed that in practice DO does not constitute any detrimental effect on the reactor treatment performance. Lowering the temperature down to 15°C in EGSB reactors also showed that the potentials of anaerobic technology can be further explored in the treatment of dilute wastewaters.
• Lettinga, G., Field, J., Lier, J. V., Zeeman, G., & Pol, L. H. (1997). Advanced anaerobic wastewater treatment in the near future. Water Science and Technology, 35(10), 5-12.
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  Abstract: New insights into the anaerobic degradation of very different categories of compounds, and into process and reactor technology will lead to very promising new generations of anaerobic treatment system, such as 'Expanded Granular Sludge Bed' (EGSB) and 'Staged Multi-Phase Anaerobic' (MPSA) reactor systems. These concepts will provide a higher efficiency at higher loading rates, are applicable for extreme environmental conditions (e.g. low and high temperatures) and to inhibitory compounds. Moreover, by integrating the anaerobic process with other biological methods (sulphate reduction, micro-aerophilic organisms) and with physical-chemical methods, a complete treatment of the wastewater can be accomplished at very low costs, while at the same time valuable components can be recovered for reuse.
• Mester, T., & Field, J. A. (1997). Optimization of manganese peroxidase production by the white rot fungus Bjerkandera sp. strain BOS55. FEMS Microbiology Letters, 155(2), 161-168.
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  Abstract: Manganese dependent peroxidase (MnP) is the most ubiquitous peroxidase produced by white rot fungi. MnP is known to be involved in lignin degradation, biobleaching and in the oxidation of hazardous organopollutants. Bjerkandera sp. strain BOS55 is a nitrogen-unregulated white rot fungus which produces high amounts of MnP in the excess of N-nutrients due to increased biomass yield. Therefore, the strain is a good candidate for use in large scale production of this enzyme. The objective of this study was to optimize the MnP production in N-sufficient cultures by varying different physiological factors such as Mn concentration, culture pH, incubation temperature and the addition of organic acids. The fungus produced the highest level of MnP (up to 900 U 1-1) when the Mn concentration was 0.2 to 1 mM, the pH value was 5.2, and the incubation temperature was 30°C. A noteworthy finding was that MnP was also produced at lower levels in the complete absence of Mn. The addition of organic acids like glycolate, malonate, glucuronate, gluconate, 2-hydroxybutyrate to the culture medium increased the peak titres of MnP up to 1250 U 1-1. FPLC profiles indicated that the organic acids stimulated the production of all MnP isoenzymes present in the extracellular fluid of the fungus.
• Mester, T., Swarts, H. J., Romero, S., A.M., J., & Field, J. A. (1997). Stimulation of aryl metabolite production in the basidiomycete Bjerkandera sp. strain BOS55 with biosynthetic precursors and lignin degradation products. Applied and Environmental Microbiology, 63(5), 1987-1994.
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  PMID: 9143129;PMCID: PMC168489;Abstract: Aryl metabolites are known to have an important role in the ligninolytic system of white rot fungi. The addition of known precursors and aromatic acids representing lignin degradation products stimulated the production of aryl metabolites (veratryl alcohol, veratraldehyde, p-anisaldehyde, and 3- chloro-p-anisaldehyde) in the white rot fungus Bjerkandera sp. strain BOS55. The presence of manganese (Mn) is known to inhibit the biosynthesis of veratryl alcohol (T. Mester, E. de Jong, and J. A. Field, Appl. Environ. Microbiol. 61:1881-1887, 1995). A new finding of this study was that the production of the other aryl metabolites, p-anisaldehyde and 3-chloro-p- anisaldehyde, was also inhibited by Mn. We attempted to bypass the Mn- inhibited step in the biosynthesis of aryl metabolites by the addition of known and suspected precursors. Most of these compounds were not able to bypass the inhibiting effect of Mn. Only the fully methylated precursors (veratrate, p-anisate, and 3-chloro-p-anisate) provided similar concentrations of aryl metabolites in the presence and absence of Mn, indicating that Mn does not influence the reduction of the benzylic acid group. The addition of deuterated benzoate and 4-hydroxybenzoate resulted in the formation of deuterated aryl metabolites, indicating that these aromatic acids entered into the biosynthetic pathway and were common intermediates to all aryl metabolites. Only deuterated chlorinated anisyl metabolites were produced when the cultures were supplemented with deuterated 3-chloro-4- hydroxybenzoate. This observation combined with the fact that 3-chloro-4- hydroxybenzoate is a natural product of Bjerkandera spp. (H. J. Swarts, F. J. M. Verhagen, J. A. Field, and J. B. P. A. Wijnberg, Phytochemistry 42:1699- 1701, 1996) suggests that it is a possible intermediate in chlorinated anisyl metabolite biosynthesis.
• Meulenberg, R., Rijnaarts, H. H., Doddema, H. J., & Field, J. A. (1997). Partially oxidized polycyclic aromatic hydrocarbons show an increased bioavailability and biodegradability. FEMS Microbiology Letters, 152(1), 45-49.
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  PMID: 9228769;Abstract: Polycyclic aromatic hydrocarbons have a low water solubility and tend to adsorb on soil particles, which both result in slow bioremediation processes. Many microorganisms, known for their ability to degrade polycyclic aromatic hydrocarbons, only partially oxidize these compounds. White rot fungi, for instance, convert polycyclic aromatic hydrocarbons to more water soluble and bioavailable products. Polycyclic aromatic hydrocarbon metabolites were more readily mineralized by natural mixed bacterial cultures, like activated sludge and soil, than the parent polycyclic aromatic hydrocarbon compounds. These results suggest that sequential breakdown by white rot fungi followed by indigenous bacteria leads to an effective polycyclic aromatic hydrocarbon bioremediation process.
• Razo-Flores, E., Donlon, B., Lettinga, G., & Field, J. A. (1997). Biotransformation and biodegradation of N-substituted aromatics in methanogenic granular sludge. FEMS Microbiology Reviews, 20(3-4), 525-538.
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  PMID: 9340000;Abstract: N-Substituted aromatic compounds are environmental contaminants associated with the production and use of dyes, explosives, pesticides and pharmaceuticals. In this article, we examine the potential of anaerobic granular sludge from anaerobic treatment systems towards the detoxification, transformation, and mineralization of nitroaromatic and azo compounds, Nitroaromatics and azo dyes with strong electron withdrawing are highly inhibitory to acetoclastic methanogenic bacteria. However, nitro and azo substituted aromatics are readily reductively detoxified in methanogenic consortia to their respective aromatic amines, which are several orders of magnitude less toxic. This reductive detoxification has allowed the successful operation of anaerobic reactors for the treatment of highly toxic aromatic compounds. In the course of the experiments it was discovered that some aromatic airlines were mineralized. These results indicate that some N- substituted aromatic compounds can be completely mineralized and serve as a carbon and energy source for anaerobic bacteria.
• Razo-Flores, E., Luijten, M., Donlon, B. A., Lettinga, G., & Field, J. A. (1997). Complete biodegradation of the azo dye azodisalicylate under anaerobic conditions. Environmental Science and Technology, 31(7), 2098-2103.
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  Abstract: Azo dyes are a widespread class of poorly biodegradable industrial pollutants. In anaerobic environments, azo bonds are reductively cleaved yielding carcinogenic aromatic amines, many of which are assumed to resist further metabolism by anaerobes. Here we report for the first time that an azo dye compound is completely biodegradable in the absence of oxygen. A pharmaceutical azo dye, azodisalicylate, constructed from two 5- aminosalicylic acid (5ASA) molecules was mineralized in an adapted methanogenic consortium to CH4 and NH3 with transient accumulation of 5ASA as e degradation intermediate in both batch assays and continuous bioreactors. The anaerobic metabolism of 5ASA was shown to provide the electrons required for the initial reductive cleavage of the azo group. Our results suggest that it is possible to design azo dyes that are fully mineralized in the environment; thereby, avoiding accumulation of notorious toxic intermediates.
• Razo-Flores, E., Luijten, M., Donlon, B., Lettinga, G., & Field, J. (1997). Biodegradation of selected azo dyes under methanogenic conditions. Water Science and Technology, 36(6-7), 65-72.
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  Abstract: Biological treatment of wastewaters discharged by the textile industry could potentially be problematic due to the high toxicity and recalcitrance of the commonly-used azo dye compounds. In the present report, the fate of two azo dyes under methanogenic conditions was studied. Mordant Orange 1 (MO1) and Azodisalicylate (ADS) were completely reduced and decolorised in continuous UASB reactors in the presence of cosubstrates. In the MO1 reactor, both 5-aminosalicylic acid (5-ASA) and 1,4-phenylenediamine were identified as products of azo cleavage. After long adaptation periods, 5-ASA was detected at trace levels, indicating further mineralization. ADS, a pharmaceutical azo dye constructed from two 5-ASA units, was completely mineralized even in the absence of cosubstrate, indicating that the metabolism of 5-ASA could provide the reducing equivalents needed for the azo reduction. Batch experiments confirmed the ADS mineralization. These results demonstrate that some azo dyes could serve as a carbon, energy, and nitrogen source for anaerobic bacteria.
• Swarts, H. J., J., P., J., F., Field, J. A., & B., J. (1997). Chlorinated anisyl metabolites produced by basidiomycetes. Mycological Research, 101(3), 372-374.
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  Abstract: Chlorinated anisyl metabolites (CAM) were detected in ethyl acetate extracts from the culture medium of species of seven basidiomycete genera including, for the first time, Mycena, Peniophora, Phellinus and Phylloporia. Extension of CAM biosynthesis in Bjerkandera (B. fumosa), Hypholoma (H. elongatum) and Pholiota (P. adiposa) is a new finding. The selective and high-yield production of 3,5-dichloro-p-anisyl alcohol by H. elongatum is remarkable.
• Swarts, H. J., Mester, T., J., F., Field, J. A., & B., J. (1997). The formation of veratryl chloride by Bjerkandera sp. Strain BOS55. Phytochemistry, 46(6), 1011-1013.
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  Abstract: In the ethyl acetate extract from the extracellular fluid of the mycelium of Bjerkandera sp. BOS55 the presence of veratryl chloride was detected. Addition of deuterated benzoic acid or 4-hydroxybenzoic acid to the culture resulted in the formation of deuterated veratryl chloride. The detection of veratryl chloride constitutes the first report of a biologically derived natural product which contains a halogenated benzylic carbon. Since veratryl chloride is not very stable in aqueous medium, its detection also suggests a continuous production.
• Teunissen, P. J., Swarts, H. J., & Field, J. A. (1997). The de novo production of drosophilin A (tetrachloro-4-methoxyphenol) and drosophilin A methyl ether (tetrachloro-1,4-dimethoxybenzene) by ligninolytic basidiomycetes. Applied Microbiology and Biotechnology, 47(6), 695-700.
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  PMID: 9237391;Abstract: Ligninolytic basidiomycetes were screened for their ability to produce the tetrachlorinated hydroquinone metabolites drosophilin A (DA, tetrachloro- 4-methoxyphenol) and drosophilin A methyl ether (DAME, tetrachloro-1,4- dimethoxybenzene). Five fungal strains produced these metabolites in detectable amounts, including strains from Bjerkandera and Peniophora, which are genera not previously known for DA or DAME production. Phellinus fastuosus ATCC26.125 had the highest and most reliable production of DA and DAM E in peptone medium, respectively 15-60 μM and 4-40 μM. This fungus was used to study culture conditions that could increase DAME production. A fourfold increase in DAME production was found after the addition of hydroquinone to growing cultures of P. fastuosus. Therefore, hydroquinone is postulated to be a possible biosynthetic precursor of DAME in the fungus. Antagonising P. fastuosus by adding filter-sterilised culture fluid of a competing fungus. Phlebia radiata, increased DAME production significantly by tenfold. This result suggests that DAM E production is elicited by compounds present in the culture fluid of P. radiata, indicating that DAME has an antibiotic function in P. fastuosus.
• Vidal, G., Soto, M., Field, J., Méndez-Pampín, R., & Lema, J. M. (1997). Anaerobic biodegradability and toxicity of wastewaters from chlorine and total chlorine-free bleaching of eucalyptus kraft pulps. Water Research, 31(10), 2487-2494.
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  Abstract: Chlorine bleaching effluents are problematic for anaerobic wastewater treatment due to their high methanogenic toxicity and low biodegradability. Presently, alternative bleaching processes are being introduced, such as elemental chlorine-free (ECF) and total chlorine-free (TCF) bleaching. The methanogenic toxicity and anaerobic biodegradability of ECF and TCF bleaching effluents from oxygen-delignified eucalyptus Kraft pulp were compared with those of different chlorine bleaching sequences. The effluents from chlorine and ECF bleaching sequences had similar methanogenic toxicities, with 50% inhibiting concentrations (50% IC) of 0.65-1.48 g-COD/litre. Only the TCF bleaching effluent was distinctly less toxic, with a 50% IC of 2.3 g-COD/litre. The fact that the ECF bleaching effluent was not less toxic than that of chlorine bleaching, as well as the residual toxicity of TCF, indicate that other substances aside from organohalogens contribute to the high methanogenic toxicity in bleaching effluents. Literature data is presented suggesting that wood resin compounds released by alkaline extraction stages might be important inhibitory substances in all bleaching processes. Oxygen-delignified! bleaching effluents of eucalyptus were found to be highly biodegradable. When tested at subtoxic concentrations, the chlorine and TCF bleaching effluent COD were anaerobically degraded by 67 and 75%, respectively: In both cases, methane production accounted for most of the COD removed. Therefore, the TCF process also enhances the anaerobic biodegradability of bleaching effluents to a small extent.
• Collins, P. J., J., M., Field, J. A., & D., A. (1996). Oxidation of anthracene and benzo[a]pyrene by laccases from Trametes versicolor. Applied and Environmental Microbiology, 62(12), 4563-4567.
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  PMID: 16535468;PMCID: PMC1389006;Abstract: The in vitro oxidation of the two polycyclic aromatic hydrocarbons anthracene and benzo[a]pyrene, which have ionization potentials of ≤7.45 eV, is catalyzed by laccases from Trametes versicolor. Crude laccase preparations were able to oxidize both anthracene and the potent carcinogen benzo[a]pyrene. Oxidation of benzo[a]pyrene was enhanced by the addition of the cooxidant 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), while an increased anthracene oxidizing ability was observed in the presence of the low-molecular-weight culture fluid ultrafiltrate. Two purified laccase isozymes from T. versicolor were found to have similar oxidative activities towards anthracene and benzo[a]pyrene. Oxidation of anthracene by the purified isozymes was enhanced in the presence of ABTS, while ABTS was essential for the oxidation of benzo[a]pyrene. In all cases anthraquinone was identified as the major end product of anthracene oxidation. These findings indicate that laccases may have a role in the oxidation of polycyclic aromatic hydrocarbons by white rut fungi.
• Donlon, B. A., Razo-Flores, E., Lettinga, G., & Field, J. A. (1996). Continuous detoxification, transformation, and degradation of nitrophenols in upflow anaerobic sludge blanket (UASB) reactors. Biotechnology and Bioengineering, 51(4), 439-449.
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  PMID: 18629796;Abstract: The anaerobic transformation and degradation of nitrophenols by granular sludge was investigated in upflow anaerobic sludge blanket (UASB) reactors continuously fed with a volatile fatty acid (VFA) mixture as the primary substrate. During the start-up, subtoxic concentrations of 2-nitrephenol (2- NP), 4-nitrophenol (4-NP), and 2, 4-dinitrophenol (2, 4-DNP) were utilized. 4-NP and 2, 4-DNP were readily converted to the corresponding aromatic amine; whereas 2-NP was converted to nonaromatic products via intermediate formation of 2-aminophenol (2-AP). These conversions led to a dramatic detoxification of the mononitrophenols because the reactors treated the nitrophenolics at the concentrations which were over 25 times higher than those that caused severe inhibition. VFA removal efficiencies greater than 99% were achieved in both reactors at loading rates greater than 11.4 g COD per liter of reactor volume per day even at volumetric loading of mononitrophenols up to 910 mg/L · d. The sludges obtained from each of the reactors at the end of the continuous experiments were assayed for their specific nitrophenol reducing activity in the presence of different primary substrates. Reduction rates of 45 and 26 mg/g volatile suspended solids per day were observed for 2-NP and 4-NP, respectively, when utilizing the VFA mixture as primary substrate. Hydrogen, an interspecies-reduced compound, and substrates that provide interspecies-reducing equivalents-such as butyrate, propionate, and ethanol stimulated nitrephenol reduction, whereas acetate and methanol did not. Anaerobic batch biodegradability tests with the 2-NP-adapted sludge revealed that its corresponding aromatic amine, 2-AP, was degraded to methane at a specific rate of 14.5 mg/g VSS · d. Acetate was observed to be the major intermediate during 2-AP degradation in the presence of a specific methanogenic inhibitor 2-bromoethanesulfonate. The results of this study indicate that UASB reactors can be applied to rapidly detoxify and, under certain circumstances, degrade nitroaromatic compounds.
• Field, J. A., Baten, H., Boelsma, F., & Rulkens, W. H. (1996). Biological elimination of polycyclic aromatic hydrocarbons in solvent extracts of polluted soil by the white rot fungus, Bjerkandera sp. strain BOS55. Environmental Technology, 17(3), 317-323.
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  Abstract: Polycyclic aromatic hydrocarbons (PAH) are persistent priority pollutants of soil and sediments. Miscible solvent extraction of the soil has previously been shown to effectively clean-up PAH pollution. The purpose of this study was to evaluate the use of the white rot fungus, Bjerkundera sp. strain BOS55 in initiating the degradation of PAH present in solvent extracts. The PAH in extracts of polluted soil added to 9-day-old liquid fungal cultures were removed by 80% in 7 days with 2% (v/v) ethanol in the medium. Smaller PAH compounds of only a few rings were degraded more rapidly than complexer PAH compounds having more rings. Phenanthrene, anthracene and pyrene with 3 to 4 rings were eliminated by 96.8 to 99.5%; whereas, benzo[b]fluoranthene and indeno[123cd]pyrene of 5 and 6 rings were eliminated by 52 and 20% in 7 days, respectively. When the PAH extract of polluted soil was incubated in fungal cultures together with 20% acetone or ethanol, the cells were killed by the solvent. Nonetheless, partial bioconversion of PAH occurred which was most likely due to the presence of extracellular peroxidases.
• Field, J. A., Vledder, R. H., G., J., & Rulkens, W. H. (1996). The tolerance of lignin peroxidase and manganese-dependent peroxidase to miscible solvents and the in vitro oxidation of anthracene in solvent: Water mixtures. Enzyme and Microbial Technology, 18(4), 300-308.
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  Abstract: Lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) titers in extracellular fluids of five-day-old Bjerkandera sp. strain BOS55 cultures and semipurified preparations from Phanerochaete chrysosporium were evaluated for their tolerance to miscible solvents. MnP assayed with 2,6-dimethoxyphenol (DMP) was found to retain 20% of its normal activity in the presence of 45-58% v/v acetone, 31-34% v/v ethanol, and 29% 2-propanol. LiP assayed with veratryl alcohol (VA) was about 2-3 times less tolerant to solvents compared to MnP. Methanol and 2-methoxyethanol were extremely inhibitory to LiP. The subtoxic concentrations of acetone were sufficient to remarkably increase the solubility of the model apolar pollutant, anthracene. Each 10% increment in the solvent concentration could be correlated to a 6.4-fold increase in the soluble anthracene concentration. When the peroxidases were incubated in vitro with anthracene as a substrate, acetone was essential for the elimination of anthracene and concomitant formation of anthraquinone. We observed for the first time a H2O2-enhanced oxidation of a polycyclic aromatic hydrocarbon, anthracene, by MnP when enough acetone was used (40% v/v). At acetone concentrations ranging from 5-60%, MnP was also able to oxidize anthracene in the absence of exogenous H2O2 additions but to a lesser extent. The optimum acetone concentration for the LiP-mediated oxidation of anthracene was 10% v/v. The in vitro oxidation rates with semipurified LiP (65 nmol VA min-1 ml-1) and MnP (33 nmol DMP min-1 ml-1) at the optimal solvent concentration was 4.3 and 4.0 mg anthracene l-1 d-1, respectively. The rate of anthracene conversion by LiP was similar to the anthracene dissolution rate in 10% acetone. In contrast, the conversion by MnP was estimated to be several orders of magnitude slower than the dissolution kinetics in 40% acetone.
• Florencio, L., Field, J. A., Langerak, A. V., & Lettinga, G. (1996). pH-stability in anaerobic bioreactors treating methanolic wastewaters. Water Science and Technology, 33(3), 177-184.
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  Abstract: Methanol is the main pollutant in evaporator condensate wastewaters from the kraft pulping industry. These wastewaters have no alkalinity. As methanogenesis is known to work best at neutral pH, additional alkalinity is needed to prevent the pH from dropping, and consequently, cause reactor instability. In anaerobic environments, methanol can either be directly converted to methane by methylotrophic methanogens or be converted to acetate by acetogens. The COD removal efficiency and stability of anaerobic reactor treating methanolic wastewaters are dependent on which route methanol is degraded. In this study, a mathematical model was made and tested. The model estimates pCO2 and pH expected from a certain stoichiometry yield of acetic acid and methane from methanol and the amount of alkalinity added. Continuous experiments were performed in five UASB (Upflow Anaerobic Sludge Blanket) reactors to check if the theoretical and experimental data matched. Methanol (5 g COD/l) was the only substrate used and NaHCO3 and K2HPO4 were the sources of added alkalinity. The amount of added alkalinity varied from 0 to 50 meq/l. The experimental data fitted very well with the model. With this model it is possible to predict the pH in the reactor and the CO2 composition of the biogas.
• J., M., Wasseveld, R. A., & Field, J. A. (1996). Hydrogen peroxide production as a limiting factor in xenobiotic compound oxidation by nitrogen-sufficient cultures of Bjerkandera sp. strain BOS55 overproducing peroxidases. Applied and Environmental Microbiology, 62(3), 880-885.
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  PMID: 16535276;PMCID: PMC1388801;Abstract: The overproduction of ligninolytic peroxidase by the N-deregulated white rot fungus Bjerkandera sp. strain BOS55 under nitrogen-sufficient conditions had no noteworthy effect on the oxidation of anthracene or the decolorization of the polymeric aromatic dye Poly R-478 in 6-day-old cultures. Only when the endogenous production of H2O2 was increased by the addition of extra oxygen and glucose could a 2.5-fold increase in the anthracene oxidation rate and a 6-fold increase in the Poly R-478 decolorization rate be observed in high-N cultures with 10- to 35-fold higher peroxidase activities than N-limited cultures. Further increase of the H2O2 generation rate in high-N cultures with glucose oxidase led to an additional 3.5-fold increase in the anthracene oxidation rate (350 mg liter-1 day-1) and a 10-fold increase in the Poly R-478 decolorization rate. These results indicate that xenobiotic compound oxidation by white rot fungi cannot be improved by overproducing peroxidases without increasing the endogenous production of H2O2. The absence of Mn, which decreased the manganese peroxidase titers and increased the lignin peroxidase titers, was associated with up to 95% improvements in the anthracene oxidation rate. The simultaneous presence of Mn and veratryl alcohol was observed to have a synergistic negative effect on the oxidation of anthracene and the decolorization of Poly R-478.
• Mester, T., Peña, M., & Field, J. A. (1996). Nutrient regulation of extracellular peroxidases in the white rot fungus, Bjerkandera sp. strain BOS55. Applied Microbiology and Biotechnology, 44(6), 778-784.
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  Abstract: Previous research indicated that the production of peroxidases in Bjerkandera sp. strain BOS55 increased under N-sufficient conditions. In this study different organic N sources (amino acids, peptides, proteins) were tested as N supplements. Most N sources significantly enhanced the titres of peroxidases, which increased in parallel with growth. While peroxidase production by Bjerkandera sp. strain BOS55 was not repressed by the addition of sufficient or excess nitrogen, the occurrence of ligninolytic activity was a secondary metabolic event triggered by the cessation of growth. Among the factors studied (O2, macronutrients, micronutrients, and vitamins), only those supplements with additional carbon or carbon and nitrogen increased the growth, indicating that the secondary metabolism was triggered by carbon limitation.
• Razo-Flores, E., Donlon, B., Field, J., & Lettinga, G. (1996). Biodegradability of N-substituted aromatics and alkylphenols under methanogenic conditions using granular sludge. Water Science and Technology, 33(3), 47-57.
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  Abstract: The biodegradability of seventeen N-substituted aromatic and six alkylphenol compounds were evaluated under methanogenic conditions. Biodegradation was assessed in batch assays inoculated with unacclimated and predigested anaerobic granular sludge at 30°C under agitated conditions over a 150 day period. The compounds were supplied at sub-toxic concentrations in the assays in order to prevent inhibition to the methanogens. The biodegradability test was performed by the measurement of the methane composition in the headspace of the serum flasks. The methanogenic consortia completely mineralized 2-, 3-aminobenzoate, 2-aminophenol and 4-cresol; whereas, 4-aminobenzoate was only partially degraded. The other N-substituted compounds and the alkylphenols tested were not biodegradable under the experimental conditions employed. An additional biodegradability assay was conducted with sludge from an upward-flow anaerobic sludge bed reactor adapted to the degradation of 2-nitrophenol. This sludge mineralized 2-aminophenol without any lag phase while the unadapted sludge required 110 days of acclimation. The three aminobenzoate isomers were fully mineralized by the adapted sludge after similar lag periods observed in the unadapted sludge. The 2-nitrophenol adapted sludge cross-acclimatized to the mineralization of 5-aminosalicylate and 4-aminophenol. This constitutes the first report demonstrating the anaerobic mineralization of 5-aminosalicylate, which indicates that at least some azo dye cleavage products can be degraded in methanogenic consortia.
• Razo-Flores, E., Svitelskaya, A., Donlon, B., Field, J., & Lettinga, G. (1996). The effect of granular sludge source on the anaerobic biodegradability of aromatic compounds. Bioresource Technology, 56(2-3), 215-220.
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  Abstract: The ability of bacteria from five different granular sludge sources to anaerobically biodegrade aromatic compounds was evaluated. The biodegradabilities of phenol 4-cresol, 2-aminobenzoate (2-AB) and 5-aminosalicylate (5-ASA) were determined by measuring compound conversion to methane in batch serum bottles at 30°C under agitated conditions over a period of at least 100 days. Phenol and 4-cresol were completely mineralized by all the granular sludges tested. This observation indicates a universal capacity of granular sludge to degrade phenol and 4-cresol; which would be expected since these compounds are intermediates in the anaerobic degradation of the commonly occurring amino acid tyrosine. In contrast, 5-ASA and 2-AB were degraded by only one or two granular sludges. Previous acclimation to an N-substituted aromatic was a prerequisite for 5-ASA degradation.
• Swarts, H. J., Verhagen, F. J., Field, J. A., & Wijnberg, J. B. (1996). Novelchlorometabolites produced by Bjerkandera species. Phytochemistry, 42(6), 1699-1701.
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  Abstract: The EtOAc extract from the extracellular fluid of the mycelium of Bjerkandera sp. BOS55 contained four novel chlorinated benzoic acid derivatives, i.e. 3-chloro-4-hydroxybenzoic acid, 3,5-dichloro-4-hydroxy- benzoic acid, methyl 3,5-dichloro-4-hydroxybenzoate and methyl 3,5-dichloro- p-anisate. 3-Chloro-4-hydroxybenzoic acid was also produced by B. adusta.
• Verhagen, F. J., Swarts, H. J., Kuyper, T. W., Wijnberg, J. B., & Field, J. A. (1996). The ubiquity of natural adsorbable organic halogen production among basidiomycetes. Applied Microbiology and Biotechnology, 45(5), 710-718.
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  Abstract: Recently, several species of basidiomycetes were shown to produce de novo high concentrations of chloroaromatic metabolites. Since these lignocellulose-degrading fungi play a major role in the ecosphere, the purpose of this study was to determine the ubiquity of organohalogen production among basidiomycetes. A total of 191 fungal strains were monitored for adsorbable organic halogen (AOX) production when grown on defined liquid media. Approximately 50% of the strains tested and 55% of the genera tested produced AOX. A low production of 0.1-0.5 mg AOX/l was observed among 25% of the strains, a moderate production of 0.5-5.0 mg AOX/l was observed among 16% of the strains and 9% of the strains produced high levels (5-67 mg AOX/l). The latter group was dominated by species belonging to the genera Hypholoma, Mycena and Bjerkandera, showing specific AOX productions in the range 1074-30 893 mg AOX/kg dry weight of mycelial biomass. Many highly ecologically significant fungal species were identified among the moderate to high producers. These species were also able to produce AOX when cultivated on natural lignocellulosic substrates. Hypholoma fasciculare and Mycena metata respectively produced up to 132 mg and 193 mg AOX/kg dry weight of forest litter substrate in 6 weeks.
• Donlon, B. A., Razo-Flores, E., Field, J. A., & Lettinga, G. (1995). Toxicity of N-substituted aromatics to acetoclastic methanogenic activity in granular sludge. Applied and Environmental Microbiology, 61(11), 3889-3893.
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  PMID: 8526501;PMCID: PMC167694;Abstract: N-substituted aromatics are important priority pollutants entering the environment primarily through anthropogenic activities associated with the industrial production of dyes, explosives, pesticides, and pharmaceuticals. Anaerobic treatment of wastewaters discharged by these industries could potentially be problematical as a result of the high toxicity of N- substituted aromatics. The objective of this study was to examine the structure-toxicity relationships of N-substituted aromatic compounds to acetoclastic methanogenic bacteria. The toxicity was assayed in serum flasks by measuring methane production in granular sludge. Unacclimated cultures were used to minimize the biotransformation of the toxic organic chemicals during the test. The nature and the degree of the aromatic substitution were observed to have a profound effect on the toxicity of the test compound. Nitroaromatic compounds were, on the average, over 500-fold more toxic than their corresponding aromatic amines. Considering the facile reduction of nitro groups by anaerobic microorganisms, a dramatic detoxification of nitroaromatics towards methanogens can be expected to occur during anaerobic wastewater treatment. While the toxicity exerted by the N-substituted aromatic compounds was closely correlated with compound apolarity (log P), it was observed that at any given log P, N-substituted phenols had a toxicity that was 2 orders of magnitude higher than that of chlorophenols and alkylphenols. This indicates that toxicity due to the chemical reactivity of nitroaromatics is much more important than partitioning effects in bacterial membranes.
• Field, J. A., Boelsma, F., Baten, H., & Rulkens, W. H. (1995). Oxidation of anthracene in water/solvent mixtures by the white-rot fungus, Bjerkandera sp. strain BOS55. Applied Microbiology and Biotechnology, 44(1-2), 234-240.
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  Abstract: Polycyclic aromatic hydrocarbons (PAH) are persistent priority pollutants of soil and sediments. The use of white-rot fungi has been proposed as a means of bioremediating PAH-polluted sites. However, higher PAH compounds of low bioavailability in polluted soil are biodegraded slowly. In order to enhance their bioavailability, PAH solubilization, can be increased in water/solvent mixtures. The oxidation of a model PAH compound, anthracene, in the presence of cosolvents by the white-rot fungus, Bjerkandera sp. strain BOS55 was investigated. Acetone and ethanol at 5% were toxic to this fungus when added at the time of inoculation. However, when solvents up to 20% (v/v) were added to 9-day-old cultures, ligninolytic activity as indicated by Poly R-478 dye decolorization and anthracene oxidation was evident for several days. Since 20% solvent was toxic to cells, the oxidation of anthracene can be attributed to extracellular peroxidases, which were shown to tolerate the solvent. Solvent additions of 11%-21% (v/v) acetone or ethanol increased the rate of anthracene bioconversion to anthraquinone in liquid medium by a factor of 2-3 compared to fungal cultures receiving 1%-3% solvent. © 1995 Springer-Verlag.
• Field, J. A., Stams, A. J., Kato, M., & Schraa, G. (1995). Enhanced biodegradation of aromatic pollutants in cocultures of anaerobic and aerobic bacterial consortia. Antonie van Leeuwenhoek, 67(1), 47-77.
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  PMID: 7741529;Abstract: Toxic aromatic pollutants, concentrated in industrial wastes and contaminated sites, can potentially be eliminated by low cost bioremediation systems. Most commonly, the goal of these treatment systems is directed at providing optimum environmental conditions for the mineralization of the pollutants by naturally occurring microflora. Electrophilic aromatic pollutants with multiple chloro, nitro and azo groups have proven to be persistent to biodegradation by aerobic bacteria. These compounds are readily reduced by anaerobic consortia to lower chlorinated aromatics or aromatic amines but are not mineralized further. The reduction increases the susceptibility of the aromatic molecule for oxygenolytic attack. Sequencing anaerobic and aerobic biotreatment steps provide enhanced mineralization of many electrophilic aromatic pollutants. The combined activity of anaerobic and aerobic bacteria can also be obtained in a single treatment step if the bacteria are immobilized in particulate matrices (e.g. biofilm, soil aggregate, etc.). Due to the rapid uptake of oxygen by aerobes and facultative bacteria compared to the slow diffusion of oxygen, oxygen penetration into active biofilms seldom exceeds several hundred micrometers. The anaerobic microniches established inside the biofilms can be applied to the reduction of electron withdrawing functional groups in order to prepare recalcitrant aromatic compounds for further mineralization in the aerobic outer layer of the biofilm. Aside from mineralization, polyhydroxylated and chlorinated phenols as well as nitroaromatics and aromatic amines are susceptible to polymerization in aerobic environments. Consequently, an alternative approach for bioremediation systems can be directed towards incorporating these aromatic pollutants into detoxified humic-like substances. The activation of aromatic pollutants for polymerization can potentially be encouraged by an anaerobic pretreatment step prior to oxidation. Anaerobic bacteria can modify aromatic pollutants by demethylating methoxy groups and reducing nitro groups. The resulting phenols and aromatic amines are readily polymerized in a subsequent aerobic step. © 1995 Kluwer Academic Publishers.
• Field, J. A., Verhagen, F. J., & Jong, E. d. (1995). Natural organohalogen production by basidiomycetes. Trends in Biotechnology, 13(11), 451-456.
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  Abstract: Basidiomycetes are ecologically important higher fungi that synthesize three families of organohalogen metabolites: halomethanes, halogenated aromatics and haloaliphatic compounds. To date, a total of 53 halogenated metabolites have been identified in 34 genera of basidiomycetes. These organohalogen metabolites have demonstrable physiological roles as antibiotics, as methyl donors and as subsstrates for H2O2-generating oxidases. The concentration of chlorinated aromatic metabolites encountered in natural environments associated with the widespread occurrence of basidiomycetes have been shown to exceed the hazardous-waste norms that are applied to analogous anthropogenic chlorophenols in soil. © 1995 Elsevier Science Ltd.
• Florencio, L., Field, J. A., & Lettinga, G. (1995). Substrate competition between methanogens and acetogens during the degradation of methanol in UASB reactors. Water Research, 29(3), 915-922.
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  Abstract: To evaluate the effect of methanol concentration and inorganic carbon on the competition between acetogens and methanogens for methanol, eight upflow anaerobic sludge blanket reactors were operated continuously with different levels of sodium bicarbonate at variable methanol loadings. The results indicated that methylotrophic methanogens will predominate in a broad pH range (4.2-7.2) if either the methanol concentration in the effluent or inorganic carbon is low. Continuous steady state methane production resulted in chemical oxygen demand (COD) removals of 99.8-54% at organic loadings ranging from 7.6 to 22 g COD·l-1·d-1. On the other hand, significant acetogenesis occurred if exogenous inorganic carbon was supplied and if the methanol concentration in the effluent was greater than 1000 mg COD·l-1. The observation that acetogenesis only occurred in the presence of high methanol concentrations in the effluent confirms the previously determined 60-times-higher substrate affinity of methanogens. Steady state acetogenesis was only observed when unionized VFA, which inhibited the methanogens, were present at high levels for prolonged periods of time. Addition of moderate levels of NaHCO3 (15 meq·l-1) were found to create such conditions since VFA accumulation exceeded the buffering capacity. Continuous acetogenesis from methanol at organic loading rates of 21 g COD·l-1·d-1 resulted in poor COD removal (16.3%). In contrast, high levels of NaHCO3 supplementation (≥25 meq·l-1) favured methane production since the alkalinity was strong enough to prevent toxic levels of undissociated acids even in periods when VFA accumulated as a result of organic overloading. © 1995.
• Kaal, E. E., Field, J. A., & Joyce, T. W. (1995). Increasing ligninolytic enzyme activities in several white-rot Basidiomycetes by nitrogen-sufficient media. Bioresource Technology, 53(2), 133-139.
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  Abstract: Ligninolytic enzyme activities were monitored in five white-rot fungi cultivated on nitrogen (N)-limited glucose-BIII medium and were compared with the activities obtained in media supplemented with 56 mm peptone-N. Only Phanerochaete chrysosporium and two Bjerkandera strains produced detectable lignin peroxidase (LiP) activity. LiP was stimulated by either N-limitation or N-sufficiency in P. chrysosporium and Bjerkandera spp., respectively. All of the fungal strains tested produced manganese-dependent peroxidase (MnP) activity, which was consistently stimulated by the peptone supplementation. Both the manganese-independent peroxidase (MiP) activities, which were detected only in Bjerkandera spp., and the laccase activities, which were detected only in Lentinula edodes and Pleurotus ostreatus, were also enhanced by peptone. In several fungal strains, the activity of ligninolytic enzymes was likewise stimulated by supplying 56 mm NH+4-N at an initial pH of 7·3. This study indicates that several commercially important and commonly occurring white-rot fungi produce higher ligninolytic enzyme activities in response to a nitrogen-rich medium, in contrast to the physiological model proposed for P. chrysosporium. © 1995.
• Kortekaas, S., Soto, M., Vicent, T., Field, J. A., & Lettinga, G. (1995). Contribution of extractives to methanogenic toxicity of hemp black liquor. Journal of Fermentation and Bioengineering, 80(4), 383-388.
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  Abstract: The objective of this research was to identify the major sources of methanogenic toxicity in hemp black liquors. Hemp extractives were shown to be highly toxic. The stem wood extractives, which had a 50% inhibitory concentration (IC) corresponding to 0.25 g COD/l, were somewhat more toxic than the bark extractives with a 50% IC corresponding to 0.65 g COD/l. The methanogenic toxicity of black liquors from hemp was comparable with the toxicity of black liquors from woody feedstocks and ranged between 2.8 and 4.8 g COD/l. Removal of extractives from the hemp fiber prior to pulping only partly reduced methanogenic toxicity, in spite of high extractive removal efficiencies. The residual extractives remaining in the feedstocks after fiber extraction (10 to 30% of the extractives according Soxhlet determination) were apparently released into the black liquor during pulping. To improve removal of the extractives, hemp black liquor was subjected to several detoxification treatments. Ethyl ether extraction and Amberlite XAD-2 treatment achieved complete wastewater detoxification. These results indicate that the apolar extractive fraction is the major source of methanogenic toxicity in hemp black liquors. © 1995.
• Mester, T., Jong, E. D., & Field, J. A. (1995). Manganese regulation of veratryl alcohol in white rot fungi and its indirect effect on lignin peroxidase. Applied and Environmental Microbiology, 61(5), 1881-1887.
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  PMID: 16535027;PMCID: PMC1388445;Abstract: Many white rot fungi are able to produce de novo veratryl alcohol, which is known to be a cofactor involved in the degradation of lignin, lignin model compounds, and xenobiotic pollutants by lignin peroxidase (LIP). In this study, Mn nutrition was shown to strongly influence the endogenous veratryl alcohol levels in the culture fluids of N-deregulated and N-regulated white rot fungi Bjerkandera sp. strain BOS55 and Phanerochaete chrysosporium BKM- F-1767, respectively. Endogenous veratryl alcohol levels as high as 0.75 mM in Bjerkandera sp. strain BOS55 and 2.5 mM in P. chrysosporium were observed under Mn-deficient conditions. In contrast, veratryl alcohol production was dramatically decreased in cultures supplemented with 33 or 264 μM Mn. The LiP titers, which were highest in Mn-deficient media, were shown to parallel the endogenous veratryl alcohol levels, indicating that these two parameters are related. When exogenous veratryl alcohol was added to Mn-sufficient media, high LiP titers were obtained. Consequently, we concluded that Mn does not regulate LiP expression directly. Instead, LiP titers are enhanced by the increased production of veratryl alcohol. The well-known role of veratryl alcohol in protecting LiP from inactivation by physiological levels of H2O2 is postulated to be the major reason why LiP is apparently regulated by Mn. Provided that Mn was absent, LiP titers in Bjerkandera sp. strain BOS55 increased with enhanced fungal growth obtained by increasing the nutrient N concentration while veratryl alcohol levels were similar in both N-limited and N-sufficient conditions.
• Florencio, L., Field, J. A., & Lettinga, G. (1994). Importance of cobalt for individual trophic groups in an anaerobic methanol-degrading consortium. Applied and Environmental Microbiology, 60(1), 227-234.
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  PMID: 8117078;PMCID: PMC201293;Abstract: Methanol is an important anaerobic substrate in industrial wastewater treatment and the natural environment. Previous studies indicate that cobalt greatly stimulates methane formation during anaerobic treatment of methanolic wastewaters. To evaluate the effect of cobalt in a mixed culture, a sludge with low background levels of cobalt was cultivated in an upflow anaerobic sludge blanket reactor. Specific inhibitors in batch assays were then utilized to study the effect of cobalt on the growth rate and activity of different microorganisms involved in the anaerobic degradation of methanol. Only methylotrophic methanogens and acetogens were stimulated by cobalt additions, while the other trophic groups utilizing downstream intermediates, H2-CO2 or acetate, were largely unaffected. The optimal concentration of cobalt for the growth and activity of methanol-utilizing methanogens and acetogens was 0.05 mg liter-1. The higher requirement of cobalt is presumably due to the previously reported production of unique corrinoid- containing enzymes (or coenzymes) by direct utilizers of methanol. This distinctly high requirement of cobalt by methylotrophs should be considered during methanolic wastewater treatment. Methylotroph methanogens presented a 60-fold-higher affinity for methanol than acetogens. This result in combination with the fact that acetogens grow slightly faster than methanogens under optimal cobalt conditions indicates that acetogens can outcompete methanogens only when reactor methanol and cobalt concentrations are high, provided enough inorganic carbon is available.
• Jong, E. D., Cazemier, A. E., Field, J. A., & Bont, J. D. (1994). Physiological role of chlorinated aryl alcohols biosynthesized de novo by the white rot fungus Bjerkandera sp. strain BOS55. Applied and Environmental Microbiology, 60(1), 271-277.
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  PMID: 16349157;PMCID: PMC201299;Abstract: The white rot fungus Bjerkandera sp. strain BOS55 produces veratryl, anisyl, 3-chloroanisyl, and 3,5-dichloroanisyl alcohol and the corresponding aldehydes de novo from glucose. All metabolites are produced simultaneously with the extracellular ligninolytic enzymes and have an important physiological function in the fungal ligninolytic system. Both mono- and dechlorinated anisyl alcohols are distinctly better substrates for the extracellular aryl alcohol oxidases than veratryl alcohol. The aldehydes formed are readily recycled by reduction by washed fungal mycelium, thus creating an extracellular H2O2 production system regulated by intracellular enzymes. Lignin peroxidase does not oxidize the chlorinated anisyl alcohols either in the absence or in the presence of veratryl alcohol. It was therefore concluded that the chlorinated anisyl alcohols are well protected against the fungus's own aggressive ligninolytic enzymes. The relative amounts of veratryl alcohol and the chlorinated anisyl alcohols differ significantly according to the growth conditions, indicating that production of veratryl alcohol and the production of the (chlorinated) anisyl metabolites are independently regulated. We conclude that the chlorinated anisyl metabolites biosynthesized by the white rot fungus Bjerkandera sp. strain BOS55 can be purposefully produced for ecologically significant processes such as lignin degradation.
• Jong, E. D., Field, J. A., & A.M., J. (1994). Aryl alcohols in the physiology of ligninolytic fungi. FEMS Microbiology Reviews, 13(2-3), 153-188.
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  Abstract: White-rot fungi have a versatile machinery of enzymes which work in harmony with secondary aryl alcohol metabolites to degrade the recalcitrant, aromatic biopolymer lignin. This review will focus on the important physiological roles of aryl (veratryl, anisyl and chlorinated anisyl) alcohols in the ligninolytic enzyme system. Their functions include stabilization of lignin peroxidase, charge-transfer reactions and as substrate for oxidases generating extracellular H2O2. The aryl alcohol/aldehyde couple is well protected against degradation by the fungi's extracellular ligninolytic enzymes and their concentrations in the extracellular fluid are highly regulated by intracellular enzymes.
• Jong, E. D., Field, J. A., Spinnler, H. -., Wijnberg, J. B., & Bont, J. D. (1994). Significant biogenesis of chlorinated aromatics by fungi in natural environments. Applied and Environmental Microbiology, 60(1), 264-270.
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  PMID: 16349156;PMCID: PMC201298;Abstract: Common wood- and forest litter-degrading fungi produce chlorinated anisyl metabolites. These compounds, which are structurally related to xenobiotic chloroaromatics, occur at high concentrations of approximately 75 mg of chlorinated anisyl metabolites kg of wood-1 or litter-1 in the environment. The widespread ability among common fungi to produce large amounts of chlorinated aromatic compounds in the environment makes us conclude that these kinds of compounds can no longer be considered to originate mainly from anthropogenic sources.
• Kato, M. T., Field, J. A., Kleerebezem, R., & Lettinga, G. (1994). Treatment of low strength soluble wastewaters in UASB reactors. Journal of Fermentation and Bioengineering, 77(6), 679-686.
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  Abstract: Low strength wastewaters can be those with chemical oxygen demand (COD) below 2,000 mg/l. The anaerobic treatment of such wastewaters has not been fully explored so far. The suboptimal reaction rates with low substrate concentrations, and the presence of dissolved oxygen in the influent are regarded as possible constraints. In this study, the treatment of low strength soluble wastewaters containing ethanol or whey was studied in lab-scale upflow anaerobic sludged bed (UASB) reactors at 30°C. The high treatment performance obtained demonstrates that UASB reactors are viable for treating both types of wastewaters at low COD concentrations. The treatment of the ethanol containing wastewater resulted in COD removal efficiencies exceeding 95% at organic loading rates (OLR) between 0.3 to 6.8 g COD/l · d with influent concentrations in the range of 422 to 943 mg COD/l. In the case of the more complex whey containing wastewater, COD removal efficiencies exceeded 86% at OLRs up to 3.9 g COD/l · d, as long as the COD influent was above 630 mg/l. Lowering the COD influent resulted in decreased efficiency with sharper decrease at values below 200 mg/l. Acidification instead of methanogenesis was found to be the rate limiting step in the COD removal at low concentrations, which was not the case when treating ethanol. The effect of dissolved oxygen in the influent as a potential danger in anaerobic treatment was investigated in reactors fed with and without dissolved oxygen. Compared with the control reactor, the reactor receiving oxygen showed no detrimental effects in the treatment performance. Thus, the presence of dissolved oxygen in dilute wastewaters is expected to be of minor importance in practice.
• Kato, M. T., Field, J. A., Versteeg, P., & Lettinga, G. (1994). Feasibility of expanded granular sludge bed reactors for the anaerobic treatment of low-strength soluble wastewaters. Biotechnology and Bioengineering, 44(4), 469-479.
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  Abstract: The application of the expanded granular sludge bed (EGSB) reactor for the anaerobic treatment of low-strength soluble wastewaters using ethanol as a model substrate was Investigated in laboratory-scale reactors at 30°C. Chemical oxygen demand (COD) removal efficiency was above 80% at organic loading rates up to 12 g COD/L.d with influent concentrations as low as 100 to 200 mg COD/L. These results demonstrate the suitability of the EGSB reactor for the anaerobic treatment of low-strength wastewaters. The high treatment performance can be attributed to the intense mixing regime obtained by high hydraulic and organic loads. Good mixing of the bulk liquid phase for the substrate-biomass contact and adequate expansion of the sludge bed for the degassing were obtained when the liquid upflow velocity (V(up)) was greater than 2.5 m/h. Under such conditions, an extremely low apparent K(s) value for acetoclastic methanogenesis of 9.8 mg COD/L was observed. The presence of dissolved oxygen in the wastewater had no detrimental effect on the treatment performance. Sludge piston flotation from pockets of biogas accumulating under the sludge bed occurred at V(up) lower than 2.5 m/h due to poor bed expansion. This problem is expected only in small diameter laboratory-scale reactors. A more important restriction of the EGSB reactor was the sludge washout occurring at V(up) higher than 5.5 m/h and which was intensified at organic loads higher than 7 g COD/L.d due to buoyancy forces from the gas production. To achieve an equilibrium between the mixing intensity and the sludge hold-up. the operation should be limited to an organic loading rate of 7 g COD/L.d and to a liquid upflow velocity between 2.5 and 5.5 m/h.
• Kortekaas, S., Doma, H. S., Potapenko, S. A., Field, J. A., & Lettinga, G. (1994). Sequenced anaerobic-aerobic treatment of hemp black liquors. Water Science and Technology, 29(5-6), 409-419.
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  Abstract: This study evaluates the sequenced anaerobic-aerobic treatment of hemp bark and hemp stem wood black liquors with respect to COD removal efficiency and detoxification. Anaerobic toxicity assays revealed that soda pulping liquors derived from hemp are just as toxic as those derived from wood. Hemp bark and stem wood black liquors caused 50% inhibition at concentrations of 5.9 and 4.5 g COD/l, respectively. Long term experiments were conducted in lab-scale upflow anaerobic sludge blanket (UASB) reactors at 30°C. Black liquor was fed at sub-toxic concentrations (< 4 g COD/l). In the bark column loadings were applied up to 17.5 g COD/l(*)d with COD and BOD5 efficiencies of 56.0 and 87.9%, respectively. In the stem wood column loadings up to 18.4 g COD/l(*)d were reached with COD and BOD5 efficiencies of 42.3 and 81.0%, respectively. Aerobic post-treatment displayed only minor extra COD removal and a strong increase of color levels. After sequenced anaerobic-aerobic treatment, the COD removal was 70.9 and 58.4% for bark and stem wood liquor, respectively, whereas the BOD5 removal exceeded 98%. Strong detoxification was accomplished after anaerobic-aerobic treatment, as was demonstrated by anaerobic toxicity assays. After anaerobic-aerobic treatment hemp bark and stem wood black liquors caused 50% inhibition at concentrations of 13.5 and 21.5 g COD/l, respectively.
• Kotterman, M. J., Heessels, E., Jong, E. d., & Field, J. A. (1994). The physiology of anthracene biodegradation by the white-rot fungus Bjerkandera sp. strain BOS55. Applied Microbiology and Biotechnology, 42(1), 179-186.
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  Abstract: A recently isolated white-rot strain, Bjerkandera sp. strain BOS55, displays high extracellular peroxidase activity, and rapidly degrades polycyclic aromatic hydrocarbons (PAH). In this study, the culture conditions for the biodegradation of the model PAH compound, anthracene, were optimized with respect to O2, N, and C. An additional objective was to determine if the decolorization of the polymeric ligninolytic indicator dye, Poly R-478, could be correlated to anthracene biodegradation observed under a wide range of culture conditions. The supply of O2 was found to be the most important parameter in the biodegradation of anthracene. Increasing culture aeration enhanced the biodegradation of anthracene and the accumulation of its peroxidase-mediated oxidation product anthraquinone. Decolorization of Poly R-478 was less affected by inadequate aeration. Provided that ample aeration was supplied, the degradation of anthracene under different culture conditions was strongly correlated with the ligninolytic activity as indicated by the rate of Poly R-478 decolorization. Concentrations up to 22 mM NH4+ N did not repress anthracene biodegradation and only caused a 0%-40% repression of the Poly R-478 decolorizing activity in various experiments. A cosubstrate requirement of 100 mg glucose / mg anthracene biodegraded was observed in this study. © 1994 Springer-Verlag.
• Field, J. A., Jong, E. D., Feijoo-Costa, G., & Bont, J. D. (1993). Screening for ligninolytic fungi applicable to the biodegradation of xenobiotics. Trends in Biotechnology, 11(2), 44-49.
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  Abstract: It is shown that white-rot fungi constitute a promising group of microorganisms for application in the bioremediation of recalcitrant xenobiotic compounds. Decolorization of polymeric dyes has proven to be a good indicator of the initial transformation of xenobiotics mediated by the peroxidative activity of the fungi.
• Florencio, L., Jenicek, P., Field, J. A., & Lettinga, G. (1993). Effect of cobalt on the anaerobic degradation of methanol. Journal of Fermentation and Bioengineering, 75(5), 368-374.
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  Abstract: The effect of trace elements on the methanogenesis from methanol and acetate was studied utilizing granular sludge obtained from an anaerobic wastewater treatment plant. The methanogenic activity from methanol was dramatically stimulated by the addition of a cocktail of trace elements in the basal medium. When trace elements were supplied individually, cobalt greatly stimulated methanogenesis which equalled the stimulation observed with the complete trace element mixture. No remarkable influence of any trace element was observed when acetate was used as the substrate. Two Upflow Anaerobic Sludge Blanket (UASB) reactors were operated with and without supplementation of cobalt. Cobalt greatly stimulated both acetogenesis in the initial operational phase and later methanogenesis. The cobalt sufficient column provided almost 3 times the methane productivity compared to the cobalt deprived column. At an organic loading rate of 8 g COD/1 · d, 87% of the COD was converted to methane in the cobalt sufficient column. Under low cobalt concentration, methanogens compete better for cobalt than acetogens.
• Florencio, L., Nozhevnikova, A., Langerak, A. v., Stams, A. J., Field, J. A., & Lettinga, G. (1993). Acidophilic degradation of methanol by a methanogenic enrichment culture. FEMS Microbiology Letters, 109(1), 1-6.
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  Abstract: An acidophilic methanogenic enrichment culture was obtained in a continuous up-flow anaerobic sludge blanket reactor operated at pH 4.2 with methanol as the sole carbon source. The specific methylotrophic methanogenic activity of the enriched reactor sludge at pH 5 was 3.57 g COD g-1 volatile suspended solids day-1 and the apparent doubling time of the biomass was 15.8 h. Acidic conditions were obligatory, since the enrichment culture was not able to produce methane or to grow at pH 7. Based on morphological characteristics, the dominant methanogenic species in the enrichment culture was a Methanosarcina. © 1993.
• Kaal, E. E., Jong, E. D., & Field, J. A. (1993). Stimulation of ligninolytic peroxidase activity by nitrogen nutrients in the white rot fungus Bjerkandera sp. strain BOS55. Applied and Environmental Microbiology, 59(12), 4031-4036.
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  PMID: 16349104;PMCID: PMC195863;Abstract: Bjerkandera sp. strain BOS55, a newly isolated wild-type white rot fungus, produced lignin peroxidase (LiP) in nitrogen (N)-sufficient glucose-peptone medium, whereas no LiP was detectable in N-limited medium. The production of LiP was induced by the peptide-containing components of this medium and also by soy bean protein. Furthermore, the production of manganese-dependent peroxidase was stimulated by organic N sources, although lower production was also evident in N-limited medium. Further research showed that the induction of LiP depended on the combination of pH and the type of N source. An amino acid mixture and ammonium induced LiP only at either pH 6 or 7.3, respectively. Peptone induced LiP activity at all pH values tested; however, the highest activity was observed at pH 7.3. The results presented here indicate that Bjerkandera spp. are distinct from the model white rot fungus, Phanerochaete chrysosporium, which produces ligninolytic peroxidases in response to N limitation.
• Kato, M. T., Field, J. A., & Lettinga, G. (1993). High tolerance of methanogens in granular sludge to oxygen. Biotechnology and Bioengineering, 42(11), 1360-1366.
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  PMID: 18612964;Abstract: This research assessed the effect of oxygen exposure no the methanogenic activity of anaerobic granular sludges. The toxicity of oxygen to acetoelastic methanogens in five different anaerobic granular sludges was determined in serum flasks with effective gas-to-liquid volumes of 4.65 to 1. The amount of oxygen that caused 50% inhibition of the methanogenic activity after 3 days of exposure ranged from 7% to 41% oxygen in the head space. These results indicate that methanogens located in granular sludge have a high tolerance for oxygen. The most important factor contributing to the tolerance was the oxygen consumption by facultative bacteria metabolizing biodegradable substrates. Uptake of oxygen by these bacteria creates anaerobic microenvironments where the methanogenic bacteria are protected. The results also indicate that methanogens in sludge consortia still have some tolerance to oxygen, even in the absence of facultative substrate for oxygen respiration.
• Kato, M. T., Field, J. A., & Lettinga, G. (1993). Methanogenesis in granular sludge exposed to oxygen. FEMS Microbiology Letters, 114(3), 317-323.
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  Abstract: Substrate competition between methanogenic and facultative bacteria under highly aerobic conditions was investigated in batch experiments. Natural mixed cultures of anaerobic bacteria immobilized in granular sludge were able to concurrently utilize oxygen and produce methane when supplied with ethanol as substrate. The most oxygen tolerant sludge converted 3 to 25% of substrate chemical oxygen demand to methane after 3 days while 23 to 2 mg 1-1 of dissolved oxygen were present in the media. The tolerance of methanogens to oxygen and their coexistence with facultative bacteria were evident even after long periods of oxygen exposure. Eventually, methane oxidizing bacteria developed in the co-culture. The consumption of oxygen by facultative bacteria, creating anaerobic microniches inside the granules, is hypothesized to protect the methanogens. © 1993.
• Field, J. A., Jong, E. D., Costa, G. F., & Bont, J. D. (1992). Biodegradation of polycyclic aromatic hydrocarbons by new isolates of white rot fungi. Applied and Environmental Microbiology, 58(7), 2219-2226.
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  PMID: 1637159;PMCID: PMC195758;Abstract: Eight rapid Poly R-478 dye-decolorizing isolates from The Netherlands were screened in this study for the biodegradation of polycyclic aromatic hydrocarbons (PAH) supplied at 10 mg liter-1. Several well-known ligninolytic culture collection strains, Phanerochaete chrysosporium BKM-F- 1767, Trametes versicolor Paprican 52, and Bjerkandera adusta CBS 595.78 were tested in parallel. All of the strains significantly removed anthracene, and nine of the strains significantly removed benzo[a]pyrene beyond the limited losses observed in sterile liquid and HgCl2-poisoned fungus controls. One of the new isolates, Bjerkandera sp. strain Bos 55, was the best degrader of both anthracene and benzo[a]pyrene, removing 99.2 and 83.1% of these compounds after 28 days, respectively. Half of the strains, exemplified by strains of the genera Bjerkandera and Phanerochaete, converted anthracene to anthraquinone, which was found to be a dead-end metabolite, in high yields. The extracellular fluids of selected strains were shown to be implicated in this conversion. In contrast, four Trametes strains removed anthracene without significant accumulation of the quinone. The ability of Trametes strains to degrade anthraquinone was confirmed in this study. None of the strains accumulated PAH quinones during benzo[a]pyrene degradation. Biodegradation of PAH by the various strains was highly correlated to the rate by which they decolorized Poly R-478 dye, demonstrating that ligninolytic indicators are useful in screening for promising PAH-degrading white rot fungal strains.
• Jong, E. d., Field, J. A., & A.M., J. (1992). Evidence for a new extracellular peroxidase Manganese-inhibited peroxidase from the white-rot fungus Bjerkandera sp. BOS 55. FEBS Letters, 299(1), 107-110.
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  PMID: 1312029;Abstract: A novel enzyme activity was detected in the extracellular fluid of Bjerkandera sp, BOS 55. The purified enzyme could oxidize several compounds, such as Phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, ABTS and guaiacol, with H2O2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H2O2. With some substrates, a strong inhibition of the peroxidative activity by Mn2+ was observed. Phenol red oxidation was inhibited by 84% with only 1 mM of this metal ion. Because DMP oxidation by this enzyme is only slightly inhibited by Mn2+, this substrate should not be used in assays to detect manganese peroxidase. The enzyme is tentatively named 'Manganese-Inhibited Peroxidase'. © 1992.
• Jong, E. d., Field, J. A., Dings, J. A., Wijnberg, J. B., & A.M., J. (1992). De-novo biosynthesis of chlorinated aromatics by the white-rot fungus Bjerkandera sp. BOS55 Formation of 3-chloro-anisaldehyde from glucose. FEBS Letters, 305(3), 220-224.
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  PMID: 1299620;Abstract: The white-rot fungus Bjerkandera sp. BOS55 produced de-novo several aromatic metabolites. Besides veratryl alcohol and veratraldehyde, compounds which are known to be involved in the ligninolytic system of several other white-rot fungi, other metabolites were formed. These included anisaldehyde, 3-chloro-anisaldehyde and a yet unknown compound containing two chlorine atoms. Additionally GC/MS analysis revealed the production of small amounts of anisyl alcohol and 3-chloro-anisyl alcohol. After 14 days, the extracellular fluid of Bjerkandera BOS55 contained 100 μM veratraldehyde and 50 μM 3-chloro-anisaldehyde. This is the first report of de-novo biosynthesis of simple chlorinated aromatic compounds by a white-rot fungus. Anisaldehyde and 3-chloro-anisaldehyde were also produced by Bjerkandera adusta but not by Phanerochaete chrysosporium. © 1992.
• Field, J. A., & Lettinga, G. (1991). Treatment and detoxification of aqueous spruce bark extracts by Aspergillus niger. Water Science and Technology, 24(3-4), 127-137.
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  Abstract: Debarking effluents of the forest industry are severely toxic waste streams due to their high tannin content. The objectives of this study were to evaluate the treatment and detoxification of debarking wastewater with tannin tolerant fungi. For this purpose, Aspergillus niger was cultivated on aqueous bark extracts and the toxicity was assayed utilizing methanogenic bacteria as the test organism. Sterilized aqueous extracts of spruce bark were diluted to 5.2 g COD L-1 (containing 2.7 g tannin COD L-1) in either citrate or tartrate buffer and inoculated with A. niger spores. During four day aerobic fermentations with citrate buffer, the elimination of unfiltered and soluble COD was 28 and 63%, respectively. The apparent yield of biomass averaged 34% of the extract COD; however, 12% of the extract COD was tannins adsorbed on the fungal biomass. The decrease in the tannin concentration was 50%. According to gel chromatography results, the original oligomeric tannins that resisted biodegradation were those of the highest MW. The toxicity was consequently only partially reduced by the fermentation. During the tartrate buffered fermentations, the pH rose due to the metabolism of the organic acid in the buffer. The rise in pH beyond 6 promoted autoxidative reactions that caused extensive polymerization of the tannins which resisted biodegradation. This resulted not only in an enhanced elimination of the tannins but also in a complete detoxification of the extracts. Similar levels of detoxification could be obtained in sterile un-inoculated extracts by autoxidation alone (raising the pH and aerating the extracts). Likewise, extracts fermented in citrate buffer could be completely detoxified by a subsequent autoxidation treatment. The exclusion peak of the gel chromatograms (MW > 30,000 g mole-1) obtained from the highly autoxidized samples accounted for 53% of the UV absorbance, whereas no exclusion peak was present in the unoxidized extracts. For the biological treatment and detoxification of debarking wastewaters, we recommend the cultivation of tannin tolerant fungi followed by short autoxidation treatments that polymerize the oligomeric tannin fraction resisting biodegradation to non-toxic high molecular weight polymers.
• Soto, M., Field, J. A., Lettinga, G., Mendez, R., & Lema, J. M. (1991). Anaerobic biodegradability and toxicity of eucalyptus fiber board manufacturing wastewater. Journal of Chemical Technology and Biotechnology, 52(2), 163-176.
• Field, J. A., Lettinga, G., & Habets, L. H. (1990). Oxidative detoxification of aqueous bark extracts. Part I: Autoxidation. Journal of Chemical Technology and Biotechnology, 49(1), 15-33.
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  Abstract: The aqueous extracts of bark were studied as a model for wet debarking wastewater. These extracts are known to contain a high concentration of methanogenic toxic tannins. The objective of this study was to modify the native bark tannins (oligomers) with oxidative methods in order to decrease their methanogenic toxicity. The tannins were polymerized by autoxidation, forming colored high-molecular-weight tannins that were nontoxic to methanogenic bacteria. The autoxidation of pine bark extracts provided complete detoxification. In the case of spruce bark, which was responsible for extracts of higher toxicity, the detoxification was either partial or complete depending on the specific sample of spruce bark from which the extract was prepared. The autoxidation of birch bark did not result in significant detoxification. Although the oligomeric tannins were effectively polymerized, birch bark extracts contain nontannin toxins which were not affected by the autoxidation and evidence that highly toxic intermediates were formed during the high pH autoxidation of birch bark extracts is presented.
• Field, J., Lettinga, G., & Habets, L. H. (1990). Measurement of low molecular weight tannins: Indicators of methanogenic toxic tannins. Journal of Fermentation and Bioengineering, 69(3), 148-153.
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  Abstract: The effectiveness of several low molecular weight (MW) tannin measurement methods for indicating the tannin toxicity to methan bacteria was evaluated. The methanogenic toxicity of the low and high MW tannins from autoxidized bark extracts was studied by selective removal of MW fractions from the extract with active carbon adsorption and calcium precipitation treatments. The toxicity of the low MW tannin fraction and the nontoxicity of the high MW tannin fraction were demonstrated. The low MW tannin concentration, measured by HPLC and a method based on the loss of tannins by treatment with granular active carbon (AC), had a very close relationship with the methanogenic toxicity, but a poor relationship was found with the total tannin concentration. The low MW tannins detected by the HPLC and AC methods had similar peak area positions in HPLC chromatograms as the tannins that were adsorbed by polyamide (trisacryl GF05) gel beads. These gel beads have an exclusion limit of 3000 g·mol-1, indicating that this is the approximate MW boundary between toxic and non-toxic tannins. © 1990.
• Field, J. A., & Lettinga, G. (1989). The effect of oxidative coloration on the methanogenic toxicity and anaerobic biodegradability of phenols. Biological Wastes, 29(3), 161-179.
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  Abstract: Short exposures to air increased the methanogenic toxicity of monomeric phenols provided these compounds had neighboring hydroxyl groups, necessary for the coloration, and if their oxidized products lacked free carboxylic-acid groups. In contrast, a methanogenic toxic tannin, gallotannic acid (oligomeric), was detoxified by oxidative coloration. The extensive oxidation of a monomer, L-dopa, produced a dark-colored precipitate which was non-toxic. These results indicated that the initial products of the oxidation of monomers are toxic whereas highly polymerized products are non-toxic. The colorless phenolic compounds tested in this study were either partially or fully degraded.
• Field, J. A., Kortekaas, S., & Lettinga, G. (1989). The effect of autoxidation on the methanogenic toxicity and anaerobic biodegradability of pyrogallol. Biological Wastes, 30(2), 111-121.
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  Abstract: In a previous study, catechin (a condensed tannim monomer) was polymerized by autoxidation treatments. The resulting oligomeric tannins were responsible for the methanogenic toxicity observed in the autoxidized catechin solutions (Field, J. A., Kortekaas, S. & Lettinga, G. (1989). The tannin theory of methanogenic toxicity. Biol. Wastes 29(4) 241-62). In this study the autoxidation of pyrogallol (a hydrolyzable tanning monomer) did not cause extensive polymerization. Initially, some polymerization occurred, producing toxic intermediates that were later destroyed by a destructive type of oxidation caused by prolonged autoxidation treatments. The first intermediate formed, purpurogallin (a dimer), caused a high level of toxicity to both the methanogenic activity and to the anaerobic degradation of pyrogallol. Since purpurogallin is a highly toxic autoxidation product that lacks tannic features, the changes in methanogenic toxicity induced by the autoxidation of pyrogallol cannot be estimated by changes in the oligomeric tannin concentration. Regardless of the reactions that take place during the autoxidation of tannin monomeric derivatives, the initial reactions can potentially lead to colored products of increased methanogenic toxicity. These are later detoxified by prolonged autoxidation, either by polymerization to high MW compounds (with condensed tannin model compounds), or by destruction of the initially toxic intermediates to low MW compounds (with hydrolyzable tannin model compounds). © 1989.
• Field, J. A., Kortekaas, S., & Lettinga, G. (1989). The tannin theory of methanogenic toxicity. Biological Wastes, 29(4), 241-262.
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  Abstract: The tannin theory describes the effectiveness of tannic compounds as bacterial inhibitors based on their molecular weight (MW). The inhibition is postulated to result from the hydrogen-bonding of the tannins with bacterial proteins (i.e. enzymes). Oligomeric tannins are expected to be the most effective inhibitors. The oligomeric tannins form stronger hydrogen-bonds with proteins than do their monomers. The high-MW tannins are also reactive with proteins in the bulk solution; however, they are not able to penetrate to bacterial proteins. In this study, tannins were polymerized by autoxidation to colored compounds in order to investigate the role of tannin MW on methanogenic toxicity. The autoxidation of catechin, a tannin monomer, paralleled the expected toxicity as predicted by the tannin theory. The toxicity increased as it was polymerized to oligomeric tannins and decreased as the oligomers were converted to non-toxic high-MW tannins. The autoxidation of green-tea tannins, oligomeric tannins, did not lead to extensive polymerization reactions and no accumulation of high MW tannins was observed. Instead, the toxic green-tea tannins were transformed to colored non-tannic compounds of variable MW, which were non-toxic.
• Temmink, J. H., Field, J. A., Haastrecht, J. V., & Merkelbach, R. C. (1989). Acute and sub-acute toxicity of bark tannins in carp (Cyprinus carpio L.). Water Research, 23(3), 341-344.
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  Abstract: The 24-h LC(I)50 and 96-h LC(I)50 of bark extracts for carp (Cyprinus carpio L.) were estimated at approx. 65 and 50 mg/l total COD, respectively, in a semi-static system. Tannins, constituting 50-60% of total COD, are responsible for this toxicity. Considerable toxicity was also found in sub-acute studies in a flow-through system. In both systems oxidative polymerization of the tannins into high-molecular-weight polymers abolished in aquatic toxicity almost completely. Epithelial damage of the gill was the most important lesion of toxicity, supporting the hypothesis that the toxic action of oligomeric bark tannins is through binding to and cross-linking of cell membrane enzymes.
• Field, J. A., & Lettinga, G. (1987). The methanogenic toxicity and anaerobic degradability of a hydrolyzable tannin. Water Research, 21(3), 367-374.
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  Abstract: Gallotannic acid was found to be highly toxic to methanogenic activity. Concentrations, representing 50% inhibition, approximated 700 mg l** minus **1. The toxicity was persistent despite the rapid degradation of gallotannic acid to volatile fatty acids and methane. A 72. 5% loss of sludge activity was associated with a 1 day exposure of methanogenic granular sludge to 1000 mg l** minus **1 gallotannic acid. The toxicity of gallotannic acid was persistent over 2 month assay periods. The monomeric derivatives of gallotannic acid, gallic acid and pyrogallol were much less toxic. The 50% inhibition concentration of the monomers approximated 3000 mg l** minus **1 and their toxicities were not persistent. No activity losses were evident after sludge was exposed to 3000 mg l** minus **1 gallic acid for 19 days. Additional study results are discussed.
• Field, J. A., Lettinga, G., & Geurts, M. (1987). The methanogenic toxicity and anaerobic degradability of potato starch wastewater phenolic amino acids. Biological Wastes, 21(1), 37-54.
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  Abstract: Potatoes, which are important agricultural feedstocks for the starch industry, contain tyrosine and phenol oxidase. Since l-dopa can be formed from tyrosine by phenol oxidase, both tyrosine and l-dopa are presumably present in potato starch wastewaters. The purpose of this study was to evaluate the methanogenic toxicity and anaerobic degradability of these two phenolic amino acids. Tyrosine was found to be negligibly toxic to methane bacteria, while l-dopa caused from 40% to 50% inhibitions of the methanogenic activity at a concentration of 327 mg liter-1. The toxicity of l-dopa occurred only if anaerobic sludge was exposed to l-dopa in the presence of Volatile Fatty Acids (VFA). The l-dopa toxicity could be minimized by maintaining low VFA concentrations in the media and by adapting the sludge to VFA prior to l-dopa exposure. Both tyrosine and l-dopa were anaerobically degraded to C̀H4; however, only tyrosine was degradable after prolonged operation of continuously VFA-fed, granular sludge, packed columns. Phenol and p-cresol were identified as phenolic intermediates of anaerobic tyrosine degradation. Both p-cresol and m-cresol were identified as phenolic intermediates of anaerobic l-dopa degradation. © 1987.
• Brons, H. J., Field, J. A., Lexmond, W. A., & Lettinga, G. (1985). Influence of humic acids on the hydrolysis of potato protein during anaerobic digestion. Agricultural Wastes, 13(2), 105-114.
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  Abstract: Some evidence that humic compounds are capable of inhibiting enzymatic activity is reported in the literature. The influence of sodium humate on the hydrolysis of a non-soluble potato protein during anaerobic digestion was investigated. The addition of 250 and 1000 mg of humate per litre caused an inhibition of the protein hydrolysis. The inhibition could be characterized mostly as an extended lag-phase of hydrolysis (extended 8 days by 1000 mg of humate per litre). The onset of acidification and methanogenesis was dependent on the release of substrate by hydrolysis. No inhibition was observed in the anaerobic digestion of a mixture of the six most abundant amino acids in potato protein. This indicated that only the hydrolytic stage of anaerobic protein digestion was inhibited by humate. © 1985.
• Field, J. A., Reneau Jr., R. B., & Kroontje, W. (1985). Effects of anaerobically digested poultry manure on soil phosphorus adsorption and extractability. Journal of Environmental Quality, 14(1), 105-107.
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  Abstract: Research in the past has indicated that large additions of animal manures to soil can reduce soil P adsorption and increase P extractability. However, application rates used in many studies are unrealistic with respect to crop fertilization and land disposal due to the excessive quantities of N applied with the manures. The objective of this study was to evaluate effluent from a poultry manure anaerobic digester applied to a Hayesville loam soil (clayey, oxic, mesic, Typic Hapludult) on soil P adsorption and extractability after three incubation periods (0, 30, and 90 d). Effluent solids were applied at rates corresponding to 753 through 7530 mg N kg-1 soil. Langmuir equation parameters (from least square fitted data; R2 > 0.98) were used as a basis to compare the influence of treatments on P adsorption properties of the soil. The P adsorption maxima parameter was not influenced by any of the effluent treatments. The parameter related to P bonding energy of the soil was reduced 33 and 41%, respectively, by mid and high rate effluent applications. A similar reduction was not observed for the low rate treatment, which corresponded to fertilizer application of 100 mg N kg-1 soil. Extractable soil P increased in a predictable manner with increased effluent solids application for all three incubation periods. These increases in extractable P could be described by linear regression equations. The slopes of these lines indicate that a 1-g addition of effluent solids to 1-kg soil-accounts for increases in extractable P of 3 to 6 mg kg-1.
• Field, J. A., Reneau Jr., R. B., Kroontje, W., & Caldwell, J. S. (1985). Nutrient recoveries from plug-flow anaerobic digestion of poultry manure. Agricultural Wastes, 13(3), 207-216.
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  Abstract: Anaerobic digestion provides for the production of a biogas fuel while concurrently conserving the nutrient value of manures used. The nutrients placed in anaerobic digesters are, however, subject to possible transformations. The objectives of this study were to evaluate changes in the N composition following plug-flow digestion of poultry manure, and to determine the distribution of N, K, P, Ca and Mg between the outflowing effluent and the settled manure sludge which remained in the digester. Total Kjeldhal N (TKN) was completely recovered in the pilot-scale digestion operated with 6% influent TS, 35°C and 30 day retention time. Mineralization of poultry manure organic N by the digestion process was 53%. Consequently, NH4+-N in the digested manure accounted for the large majority (79·6%) of the TKN. The effluent accounted for the majority of the N and K present in the digested manure (91·2 and 95·9%, respectively). The settled manure sludge, accounted for 26·3% of the recovered TS and contained approximately 30% of the P, Ca and Mg. © 1985.
• Field, J. A., Caldwell, J. S., Jeyanayagam, S., Reneau Jr., R. B., Kroontje, W., & Collins Jr., E. R. (1984). FERTILIZER RECOVERY FROM ANAEROBIC DIGESTERS.. Transactions of the American Society of Agricultural Engineers, 27(6), 1871-1876, 1881.
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  Abstract: Agricultural wastes can be anaerobically digested to produce methane. Fertilizer nutrients in the wastes are recovered after the digestion process. The objectives of this study were to evaluate nutrient recovery, extractability and distribution between supernatant and sludge fractions.
• Field, J. A., Parker, J. C., & Powell, N. L. (1984). Comparison of field- and laboratory-measured and predicted hydraulic properties of a soil with macropores.. Soil Science, 138(6), 385-396.
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  Abstract: Soil hydraulic properties were determined in situ by the instantaneous profile method on three 3-m square plots of Dothan soil. Simultaneous measurements of matric tension and moisture content with depth during drainage were made with tensiometers and a field-calibrated neutron probe. Unsaturated conductivity values calculated form the in situ data ranged within an order of magnitude for corresponding horizons in the three plots.-from Authors

Proceedings Publications

• Field, J. A., Ramos Ruiz, A., Simon Pascual, A., Zhu, K., & Sierra Alvarez, R. (2017, May). Recovery of Critical Metal and Metalloid Resources by Anaerobic Processes. In The 14th International Water Association, Leading Edge Conference on Water and Wastewater Technologies. Innovative technology solutions to address challenges at the water-energy-food interface. May 29th – June 2nd . 2017. Florianópolis, Brazil [KEYNOTE]..
• Field, J. A., Rodriguez-Freire, L. .., & Sierra-Alvarez, R. .. (2017, June 2017). Biorremediacion anaerobia de aguas contaminadas con arsenico. In Simposio Internacional: Ri­o Tinto, aspectos fundamentales y aplicados de un analogo terrestre de Marte. 6th and 7th June 2017 Madrid, Spain [INVITED TALK]..
• Field, J. A., Sierra-Alvarez, R. .., & Ayala, P. (2017, May 2017). Novel electron donors for bioremediation of acid rock drainage.. In Water Reuse Monitoring and Treatment Technologies. May 17th – May 19th, Recife, Brazil [ORAL]..
• Field, J. A., Simon-Pascual, A. .., Zhu, K., & Sierra-Alvarez, R. .. (2017, October 2017). Recovery of platinum group metals (PGM) utilizing anaerobic microbial consortia.. In 15th IWA World Conference on Anaerobic Digestion. October 17th - 20th, 2017, Beijing, China [ORAL]..
• Madeira, C. .., Chorover, J., Sierra-Alvarez, R. .., & Field, J. A. (2017, May 2017). Biodegradation of the emerging insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) by soil microorganisms. In Platform presentation at Battelles Fourth International Symposium on Bioremediation and Sustainable Environmental Technologies. May 22nd – May 25th, 2017. Miami, Florida [ORAL]..

Presentations

• Field, J. A. (2020, December). DNAN & NTO: A Tale of Three Fates.. SERDP ESTCP Symposium 2020 Enhancing DoD’s mission Effectiveness. November 30 - December 4, 2020 . Technical SessionRange Resiliency: Fate, Transport & Mitigation of Legacy & Insensitive High Explosive Constituents Technical Session. Virtual: SERDP & ESTCP.
• Field, J. A. (2019, Sep 2019). Limits of Anaerobic Biodegradability. [Invited Lecture]. Anaerobic Treatment of High-Strength Industrial Wastes 2019 Marquette University Sept. 10-11, 2019 Milwaukee, WI USA. Presentation.
• Field, J. A. (2019, Sep 2019). Methanogenic Granular Sludge for Priority Inorganic Pollutants [Invited talk]. Anaerobic Treatment of High-Strength Industrial Wastes 2019 Marquette University Sept. 10-11, 2019 Milwaukee, WI USA. N/A.
• Field, J. A., Jog, K., Sierra-Alvarez, R. .., Nguyen, C., & Vanover, E. (2019, May 2019). Biodegradability and Microbial Toxicity of Azoles, the Forgotten Class of Emerging Contaminants, [Oral presentation], Association of Environmental Engineering and Science Professors (AEESP) Research and Education Conference; 2019 May 14-16; Tempe, AZ.. N/A.
• Field, J. A., Sierra-Alvarez, R. .., Jog, K., Nguyen, C. H., Vanover, E., & Li, J. A. (2019, Jun 2019). Biodegradability and Toxicity of Ubiquitous Azoles to Anaerobic and Post-Treatment Processes [Oral Poster Pitch]. Anaerobic Digestion Conference AD16, Accelerating natural cycles Delft University of Technology, 23-27 June 2019, Delft, The Netherlands. N/A.
• Kadoya, W. M., Sierra-Alvarez, R. .., Jagadish, B., Wong, S., Abrell, L., Mash, E. A., & Field, J. A. (2019, May 2019). Mechanism of Covalent Incorporation of Nitroaromatic Contaminants into Natural Organic Matter. [Oral presentation] Association of Environmental Engineering and Science Professors (AEESP) Research and Education Conference; 2019 May 14-16; Tempe, AZ.. N/A.
• Kalyani, V. J., Vanover, E., Chi, H. N., He, C., Field, J. A., & Sierra-Alvarez, R. (2019, Nov 2019). Mechanisms of Nitrification Inhibition by Azoles: A Framework to Promote Azole Detoxification, Biodegradation and Green Chemistry [Oral Presentation] Semiconductor Research Corporation (SRC) Environment, Safety and Health (ESH) Review, Tucson, AZ, Nov 05, 2019.. N/A.
• Lakhey, N., Sierra-Alvarez, R. .., & Field, J. A. (2019, Nov 2019). The interactions of azole compounds with an anammox enrichment culture: toxicity and biotransformation [Oral Presentation] SETAC North America 40th annual meeting, November, 3-7, 2019, Toronto, Ontario, Canada.. N/A.
• Madeira, C. L., Kadoya, W. M., Li, G., Wong, S., Sierra-Alvarez, R. .., & Field, J. A. (2019, May 2019). Biological Pretreatment to Enhance In Situ Chemical Oxidation of Insensitive Munitions Compounds. [Oral presentation] Association of Environmental Engineering and Science Professors (AEESP) Research and Education Conference; 2019 May 14-16; Tempe, AZ.. N/A.
• Niu, X. Z., Abrell, L., Sierra-Alvarez, R. .., Field, J. A., & Chorover, J. (2019, Nov 2019). Environmental, Safety & Health Properties of "Onium" Photoacid Generators and their Photodegradation Products. [Oral Presentation] Semiconductor Research Corporation (SRC) Environment, Safety and Health (ESH) Review, Tucson, AZ, Nov 05, 2019.. N/A.
• Olshansky, Y., Chorover, J., Abrell, L., Field, J. A., Gomeniuc, A., Hatton, J., & Sierra-Alvarez, R. .. (2019, March 2019). Sorption of PFAS by Cationic Hydrophobic Polymers [Oral Presentation] 2019 spring American Chemical Society National Meeting. March 31 - April 4, 2019. Orlando, FL.. N/A.
• Ayala-Parra, P. .., Field, J. A., & Sierra-Alvarez., R. 2. (2018, Fall). Nutrient recovery and biogas generation from the anaerobic digestion of waste biomass from algal biofuel production.. Sixth Int. Symp. On Environmental Biotechnology and Engineering. Ciudad Obregon, Sonora, Mexico. Nov. 5-9. [oral presentation].
• Field, J. A., & Sierra-Alvarez, R. .. (2018, Spring). Methanogenic granular sludge for the bioremediation and biorecovery of priority inorganic pollutants.. IWA Biofilms: Granular Sludge Conference , March 18-21, 2018, Delft, The Netherlands [oral talk & won best speaker award].
• Sierra Alvarez, M. R., Field, J. A., Nguyne, C. H., Zeng, C., Borrero, N., Sherwood, C. L., McCorkel, M., & Boitano, S. A. (2018, Spring). Nanoparticle toxicity: cytotoxic and sub-cytotoxic measurements using xCELLigence real time cell analysis. 4th Conference on Impedance-Based Cellular Assays, 6th-8th June 2018 (Oral presentation). Edinburgh, UK.
• Sierra-Alvarez, R. .., Olshansky, Y., Field, J. A., Abrell, L., Hatton, J., Gomeniuc, A., & Chorover, J. (2018, Fall). ER18-1052: Remediation of Per- and Polyfluoroalkyl Contaminated Groundwater Using Cationic Hydrophobic Polymers as Ultra-High Affinity Sorbents. SERDP/ESTCP Environmental Technology Technical Symposium & Workshop. Nov. 27-30, 2018. Washington DC. [Oral Presentation].
• Field, J. A., Li, G., & Sierra-Alvarez, R. .. (2016, December). Anammox for nutrient nitrogen removal from sludge liquors.. The UA WEST Center. Annual Conference and Industrial Advisory Board Meeting Dec 12 & 13, 2016, Tucson, AZ. [Invited Talk].
• Field, J. A., Madeira, C. L., Kadoya, W., Speet, S., Sierra-Alvarez, R., Abrell, L., Chorover, J., & Krzmarzick, M. (2016, June 2016). Biodegradation of insensitive munitions compound, 3-nitro-1,2,4-triazol-5-one (NTO), to mineral products via 3-amino-1,2,4-triazol-5-one (ATO) as the central intermediate.. SERDP ESTCP Webinar # 34. Insensitive munitions. Environmental Health Criteria, Fate and Transport, June 2nd, 2016 [Invited Talk]..
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  The Strategic Environmental Research and Development Program (SERDP) and the Environmental Security Technology Certification Program (ESTCP) have launched a webinar series to promote the transfer of innovative, cost-effective and sustainable solutions developed through projects funded by these programs. The webinar series targets Department of Defense and Department of Energy practitioners, the regulatory community and environmental researchers with the goal of providing cutting edge and practical information that is easily accessible at no cost.
• Field, J. A., Madeira, C., Olivares, C., Abrell, L., Krzmarzick, M., Chorover, J., & Sierra-Alvarez, R. (2016, August). Biotransformation and biodegradation of insensitive munitions compounds in soil. Environmental Chemistry Session, Microbial and Molecular Tools to Determine the Fate and Biotransformation of Emerging Contaminants.. 252nd American Chemical Society National Meeting , August 21-25, 2016, Philadelphia, PA [Keynote talk].
• Field, J. A., Rodriguez-Freire, L. .., & Sierra-Alvarez, R. (2016, April). Arsenic remediation by formation of arsenic sulfide minerals in a continuous anaerobic bioreactor.. Department of Metallurgical and Materials Engineering and the Department of Sanitation and Environmental Engineering, Universidade Federal Minas Gerais, , Belo Horizonte, Brazil April 7th, 2016 [Invited Talk].
• Field, J. A., Sierra-Alvarez, R. .., & Ayala, P. (2016, March). Eliminacion de Metales Pesados y Azufre con Bacterias Sulfato-Reductoras en una Barrera Permeable Reactivo Relleno con Hierro Metálico.. Developing a Pan American Hub for Environmentally and Socially Compatible Mining. Pontificia Universidad Catolica de Peru (PUCP) March 7-9, 2016, Lima, Peru [Invited talk]..
• Li, G., Carvajal-Arroyo, J. M., Sierra-Alvarez, R., & Field, J. A. (2016, July). Mechanisms and control of nitrite inhibition of anaerobic ammonium oxidation (anammox).. Water Environmental Federation/International Water Association, Nutrient Removal and Recovery Conference. Denver, CO, USA, July 10-13, 2016 [Oral Presentation].
• Olivares, C. I., Field, J. A., Sierra-Alvarez, R., Krzmarzick, M., Madeira, C. L., & Chorover, J. (2016, July). Invited Talk: Biotransformation pathways dictating the fate in soil of insensitive munition compounds, 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazole-5-one (NTO).. Session on the Bioremediation of Explosive-Contaminated Soil and Water at the Society of Industrial Microbiology and Biotechnology Annual Meeting in New Orleans, LA July 24-28, 2016. [Invited Talk].

Poster Presentations

• Field, J. A. (2021, November). Complete Biodegradation of Insensitive High Explosive Compounds. .. November 29 – December 3, 2021. Menezes, O.; Rios-Valenciana, E. E.; Krzmarzick, M. j.; Spain, J. C.; Konstantinidis, K. T.; Sierra-Alvarez, R. , Jim A. Field: SERDP and ESTCP Symposium..
• Field, J. A. (2021, November). High Rate Degradation of 3-Nitro-1,2,4-triazol-5-one (NTO) to Environmentally Benign End Products in Sequential Reducing-Oxidizing Reactive Mineral Packed Bed Reactor,. SERDP & ESTCP Symposium, Nov 30th, 2021. High Rate Degradation of 3-Nitro-1,2,4-triazol-5-one (NTO) to Environmentally Benign End Products in Sequential Reactorducing-Oxidizing Reactive Mineral Packed Bed Reactor,: Youngjae Yu, Mitchell Miller, Osmar Menezes, Rob Root, Jon Chorover, Reyes Sierra-Alvarez, and Jim A. Field.
• Lakhey, N., Swartzendruber, D., Ding, H., Sierra-Alvarez, R. .., & Field, J. A. (2019, May 2019). Effects of common wastewater constituents and environmental conditions on Anammox bacterial growth kinetics [Poster Presentation], 2019 AEESP Research and Education Conference, May 14-16; 2019, Tempe, AZ, USA.. N/A.
• Madeira, C. L., Menezes, O., Kalyani, V. J., Brooks, M., Park, D., Konstantinidis, K., Krzmarzick, M. J., Spain, J. C., Sierra-Alvarez, R., & Field, J. A. (2019, Dec 2019). Complete Biodegradation of Insensitive High Explosive Compounds. [Poster]. 2019 SERDP and ESTCP Symposium, Dec 3 - Dec 5, 2019, Washington DC.. N/A.
• Yu, Y., Castrejon, M. R., Madeira, C. L., Sierra-Alvarez, R., Field, J. A., Chorover, J., & Root, R. (2019, Dec 2019). High rate degradation of 3-Nitro-1,2,4-triazol-5-one (NTO) to environmentally benign end products in sequential reducing-oxidizing reactive mineral packed bed reactor [Poster]. 2019 SERDP and ESTCP Symposium, Dec 3 - Dec 5, 2019, Washington DC.. N/A.
• Abrell, L., Kadoya, W. M., Madeira, C. L., Sierra-Alvarez, R. .., Wong, S., Mash Jr, ,. E., Field, J. A., & ., . (2018, Fall). Nitroaromatic pollutants environmental fate characterized by HRAM; Understanding anaerobic formation of azo coupling products.. 66th Am. Soc. For Mass Spectrometry (ASMS) Conference on Mass Spectrometry and Allied Topics. June 2-3, San Diego, CA. [poster].
• Field, J. A., Dontsova, K. M., Taylor, S., Beal, S., & Kadoya, W. (2018, November). Phototransformation Kinetics and Products of Aqueous 2,4-Dinitroanisole (DNAN).. 2018 SERDP & ESTCP Symposium. Washington, DC: Strategic Environmental Research and Development Program (SERDP).
• Jog, K., Speed, D., Li, G., Nguyen, C. H., Field, J. A., & Sierra-Alvarez, R. .. (2018, Fall). Azoles as emerging contaminants and their role in nitrification inhibition.. Semiconductor Environmental Safety & Health Conference (SESHA)/ Semiconductor Industry Association (SIA) International High Technology ESH Symposium and Exhibition, April 16-20, Scottsdale, Arizona. [Poster, 3rd prize].
• Kadoya, W. M., Sierra, R., Jagadish, B., Wong, S., Abrell, L., Mash, E. A., & Field, J. A. (2018, Fall). Anaerobic Adduct Formation between Reduced Intermediates of 2,4-Dinitroanisole (DNAN) and Humic Model Compounds.. Symposium of the Strategic Environmental Research and Development Program (SERDP) and Environmental Security Technology Certification Program (ESTCP); November 27-29; Washington, DC [poster].
• Madeira, C. L., Jog, K. V., Vanover, E. T., Brooks, M. D., Taylor, D. K., Sierra-Alvarez, R., Spain, J. C., Krzmarzick, M. J., & Field, J. A. (2018, Fall). Enrichment culture that biodegrades the reduced metabolite of the insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) to inorganic products.. SERDP/ESTCP Environmental Technology Technical Symposium & Workshop. Nov. 27-30, 2018. Washington DC. [poster].
• Madeira, C. L., Kadoya, W. M., Li, G., Wong, S., Sierra-Alvarez, R. .., & Field, J. (2018, Fall). Reductive Biological Pretreatment to Enhance In Situ Chemical Oxidation of Insensitive Munitions Compounds.. SERDP/ESTCP Environmental Technology Technical Symposium & Workshop. Nov. 27-30, Washington DC. [poster].
• Nguyen, C. H., Zeng, C., Boitano, S., Shadman, F., Field, J. A., & Sierra-Alvarez, R. .. (2018, Fall). Cytotoxicity of gallium arsenide (GaAs) and indium arsenide (InAs) nanoparticles to human lung epithelial cells.. Semiconductor Environmental Safety & Health Conference (SESHA)/ Semiconductor Industry Association (SIA) International High Technology ESH Symposium and Exhibition, April 16-20, Scottsdale, Arizona. [Poster award, 1st prize].
• Vanover, E., Madeira, C. L., Speet, S. A., Abrell, A., Chorover, J., Sierra-Alvarez, R. .., & Field, J. A. (2018, Fall). Remediation of Insensitive Munitions Compound 3-nitro-1,2,4-triazol-5-one (NTO).. 2018 Student Western Alliance to Expand Student Opportunity (WAESO) Conference. March 2nd. Tempe, AZ. [poster, 3rd prize].
• Abrell, L., Olivares, C. I., Chorover, J., & Sierra-Alvarez, R. .. (2016, June). Field, J. A. New 2,4-dinitroanisole (DNAN; munitions chemical) (bio)transformation products discovered and bioassayed using high resolution UPLC-QToFMS.. 64th ASMS Conference on Mass Spectrometry, San Antonio, TX, USA, Jun 5-9, 2016 [Poster].
• Madeira, C. L., Speet, S. A., Abrell, L., Chorover, J., Sierra-Alvarez, R. .., & Field, J. A. (2016, November). Biodegradation of the Insensitive Munitions Compound 3-nitro-1,2,4-triazol-5-one (NTO).. 7th SETAC World Congress/37th SETAC North America Annual Meeting, Nov. 6-10, 2016. Orlando, FL [Poster].

Others

• Dontsova, K. M., Field, J. A., & Johnson, M. (2016, June 2016). SERDP ESTCP Webinar # 34. Insensitive munitions. SERDP ESTCP webinar series. https://www.serdp-estcp.org/Tools-and-Training/Webinar-Series/06-02-2016
  More info

  The Strategic Environmental Research and Development Program (SERDP) and the Environmental Security Technology Certification Program (ESTCP) have launched a webinar series to promote the transfer of innovative, cost-effective and sustainable solutions developed through projects funded by these programs. The webinar series targets Department of Defense and Department of Energy practitioners, the regulatory community and environmental researchers with the goal of providing cutting edge and practical information that is easily accessible at no cost.