Ian L Pepper

Interests

Research

Environmental microbiologist working at the interface of human health and soils, potable water and municipal wastes: water quality and "smart water distribution system;" fate and transport of microbial pathogens in soil, air, and water; land application of biosolids

Courses

Scholarly Contributions

Books

• Pepper, I. L., Brusseau, M. L., & Gerba, C. P. (2019). Environmental and Pollution Science 3rd Edition. Academic Press.
• Pepper, I., Gerba, C., & Gentry, T. (2012). Environmental Microbiology, 3rd Edition.

Chapters

• Ramirez, M., Brusseau, M. L., Pepper, I. L., & Maximillian, J. (2019). Chapter 26 Environmental Human Health. In Environmental and Pollution Science 3rd Edition. Elsevier.
• Brooks, J. P., Gerba, C. P., & Pepper, I. L. (2014). Land Application of Organic Residuals: Municipal Biosolids and Animal Manures. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Gentry, T. J., Pepper, I. L., & Pierson III, L. (2014). Microbial Diversity and Interactions in Natural Ecosystems. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Gentry, T. J., Pepper, I. L., & Roane, T. (2014). Microorganisms and Metal Pollutants. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Gerba, C. P., & Pepper, I. L. (2014). Domestic and Indoor Microbiology. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Gerba, C. P., & Pepper, I. L. (2014). Drinking Water Treatment and Distribution. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Gerba, C. P., & Pepper, I. L. (2014). Municipal Wastewater Treatment. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Gerbaq, C. P., Pepper, I. L., & Newby, D. (2014). Microbial Transport in the Subsurface. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Maier, R. M., Pepper, I. L., Maier, R. M., & Pepper, I. L. (2014). Bacterial Growth. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Pepper, I. L., & Gentry, T. J. (2014). Earth Environments. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Pepper, I. L., & Gentry, T. J. (2014). Microorganisms Found in the Environment. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Pepper, I. L., & Gerba, C. P. (2014). Aeromicrobiology. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Pepper, I. L., & Gerba, C. P. (2014). Cultural Methods. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Pepper, I. L., & Gerba, C. P. (2014). Environmental Sample Collection and Processing. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Pepper, I. L., Gerba, C. P., & Gentry, T. J. (2014). Global Emerging Microbial Issues in the Anthropocene Era. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Pepper, I. L., Gerba, C. P., & Gentry, T. J. (2014). Introduction to Environmental Microbiology. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Pierson III, L., Maier, R. M., & Pepper, I. L. (2014). Microbial Communication: Bacteria-Bacteria and Bacteria-Host. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Roane, T., & Pepper, I. L. (2014). Microscopic Techniques. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Rock, C., Gerba, C. P., & Pepper, I. L. (2014). Recycled Water Treatment and Distribution. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.
• Yu, H., Halonem, M., & Pepper, I. L. (2014). Immunological Methods. In Environmental Microbiology, 3rd Ed.. Elsevier/Academic Press.

Journals/Publications

• Pepper, I. L. (2021). Influence of demographic factors and stages of the COVID-19 Pandemic on SARS-CoV-2 fecal shedding rates in wastewater. Science of the Total Environment.
• Pepper, I. L. (2021). Pilot scale investigation of the effects of a magnetic water treatment device in pool systems on chlorine demand. Journal of Water Process Engineering.
• Betancourt, W. Q., Schmitz, B. W., Innes, G. K., Prasek, S. M., Pogreba Brown, K. M., Stark, E. R., Foster, A. R., Sprissler, R. S., Harris, D. T., Sherchan, S. P., Gerba, C. P., & Pepper, I. L. (2021). COVID-19 containment on a college campus via wastewater-based epidemiology, targeted clinical testing and an intervention. Science of the Total Environment.
• Gerba, C. P., Pepper, I. L., Lopez, G. U., Quintanar, D. R., Betancourt, W. Q., & Morrison, C. M. (2019). Potential Indicators of Virus Transport and Removal during Soil Aquifer Treatment of Treated Wastewater Effluent. International Water Association.
• Pepper, I. L. (2021). Enumerating asymptomatic Covid cases and estimating SARS-CoV-2 fecal shedding rates via wastewater based epidemiology. Science of the Total Environment, 801(149794).
• Pepper, I. L., Brusseau, M. L., & Prevatt, F. J. (2021). Incidence of PFAS in soil following long-term application of Class B biosolids: A Southern Arizona Case Study. Science of the Total Environment.
• Schmitz, B. W., Innes, G. K., Xue, J., Gerba, C. P., Pepper, I. L., & Sherchan, S. (2020). Reduction of erythromycin resistance gene erm (F) and Class 1 integran-intergrase genes in wastewater by Bardenpho treatment. Water Environment Research. doi:10.1002/wer.1299
• Bright, K. R., Pepper, I. L., Gerba, C. P., Castro-Del Campo, N., & Abd-Elmaksoud, S. (2019). Comparative Assessment of BGM and PLC/PRF/5 Cell Lines for Enteric Virus Detection in Biosolids. FOOD AND ENVIRONMENTAL VIROLOGY, 11(1), 32-39. doi:https://doi.org/10.1007/s12560-019-09366-4
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  The buffalo green monkey (BGM) cell line is required for the detection of enteric viruses in biosolids through a total culturable viral assay (TCVA) by the United States Environmental Protection Agency. In the present study, BGM and PLC/PRF/5 cell lines were evaluated for TCVA and for their use in determining the incidence of adenoviruses and enteroviruses in raw sludge and Class B biosolids. Six raw sludge and 17 Class B biosolid samples were collected from 13 wastewater treatment plants from seven U.S. states. Samples were processed via organic flocculation and concentrate volumes equivalent to 4 g total solids were assayed on BGM and PLC/PRF/5 cells. Cell monolayers were observed for cytopathic effect (CPE) after two 14-days passages. Cell lysates were tested for the presence of adenoviruses and enteroviruses by PCR or RT-PCR. The PLC/PRF/5 cells detected more culturable viruses than the BGM cells by CPE (73.9% vs. 56.5%, respectively). 52% of the samples were positive for CPE using both cell lines. No viruses were detected in either cell line by PCR in flasks in which CPE was not observed. No adenoviruses were detected in 13 CPE-positive samples from BGM lysates. In contrast, of the 17 samples exhibiting CPE on PLC/PRF/5 cells, 14 were positive for adenoviruses (82.4%). In conclusion, PLC/PRF/5 cells were superior for the detection of adenoviruses in both raw sludge and Class B biosolids. Thus, the use of BGM cells alone for TCVA may underestimate the viral concentration in sludge/biosolid samples.
• Lamori, J. G., Xue, J., Taruna, A., Lopez, G. U., Kitajima, M., Gerba, C. P., Pepper, I. L., & Sherchan, S. (2019). Removal of fecal indicator bacteria and antibiotic resistant genes in constructed wetlands. Environmental Science and Pollution Research.
• Pepper, I. L. (2019). Assessing the spatial and temporal variability of bacterial communities in two Bardenpho wasterwater treatment systems via Illumini MiSeq sequencing. Sci Tot Environ, 657, 1543-4552.
• Pepper, I. L. (2019). Comparative Assessment of BGM and PLC/PRF/5 cell lines for enteric virus detection in biosolids. Food & Environ Virol., 11, 32-39.
• Lamori, J. G., Xue, J., Taruna, A., Lopez, G. U., Kitajima, M., Gerba, C. P., Pepper, I. L., & Sherchan, S. (2018). Removal of fecal indicator bacteria and antibiotic resistant genes in constructed wetlands. Journal of Environmental Management.
• Pepper, I. L. (2018). Comparative survival of viruses during thermophilic and mesophilic anaerobic digestion. Science of the Total Environment, 615, 15-19.
• Pepper, I. L., & Gerba, C. P. (2016). Evaluating the Efficacy of Ebola Human Waste Management Practices for Disposal in Toilets. Journal of Residual Science & Technology.
• Pepper, I. L., & Gerba, C. P. (2018). Risk of infection from legionella associated with spray irrigation of reclaimed water. Water Research, 139, 101 107.
• Pepper, I. L., Brooks, J. P., & Gerba, C. P. (2018). Antibiotic Resistant Bacteria in Municipal Wastes: Is there reason for concern?. Environmental Science and Technology, In Review.
• Pepper, I. L., Brooks, J. P., Tewolde, H., Adeli, A., Shankle, M. W., Way, T. R., & Smith, R. K. (2018). Effects ofsubsurface banding and broadcst of poultry litter and cover crop on soil microbial populations. Journal of Environmental Quality, 47, 427-435.
• Pepper, I. L., Miles, S., Ikner, L., Snyder, S., Yu, H., & Sherchan, S. (2018). Near real-time detection of E.coli in reclaimed water. Sensors, 18(7), 2303.
• Pepper, I. L., Morizama, H., Haramota, E., Kitajima, M., Sherchan, S., & Gerba, C. P. (2018). reduction of Crypyosporidium, giardia, and fecal indicators by Bardenpho wastewater treatment. Environmental Science and Technology, 52, 7015 - 7023.
• Pepper, I. L., Sassi, H. P., Reynolds, K. P., & Gerba, C. P. (2018). Evaluation of hospital -grade disinfectants on viral deposition on surfaces after toilet flushing. American Journal Infection Control, 46, 507 - 511.
• Pepper, I. L., Yu, H. -., Anumol, T., Park, M., Scheideler, J., & Snyder, S. (2018). Online sensor monitoring for chemical contaminant attenuation during UV/H2O2 advanced oxidation process. Water Research, 81(2o15), 250- 260.
• Sassi, H. P., Ikner, L. A., Maksoud, S. A., Gerba, C. P., & Pepper, I. L. (2018). Comparative Survival of Viruses during Thermophillic and Mesophillic Anaerobic Digestion. Sci Tot Environ, 615, 15-19.
• Rachmadi, A. T., Kitajima, M., Pepper, I. L., & Gerba, C. (2016). Enteric and indicator virus removal by surface flow wetlands. Sci Tot Environ, 542, 976-982.
• Schmitz, B., Kitajima, M., Campillo, M., Gerba, C., & Pepper, I. L. (2016). Virus reduction during advanced Bardenpho and conventional wastewater treatment processes. Environmental Science & Technology, 50, 9524-9532.
• Schmitz, B., Pearce-Walker, J., Gerba, C., & Pepper, I. L. (2016). A method for determining Ascaris viability based on early-to-late stage in vitro ova development. Journal Residual Science & Technology, 13, 275-286.
• Betancourt, W. Q., Kitajima, M. K., Wing, A. D., & Pepper, I. L. (2014). Assessment of virus removal by managed aquifer recharge at three full-scale operations. J Env Sci Hlth, Part A, 49(14), 1685-1692.
• Kitajima, M., Iker, B. C., Pepper, I. L., & Gerba, C. P. (2014). Relative Abundance and Treatment Reduction of Viruses During Wastewater Treatment Processes - Identification of Potential Viral Indicators. Science of the Total Environment, 488-489, 290-296.
• Sherchan, S., Kitajima, M., Gerba, C. P., & Pepper, I. L. (2014). Rapid Detection Technologies for Monitoring MIcroorganisms in Water. Biosensors J, 3, 109.
• Sherchan, S., Snyder, S. S., Gerba, C. P., & Pepper, I. L. (2014). Inactivation of MS2 Coliphage by UV and Hydrogen Peroxide: Comparison by Cultural and Molecular Methodologies. J Env Sci Hlth, Part A, 49, 397-403.
• Sherchan, S., Snyder, S., Gerba, C., & Pepper, I. (2014). Online monitoring of Escherichia coli and Bacillus thuringiensis spore inactivation after advanced oxidation treatment. J Env Sci Hlth, Part A, 49(8), 933-939.
• Ajibode, A. M., Ajibode, A. M., Rock, C. M., Rock, C. M., Bright, K., Bright, K., Mclain, J. E., Mclain, J. E., Gerba, C. P., Gerba, C. P., Pepper, I. L., Pepper, I. L., Ajibode, A. M., Rock, C. M., Bright, K., Mclain, J. E., Gerba, C. P., & Pepper, I. L. (2013). Influence of residence time of reclaimed water within distribution systems on water quality. Journal of Water Reuse and Desalination, 12.
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  The abstract for the publication can be found at the above URL. My participation in this manuscript was to serve as a co-PI to procure funding for the project and to aid in microbiological analyses of samples and data analysis.
• Ajibode, O. J., Rock, C., Bright, K., Mclain, J. E., Gerba, C. P., & Pepper, I. L. (2013). Influence of Residence Time of Reclaimed Water Within Distribution Systems on Water Quality. J Wat Reuse & Desal, 3, 185-196.
• Ajibode, O. M., Rock, C., Bright, K., E., J., Gerba, C. P., & Pepper, I. L. (2013). Influence of residence time of reclaimed water within distribution systems on water quality. Journal of Water Reuse and Desalination, 3(3), 185-196.
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  Abstract: The influence of residence time of reclaimed water within water distribution systems on microbial water quality was evaluated in two wastewater reclamation facilities in southern Arizona over a 15-month period. These utilities differed in age, geographic location, means of treatment, and disinfection (i.e. UV versus chlorine). At both facilities, samples were collected from the point of compliance (POC) directly after disinfection, and at discrete locations with increasing distance from the POC. Following entry into reclaimed water distribution systems, overall microbial water quality decreased rapidly due to microbial regrowth. However, following such regrowth, microbial concentrations remained relatively constant. Water-based opportunistic pathogens (Legionella, Mycobacterium, and Aeromonas) were frequently detected in both reclaimed water systems. In contrast, waterborne indicators such as Escherichia coli and Enterococcus were rarely detected, and only at low concentrations. These dates suggest the need for new indicators of water-based pathogens to be developed. Rechlorination in one of the distribution systems only reduced the concentration of bacteria temporarily due to rapid dissipation of chlorine, and subsequent regrowth of both water-based pathogens and indicators. Amoebic activity was detected in approximately one-third of all samples tested from both utilities, but was not correlated with either water-based pathogens or indicators. © IWA Publishing 2013.
• Iker, B. C., Bright, K. R., Pepper, I. L., Gerba, C. P., & Kitajima, M. (2013). Evaluation of Commercial Kits for the Extraction aqnd Purification of Viral Nucleic cids from Environmental and Fecal Samples. J. Virol. Meth., 141-145.
• Iker, B. C., Bright, K. R., Pepper, I. L., Gerba, C. P., & Kitajima, M. (2013). Evaluation of commercial kits for the extraction and purification of viral nucleic acids from environmental and fecal samples. Journal of Virological Methods, 191(1), 24-30.
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  PMID: 23578704;Abstract: The extraction and purification of nucleic acids is a critical step in the molecular detection of enteric viruses from environmental or fecal samples. In the present study, the performance of three commercially available kits was assessed: the MO BIO PowerViral Environmental DNA/RNA Isolation kit, the Qiagen QIAamp Viral RNA Mini kit, and the Zymo ZR Virus DNA/RNA Extraction kit. Viral particles of adenovirus 2 (AdV), murine norovirus (MNV), and poliovirus type 1 (PV1) were spiked in molecular grade water and three different types of sample matrices (i.e., biosolids, feces, and surface water concentrates), extracted with the kits, and the yields of the nucleic acids were determined by quantitative PCR (qPCR). The MO BIO kit performed the best with the biosolids, which were considered to contain the highest level of inhibitors and provided the most consistent detection of spiked virus from all of the samples. A qPCR inhibition test using an internal control plasmid DNA and a nucleic acid purity test using an absorbance at 230. nm for the nucleic acid extracts demonstrated that the MO BIO kit was able to remove qPCR inhibitors more effectively than the Qiagen and Zymo kits. These results suggest that the MO BIO kit is appropriate for the extraction and purification of viral nucleic acids from environmental and clinical samples that contain high levels of inhibitors. © 2013 Elsevier B.V.
• Pepper, I. L. (2013). Evaluation of Real-time Water Quality Sensors for the Detection of Intentional Spore Contamination of Potable Water. J. Biosensors & Bioelectronics, 4, 141-145.
• Pepper, I. L. (2013). Revegetation of Copper Mine Tailings through Land Application of Biosolids: Long-term Monitoring. Arid Land Res. Manag, 27, 1-12.
• Pepper, I. L. (2013). Survival of Infectious Prions During Anaerobic Digestion of Municipal Sewage Sludge and Lime Stabilization of Class B Biosolids. J. Res. Sci. Technol, 10, 69-85.
• Pepper, I. L. (2013). The Soil Health - Human Health Nexus. Crit. Rvw. Environ. Sci. & Technol, 43, 2617-2652.
• Pepper, I. L. (2013). The Soil Health-Human Health Nexus. Crit Rvw Environ Sci & Technol, 43, 2617-2652.
• Pepper, I. L. (2013). The soil health-human health nexus. Critical Reviews in Environmental Science and Technology, 43(24), 2617-2652.
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  Abstract: Soils can beneficially or adversely affect human health, and likewise human activity can improve or destroy soil health. In the new anthropogenic era, it is worth examining the soil health-human health nexus. To do this, the author evaluates soil from the perspective of what infects us, what heals us, what contaminates us, what nourishes us, and what we breathe. Likewise, the author examines the impact of humans on soil using a similar matrix and suggests strategies to improve human health by maintaining or improving soil health. © 2013 Taylor & Francis Group, LLC.
• Pepper, I. L., Zerzghi, H. G., Bengson, S. A., & Glenn, E. P. (2013). Revegetation of Copper Mine Tailings Through Land Application of Biosolids: Long-Term Monitoring. Arid Land Research and Management, 27(3), 245-256.
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  Abstract: The effects of biosolid amendment on revegetation of two copper mine tailing sites at a commercial copper mine were monitored. The two sites were amended with Class A biosolids in December 1998 (Site 1) and December 2000 (Site 2). Sites were located within 1 km of each other and were physically and chemically similar. At Site 1, biosolids were incorporated by disking into the tailings. At Site 2, biosolids were not incorporated, resulting in a 12-15 cm layer of biosolids on top of the tailings. The high moisture holding capacity of this layer of biosolids resulted in approximately twice as much soil moisture within the surface (0-30 cm tailing depth) at Site 2 than Site 1, when averaged over the period 2001-2007. This increased soil moisture persisted throughout the study period. The higher soil moisture at Site 2 resulted in significantly higher revegetation as estimated by Landsat data (Enhanced Vegetation Indices). It also resulted in enhanced microbial activities including denitrification activity, which caused soil nitrate concentrations at Site 2 to be minimal in December 2008. In contrast, nitrate levels at Site 1 at this time were excessively high. In addition, high tailing concentrations of Cu and Mo at both sites resulted in vegetation high in Cu and particularly Mo. In December 2008, 10 years after Site 1 was amended and 8 years after Site 2 was amended, the percent vegetative cover at the sites was 59.0% and 56.8%, respectively. © 2013 Copyright Taylor and Francis Group, LLC.
• Pepper, I., Pepper, I. L., Zerzghi, H., Gerba, C. P., & Brooks, J. P. (2013). Long-term effects of land application of class B biosolids on the soil microbial populations, pathogens, and activity. Journal of environmental quality, 39(1).
• Sherchan, S., Gerba, C. P., & Pepper, I. L. (2013). Evaluation of Real-time Water Quality sensors for the Detection of Intentional Spore Contamination of Potable Water. J Biosensors & Bioelectronics, 4, 141-145.
• Brooks, J. P., McLaughlin, M. R., Gerba, C. P., & Pepper, I. L. (2012). Land application of manure and class B biosolids: An occupational and public quantitative microbial risk assessment. Journal of Environmental Quality, 41(6), 2009-2023.
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  PMID: 23128758;Abstract: Land application is a practical use of municipal Class B biosolids and manure that also promotes soil fertility and productivity. To date, no study exists comparing biosolids to manure microbial risks. Th is study used quantitative microbial risk assessment to estimate pathogen risks from occupational and public exposures during scenarios involving fomite, soil, crop, and aerosol exposures. Greatest one-time risks were from direct consumption of contaminated soil or exposure to fomites, with one-time risks greater than 10-1.Recent contamination and high exposures doses increased most risks. Campylobacter jejuni and enteric viruses provided the greatest single risks for most scenarios, particularly in the short term. All pathogen risks were decreased with time, 1 d to14 mo between land application and exposure; decreases in risk were typically over six orders of magnitude beyond 30 d. Nearly all risks were reduced to below 10-4 when using a 4-mo harvest delay for crop consumption. Occupational, more direct risks were greater than indirect public risks, which oft en occur aft er time and dilution have reduced pathogen loads to tolerablelevels. Comparison of risks by pathogen group confirmed greater bacterial risks from manure, whereas viral risks were exclusive to biosolids. A direct comparison of the two residual types showed that biosolids use had greater risk because of the high infectivity of viruses, whereas the presence of environmentally recalcitrant pathogens such as Cryptosporidium and Listeria maintained manure risk. Direct comparisons of shared pathogens resulted in greater manure risks. Overall, it appears thatin the short term, risks were high for both types of residuals, but given treatment, attenuation, and dilution, risks can be reduced to nearinsignificant levels Thatbeing said, limited data sets, dose exposures, site-specific inactivation rates, pathogen spikes, environmental change, regrowth, and wildlife will increase risk anduncertainty and remain areas poorly understood. © American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America.
• Brooks, J., McLaughlin, M., Gerba, C., & Pepper, I. (2012). Land Application of Manure and Class B Biosolids: An Occupational and Public Quantitative Microbial Risk Assessment. J. Environ. Qual., 41, 2009-2023.
• Miles, S. L., Pepper, I. L., Sinclair, R. G., & Riley, M. R. (2012). Real time monitoring for pathogens in water. Water Distribution Systems Analysis 2010 - Proceedings of the 12th International Conference, WDSA 2010, 303-308.
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  Abstract: Recent concerns over the potential for bioterrorism and infrastructure resiliency have stimulated the development of sensors for real-time monitoring of biological contaminants in water distribution systems. Private and government water utilities are responding to these concerns and putting in place commercially available sensors. These sensors need to be evaluated independently (without bias) for optimization of their use, evaluation of their claimed capabilities, and their potential for integration into a supervisory control and data acquisition (SCADA) system. This research effort evaluated real-time sensors for their ability to monitor microbial contaminants in water distribution systems through rigorous evaluation protocols in afield scale test-bed laboratory specifically constructed for this purpose. Water quality sensors including the HACH Guardian Blue Monitoring Platform; the JMAR BioSentry unit; the S::CAN spectro::lyser technology; and the GE 5310 total organic carbon unit were evaluated using intrusion of Escherichia coli (E. coli) to determine the sensor's response to a microbial contaminant. Detection capabilities were examined with the contaminating E. coli bacteria suspended in deionized water or tap water with concentrations ranging from 10 3 to 10 6 cfu/mL. Most sensors were responsive to an increase in E. coli concentrations with an observed useful dynamic range between 10 3 cfu/mL to 10 6 cfu/mL. Below this range, sensors provided signals not distinguishable from noise while exceeding the threshold caused saturation and mis-classification by some sensors. The data effectively shows that select sensors can detect microbial water quality changes and thus be utilized in part of an early warning system for monitoring intrusion events in water distribution systems. © 2012 ASCE.
• Miles, S. L., Takizawa, K., Gerba, C. P., & Pepper, I. L. (2012). Survival of infectious prions in water. Water Distribution Systems Analysis 2010 - Proceedings of the 12th International Conference, WDSA 2010, 454-458.
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  Abstract: The objective of this study was to evaluate the fate of infectious prions in water. Prion diseases such as bovine spongiform encephalopathy also known as "mad cow disease", can be spread by ingestion of animal tissue and feces, suggesting that transmission via water may be possible. Few studies have evaluated the fate of prions in water and most have used assays that were not capable of determining whether or not the prions were infectious. In this study, known concentrations of infectious prions (PrP Sc) were added to deionized water, tap water, and treated wastewater. Water samples were incubated at mesophilic and thermophilic temperatures for up to for 8 weeks. The standard scrapie cell assay (SSCA) and the ELISPOT (Enzyme Linked Immuno-Spot) reaction was performed to determine the quantity of infectious prions that survived incubation. A large reduction of PrP Sc was observed during the course of the experiments at every temperature. A maximum rate of inactivation in water occurred under thermophilic conditions. The results suggest that temperature and dissolved organic matter influence inactivation. Previous studies report that prions were very resistant to degradation; however, the methods used did not distinguish whether or not prions were infectious. Our data show that infectious prions are susceptible to inactivation in water. © 2012 ASCE.
• Pepper, I. L., Zerzghi, H. G., Bengson, S. A., Iker, B. C., Banerjee, M. J., & Brooks, J. P. (2012). Bacterial populations within copper mine tailings: Long-term effects of amendment with Class A biosolids. Journal of Applied Microbiology, 113(3), 569-577.
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  PMID: 22738811;Abstract: Aim: This study evaluates the effect of surface application of dried Class A biosolids on microbial populations within copper mine tailings. Methods and Results: Mine tailing sites were established at ASARCO Mission Mine close to Sahuarita Arizona. Site 1 (December 1998) was amended with 248 tons ha-1 of Class A biosolids. Sites 2 (December 2000) and 3 (April 2006) were amended with 371 and 270 tons ha-1, respectively. Site D, a neighbouring native desert soil, acted as a control for the evaluation of soil microbial characteristics. Surface amendment of Class A biosolids showed a 4 log10 increase in heterotrophic plate counts (HPCs) compared to unamended tailings, with the increase being maintained for 10-year period. Microbial activities such as nitrification, sulphur oxidation and dehydrogenase activity were also sustained throughout the study period. 16S rRNA clone libraries obtained from community DNA suggest that mine tailings amended with biosolids achieve diversity and bacterial populations similar to native soil bacterial phyla, 10 years postapplication. Conclusion: Addition of Class A biosolids to copper mine tailings in the desert south-west increased soil microbial numbers, activity and diversity relative to unamended mine tailings. Significance and Impact of the Study: The amended tailings resulted in a functional soil with respect to microbial characteristics, which were sustainable over a 10-year period enabling the development of appropriate vegetation. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.
• Pepper, I., Arabasz, W., Cole, J., Ernst, W., Huenneke, L., Illangasekare, T., Rendu, J., & Thorleifson, H. (2012). International Science in the National Interest at the U.S. Geological Survey. National Research Council.
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  The National Academies Press, Washington, DC.
• Pepper, I., Zerzghi, H., Bengson, S., & Glenn, E. (2012). Revegetation of Copper Mine Tailings Through Land Application of Biosolids: Long-Term Monitoring. Arid Land Res. Manag..
• Pepper, I., Zerzghi, H., Bengson, S., Iker, B., Banerjee, M., & Brooks, J. (2012). Bacterial Populations within Copper Mine Tailings: Long-Term Effects of Amendments with Class A Biosolids. J. Appl. Microbiol., 113, 569-577.
• Williams, D. L., Pepper, I. L., & Gerba, C. P. (2012). Survival of Ascaris ova in desert soils: A risk assessment. Journal of Residuals Science and Technology, 9(4), 151-157.
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  Abstract: The goal of this study was to determine effects of temperature and soil type on survival of Ascaris ova in two biosolid-amended, desert soils. Results of this study suggest that a waiting period of 120 days at average soil temperatures of 25°C or 90 days at 37°C after land application of biosolids on fields to which lettuce is planted would result in annual risks of less than 1:10,000 for Ascaris from consumption of lettuce with an ova concentration of 4 ova g dry solids. © 2012 DEStech Publications, Inc.
• Galada, H., Gerba, C., Joe, A., Kumar, A., Marquez, E., Olson, M. S., Pepper, I., Richter, E., Teng, J., & Gurian, P. L. (2011). The SMART parameter database for quantitative microbial risk assessments. American Water Works Association Annual Conference and Exposition 2011, ACE 2011, 4195-4207.
• Gerba, C. P., Ross, A., Takizawa, K., & Pepper, I. L. (2011). Efficiency of ASTM Method D4994-89 for Recovery of Enteric Viruses from Biosolids. Food and Environmental Virology, 3(1), 43-45.
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  Abstract: The ASTM Method D4994-89 has been used in the United States for almost two decades to assess the virological quality of biosolids. However, the efficiency of this method for recovery of different enteric viruses has never been determined. The method was found to recover several different enteroviruses and adenovirus 2 with an efficiency ranging from 18.1 ± 5.5 to 24.6 ± 7.8%. © 2011 Springer Science + Business Media, LLC.
• Miles, S. L., Sinclair, R. G., Riley, M. R., & Pepper, I. L. (2011). Evaluation of select sensors for real-time monitoring of Escherichia coli in water distribution systems. Applied and Environmental Microbiology, 77(8), 2813-2816.
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  PMID: 21357435;PMCID: PMC3126364;Abstract: This study evaluated real-time sensing of Escherichia coli as a microbial contaminant in water dis- tribution systems. Most sensors responded to increased E. coli concentrations, showing that select sensors can detect microbial water quality changes and be utilized as part of a contaminant warning system. © 2011, American Society for Microbiology.
• Miles, S. L., Takizawa, K., Gerba, C. P., & Pepper, I. L. (2011). Survival of infectious prions in class B biosolids. Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and Environmental Engineering, 46(4), 364-370.
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  PMID: 21391030;Abstract: This study developed a method for extracting infectious prions from Class B biosolids and subsequently evaluated the survival of infectious prions under the influence of mesophilic (37°C) and thermophilic (60°C) temperatures in Class B biosolids. Unlike other studies, this study utilized a scrapie cell assay to determine infectivity and quantity of infectious prions. The best method for extraction was exposing the biosolids to 4Murea at 80°C for 10 minutes followed by a membrane centrifugation to reduce the concentration of urea. The recovery efficiency of the infectious prions from the biosolids for this method was 17.2%. In the survival study, a 2.43-log10reduction in prion infectivity was observed under mesophilic temperatures after 15 days and a 3.41-log10 reduction after 10 days under thermophilic conditions. The reduction of infectious prions was greater in the biosolids than the control in phosphate buffered saline, suggesting factors other than temperature were also playing a role in the loss of infectivity of the prions in the biosolids. Copyright © Taylor & Francis Group, LLC.
• Miles, S. L., Takizawa, K., Gerba, C. P., & Pepper, I. L. (2011). Survival of infectious prions in water. Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and Environmental Engineering, 46(9), 938-943.
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  PMID: 21707419;Abstract: The objective of this study was to evaluate the fate of infectious prions in water. Known concentrations of infectious prions were added to deionized water, tap water, and wastewater. Samples were incubated at 25°C, 37°C, and 50°C for 1 to 8 weeks. The standard scrapie cell assay (SSCA) which includes the ELISPOT (Enzyme Linked Immuno-Spot) reaction was performed to determine prion infectivity and quantity as a function of time. A reduction of infectious prions was observed at 25°C, 37°C, and 50°C ranging between 0.5-log 10 and 1.4-log 10 in one week. Results suggest that organic matter was instrumental in protecting infectious prions, allowing them to remain infectious for a longer period of time. Thus, our data effectively show a quantifiable reduction of infectious prions in water and identifies some of the components that may influence infectivity. Copyright © Taylor & Francis Group, LLC.
• Miles, S., Sinclair, R., Riley, M., & Pepper, I. (2011). Evaluation of Select Sensors for Real-time Monitoring of Escherichia coli in Water Distribution Systems. Appl. Environ. Microbiol., 77, 2813-2816.
• Pepper, I. L., Miles, S., Takizawa, K., Gerba, C., & Pepper, L. (2011). Survival of Infectious Prions in Class B Biosolids. J. Env. Sci. & Hlth., 46, 364-370.
• Pepper, I. L., Miles, S., Takizawa, K., Pepper, L., & Gerba, C. (2011). Survival of Infectious Prions in Water. J. Env. Sci. & Hlth., 46, 938-943.
• Pepper, I., Miles, S. L., Sinclair, R. G., Riley, M. R., & Pepper, I. L. (2011). Evaluation of select sensors for real-time monitoring of Escherichia coli in water distribution systems. Applied and environmental microbiology, 77(8).
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  This study evaluated real-time sensing of Escherichia coli as a microbial contaminant in water distribution systems. Most sensors responded to increased E. coli concentrations, showing that select sensors can detect microbial water quality changes and be utilized as part of a contaminant warning system.
• Pepper, I., Miles, S. L., Takizawa, K., Gerba, C. P., & Pepper, I. L. (2011). Survival of infectious prions in Class B biosolids. Journal of environmental science and health. Part A, Toxic/hazardous substances & environmental engineering, 46(4).
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  This study developed a method for extracting infectious prions from Class B biosolids and subsequently evaluated the survival of infectious prions under the influence of mesophilic (37°C) and thermophilic (60°C) temperatures in Class B biosolids. Unlike other studies, this study utilized a scrapie cell assay to determine infectivity and quantity of infectious prions. The best method for extraction was exposing the biosolids to 4 M urea at 80°C for 10 minutes followed by a membrane centrifugation to reduce the concentration of urea. The recovery efficiency of the infectious prions from the biosolids for this method was 17.2%. In the survival study, a 2.43-log(10) reduction in prion infectivity was observed under mesophilic temperatures after 15 days and a 3.41-log(10) reduction after 10 days under thermophilic conditions. The reduction of infectious prions was greater in the biosolids than the control in phosphate buffered saline, suggesting factors other than temperature were also playing a role in the loss of infectivity of the prions in the biosolids.
• Pepper, I., Miles, S. L., Takizawa, K., Gerba, C. P., & Pepper, I. L. (2011). Survival of infectious prions in water. Journal of environmental science and health. Part A, Toxic/hazardous substances & environmental engineering, 46(9).
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  The objective of this study was to evaluate the fate of infectious prions in water. Known concentrations of infectious prions were added to deionized water, tap water, and wastewater. Samples were incubated at 25°C, 37°C, and 50°C for 1 to 8 weeks. The standard scrapie cell assay (SSCA) which includes the ELISPOT (Enzyme Linked Immuno-Spot) reaction was performed to determine prion infectivity and quantity as a function of time. A reduction of infectious prions was observed at 25°C, 37°C, and 50°C ranging between 0.5-log₁₀ and 1.4-log₁₀ in one week. Results suggest that organic matter was instrumental in protecting infectious prions, allowing them to remain infectious for a longer period of time. Thus, our data effectively show a quantifiable reduction of infectious prions in water and identifies some of the components that may influence infectivity.
• Mahalanabis, M., Reynolds, K. A., Pepper, I. L., & Gerba, C. P. (2010). Comparison of Multiple Passage Integrated Cell Culture-PCR and Cytopathogenic Effects in Cell Culture for the Assessment of Poliovirus Survival in Water. Food and Environmental Virology, 2(4), 225-230.
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  Abstract: The goal of this study was to determine if a cytopathogenic effects (CPE) cell culture assay and an integrated cell culture PCR (ICC-PCR) assay would yield similar or different results when used to assess virus survival in water. Poliovirus type 1 was added to dechlorinated tapwater and stored at room temperature (22. 5-24°C) for a total of 50 days. Samples were assayed at defined time intervals by the most probable number (MPN) method on Buffalo green monkey kidney cells (BGM) by CPE and additionally by ICC-PCR. Monolayers that were CPE negative on first passage were passed onto fresh monolayers of cells for a second and third time if still negative. By CPE assay, second passage was observed to yield a greater titer (2,300 vs. 24,000 MPN/ml) and third passage also resulted in an increased titer. ICC-PCR proved to be a more rapid and sensitive method than conventional cell culture for determining virus inactivation rates in water. Poliovirus survived in tapwater for up to 32 days, as assessed by both third passage ICC-PCR and CPE. There was no statistical difference in the inactivation rates between the two methods. To determine the total number of infectious viruses, these findings indicate the need for performing three cell culture passages or, alternatively, ICC-PCR on first passage. © 2010 Springer Science + Business Media, LLC.
• Mahalanabis, M., Reynolds, K., Pepper, I., & Gerba, C. (2010). Comparison of Multiple Passage Integrated Cell Culture-PCR and Cytopathogenic Effects in Cell Culture for the Assessment of Poliovirus Survival in Wat. Food Environ. Virol., 2, 22-230.
• Miles, S., Sinclair, R., Riley, M., & Pepper, I. (2010). Real time monitoring of E.coli within potable water distributions systems. American Water Works Association Annual Conference and Exposition 2010, ACE 2010, Papers.
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  Abstract: Majority of the parameters increases to an increase in E. coli concentrations Sensors did not respond to E. coli concentrations below 103 cfu/mL Sensors utilizing a light scattering method became saturated when the E. coli concentration was over 106 cfu/mL The BioSentry is not able to distinguish between a particulate and a microorganism TOC sensors respond to either media or high bacterial load.
• Pepper, I. L., Brooks, J. P., Sinclair, R. G., Gurian, P. L., & Gerba, C. P. (2010). Pathogens and indicators in united states class B biosolids: National and historic distributions. Journal of Environmental Quality, 39(6), 2185-2190.
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  PMID: 21284317;Abstract: This paper reports on a major study of the incidence of indicator organisms and pathogens found within Class B biosolids within 21 samplings from 18 wastewater treatment plants across the United States. This is the first major study of its kind since the promulgation of the USEPA Part 503 Rule in 1993, and includes samples before and after the Part 503 Rule was promulgated. National distributions collected between 2005 and 2008 show that the incidence of bacterial and viral pathogens in Class B mesophilic, anaerobically digested biosolids were generally low with the exception of adenoviruses, which were more prevalent than enteric viruses. No Ascaris ova were detected in any sample. In contrast, indicator organism numbers were uniformly high, regardless of whether they were bacteria (fecal coliforms) or viruses (phage). Indicators were not correlated with pathogen loads. Historic distributions were collected between 1988 and 2006 at one location in Tucson, AZ. By comparing data collected before and after 1993, the influence of the USEPA Part 503 Rule on indicator and pathogen levels within Class B biosolids can be inferred. In general, the bacterial indicators total and fecal coliforms decreased from the 1980s to present. Enteric virus concentrations after 1993 are much lower than those reported in other studies in the 1980s, although our values from 1988 to 1993 are not significantly different from our values obtained from 1994 to 2006. Presumably this is due to better and more consistent treatment of the wastewater, illustrating that the Part 503 Rule has been effective in reducing public exposure to pathogens relative to 17 yr ago. The percent reduction of both indicators and pathogens during anaerobic mesophilic digestion was between 94 and 99% for all organisms, illustrating that such treatment is effective in reducing pathogen loads. Copyright © 2010 by the American Society of Agronomy.
• Pepper, I., Scott, B. A., & Pepper, I. L. (2010). Water distribution systems as living ecosystems: impact on taste and odor. Journal of environmental science and health. Part A, Toxic/hazardous substances & environmental engineering, 45(7).
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  Six waters from different U.S. cities with known diverse taste and odor (TO) evaluations were selected for additional microbial characterization. All waters were subjected to microbial and cultural analyses, and four of the waters were further analyzed by cloning and sequencing of community 16S rRNA. The purpose of the study was to evaluate water distribution systems as living ecosystems, and the impact of these ecosystems on TO. All waters had total bacterial counts of at least 10(3) per ml. The water with lowest TO ranking had 10(6) total counts per ml. Community DNA sequence analysis identified diverse bacterial communities representing five different phyla and over forty genera. Included in this diversity were heterotrophic and autotrophic species that were both aerobic or anaerobic. Additionally, waters with the lowest TO evaluations contained significant sulfide concentrations, as well as bacteria associated with both the oxidation and reduction of inorganic sulfur compounds. Low redox conditions could have resulted in the reduced sulfur compounds and concomitant TO related problems, and an increase in redox could help alleviate these problems. Overall, data show that water distribution systems contain living ecosystems that evolve based on specific environments within particular distribution systems that impact water TO.
• Pepper, I., Zerzghi, H., Brooks, J. P., Gerba, C. P., & Pepper, I. L. (2010). Influence of long-term land application of Class B biosolids on soil bacterial diversity. Journal of applied microbiology, 109(2).
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  To evaluate the effect of long-term annual land applications of Class B biosolids on soil bacterial diversity at University of Arizona Marana Agricultural Field Center, Tucson, Arizona.
• Scott, B. A., & Pepper, I. L. (2010). Water distribution systems as living ecosystems: Impact on taste and odor. Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and Environmental Engineering, 45(7), 890-900.
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  PMID: 20432105;Abstract: Six waters from different U.S. cities with known diverse taste and odor (TO) evaluations were selected for additional microbial characterization. All waters were subjected to microbial and cultural analyses, and four of the waters were further analyzed by cloning and sequencing of community 16S rRNA. The purpose of the study was to evaluate water distribution systems as living ecosystems, and the impact of these ecosystems on TO. All waters had total bacterial counts of at least 103 per ml. The water with lowest TO ranking had 106 total counts per ml. Community DNA sequence analysis identified diverse bacterial communities representing five different phyla and over forty genera. Included in this diversity were heterotrophic and autotrophic species that were both aerobic or anaerobic. Additionally, waters with the lowest TO evaluations contained significant sulfide concentrations, as well as bacteria associated with both the oxidation and reduction of inorganic sulfur compounds. Low redox conditions could have resulted in the reduced sulfur compounds and concomitant TO related problems, and an increase in redox could help alleviate these problems. Overall, data show that water distribution systems contain living ecosystems that evolve based on specific environments within particular distribution systems that impact water TO. Copyright © Taylor & Francis Group, LLC.
• Zerzghi, H., Brooks, J. P., Gerba, C. P., & Pepper, I. L. (2010). Influence of long-term land application of Class B biosolids on soil bacterial diversity. Journal of Applied Microbiology, 109(2), 698-706.
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  PMID: 20202022;Abstract: Aim: To evaluate the effect of long-term annual land applications of Class B biosolids on soil bacterial diversity at University of Arizona Marana Agricultural Field Center, Tucson, Arizona. Methods and Results: Following the final of 20 consecutive years of application of Class B biosolids in March 2005, followed by cotton growth from April to November 2005 surface soil samples (0-30 cm) were collected from control (unamended) and biosolid-amended plots. Total bacterial community DNA was extracted, amplified using 16S rRNA primers, cloned, and sequenced. All 16S rRNA sequences were identified by 16S rRNA sequence analysis and comparison to known sequences in GenBank (NCBI BlastN and Ribosomal Database Project II, RDP). Results showed that the number of known genera (identifiable > 96%) increased in the high rate biosolid plots compared to control plots. Biosolids-amended soils had a broad phylogenetic diversity comprising more than four major phyla: Proteobacteria (32%), Acidobacteria (21%), Actinobacteria (16%), Firmicutes (7%), and Bacteroidetes (6%) which were typical to bacterial diversity found in the unamended arid southwestern soils. Conclusion: Bacterial diversity was either enhanced or was not negatively impacted following 20 years of land application of Class B biosolids. Significance and Impact of the Study: This study illustrates that long-term land application of biosolids to arid southwestern desert soils has no deleterious effect on soil microbial diversity. © 2010 The Society for Applied Microbiology.
• Zerzghi, H., Gerba, C. P., & Pepper, I. L. (2010). Long-term effects of land application of class B biosolids on soil chemical properties. Journal of Residuals Science and Technology, 7(1), 51-61.
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  Abstract: Currently about 60% of all biosolids are land applied in the United States. The long-term influence of land application has been questioned due to public concern over potential hazards. The objective of this study is to evaluate the influence of land application of Class B biosolids on the soil chemical properties by analysis of depth (0-150 cm) soil samples collected 9 months after the 20th annual land application. The study showed that land application of Class B biosolids had no significant long-term effect on soil pH and CaCO 3. However, land application significantly increased soil macro-nutrients (C, N and P). Soil nitrate values in plots that received biosolids or inorganic fertilizer amendments were high indicating the potential for groundwater contamination. In addition, total and available soil P concentrations increased to values above that necessary for plant growth but P values attenuated to background levels at a soil depth of 150 cm. Total metal concentrations attenuated rapidly with increasing soil depth, and were generally similar to values found in control soils at a depth of 150 cm. Application of biosolids for nonfood agricultural crop production at this arid southwest site seems to be sustainable with respect to soil chemical entities. © 2010 DEStech Publications, Inc.
• Zerzghi, H., Gerba, C. P., Brooks, J. P., & Pepper, I. L. (2010). Long-term effects of land application of class B biosolids on the soil microbial populations, pathogens, and activity. Journal of Environmental Quality, 39(1), 402-408.
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  PMID: 20048328;Abstract: This study evaluated the infl uence of 20 annual land applications of Class B biosolids on the soil microbial community. Th e potential benefi ts and hazards of land application were evaluated by analysis of surface soil samples collected following the 20th land application of biosolids. Th e study was initiated in 1986 at the University of Arizona Marana Agricultural Center, 21 miles north of Tucson, AZ. Th e fi nal application of biosolids was in March 2005, followed by growth of cotton (Gossypium hirsutum L.) from April through November 2005. Surface soil samples (0-30 cm) were collected monthly from March 2005, 2 wk after the fi nal biosolids application, through December 2005, and analyzed for soil microbial numbers. December samples were analyzed for additional soil microbial properties. Data show that land application of Class B biosolids had no signifi cant long-term effect on indigenous soil microbial numbers including bacteria, actinomycetes, and fungi compared to unamended control plots. Importantly, no bacterial or viral pathogens were detected in soil samples collected from biosolid amended plots in December (10 mo after the last land application) demonstrating that pathogens introduced via Class B biosolids only survived in soil transiently. However, plots that received biosolids had signifi cantly higher microbial activity or potential for microbial transformations, including nitrifi cation, sulfur oxidation, and dehydrogenase activity, than control plots and plots receiving inorganic fertilizers. Overall, the 20 annual land applications showed no long-term adverse eff ects, and therefore, this study documents that land application of biosolids at this particular site was sustainable throughout the 20-yr period, with respect to soil microbial properties. Copyright © 2010 by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. All rights reserved.
• Gerba, C. P., & Pepper, I. L. (2009). Domestic and Indoor Microbiology. Environmental Microbiology, 555-563.
• Gerba, C. P., & Pepper, I. L. (2009). Wastewater Treatment and Biosolids Reuse. Environmental Microbiology, 503-530.
• Maier, R. M., & Pepper, I. L. (2009). Bacterial Communities in Natural Ecosystems. Environmental Microbiology, 347-356.
• Maier, R. M., & Pepper, I. L. (2009). Earth Environments. Environmental Microbiology, 57-82.
• Maier, R. M., Pepper, I. L., & Gerba, C. P. (2009). Introduction to Environmental Microbiology. Environmental Microbiology, 3-7.
• Miles, S. L., Gerba, C. P., Pepper, I. L., & Reynolds, K. A. (2009). Point-of-use drinking water devices for assessing microbial contamination in finished water and distribution systems. Environmental Science and Technology, 43(5), 1425-1429.
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  PMID: 19350914;Abstract: The objective of this study was to develop a method to monitor the microbial quality of treated drinking water at the tap utilizing point-of-use filter systems that are placed in water vending machines. Such vending machines have high-volume water throughput and allow for an evaluation of the occurrence of human enteric pathogens and fecal indicator bacteria in tap water over extended time periods. Seeded experiments, using Escherichia coli and bacteriophage MS-2, were performed on (i) new filters, (ii) artificially aged filters, and (iii) filters that had been used in the field (naturally aged filters) to evaluate the efficiency of recovery of these organisms from the three-component filter set (30 μm, 5 μm, solid block carbon (SBC)) by evaluating each filter independently. SBC filters had the highest recovery of the organisms, averaging recovery of 27% and 5% for E. coli and MS-2, respectively. Subsequently, tap water supplies were monitored in vending machines throughout Southern Arizona using SBC filters as a monitoring tool. A total of 48 filters from 41 unique site locations were surveyed for the presence of total coliforms, E. coli, enterococci, Cryptosporidium, enteroviruses, and noroviruses. Organisms were detected following the passage of large volumes of water ranging from 1 000 to 17 000 L through the filters. Out of 48 SBC filters 54.2% were positive for at least one organism. The number of filters positive for total coliforms, E. coli, enterococci, and enterovirus was 13, 5, 19, and 3, respectively, corresponding to 27.1%, 10.4%, 39.6%, and 6.3% of the total filters. No filters were positive for noroviruses or Cryptosporidium. These results suggest that the SBC filter can be used to monitor large volumes of treated drinking water and detect the incidence of indicators and pathogens that may be present at low concentrations. These data show that post-treated water often contains water quality indicator and pathogenic organisms at the tap, and therefore, monitoring with this method would be beneficial to the community as it allows for an assessment of exposure to pathogens and associative risks. This monitoring tool will also aid in the tracking of outbreaks and the determination of the microbial pathogen load during all stages of an outbreak as a filter can be installed and retrieved at the point-of-use at anytime during an outbreak. © 2009 American Chemical Society.
• Newby, D. T., Pepper, I. L., & Maier, R. M. (2009). Microbial Transport. Environmental Microbiology, 365-383.
• Pepper, I. L., & Dowd, S. E. (2009). Aeromicrobiology. Environmental Microbiology, 83-102.
• Pepper, I. L., & Gerba, C. P. (2009). Cultural Methods. Environmental Microbiology, 173-189.
• Pepper, I. L., & Gerba, C. P. (2009). Global Change and Microbial Infectious Disease. Environmental Microbiology, 357-364.
• Pepper, I. L., Choi, C. Y., & Gerba, C. P. (2009). Microorganisms and Bioterrorism. Environmental Microbiology, 565-574.
• Pepper, I. L., Gerba, C. F., Gentry, T., & Maier, R. M. (2009). Environmental Microbiology. Environmental Microbiology.
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  Abstract: process of asset pricing, which has developed dramatically in the last few years due to advances in financial theory and econometrics. This book covers the science of asset pricing by concentrating on the most widely used modelling technique called: Linear Factor Modelling. Linear Factor Models covers an important area for Quantitative Analysts/Investment Managers who are developing Quantitative Investment Strategies. Linear factor models (LFM) are part of modern investment processes that include asset valuation, portfolio theory and applications, linear factor models and applications, dynamic asset allocation strategies, portfolio performance measurement, risk management, international perspectives, and the use of derivatives. The book develops the building blocks for one of the most important theories of asset pricing - Linear Factor Modelling. Within this framework, we can include other asset pricing theories such as the Capital Asset Pricing Model (CAPM), arbitrage pricing theory and various pricing formulae for derivatives and option prices. As a bare minimum, the reader of this book must have a working knowledge of basic calculus, simple optimisation and elementary statistics. In particular, the reader must be comfortable with the algebraic manipulation of means, variances (and covariances) of linear combination(s) of random variables. Some topics may require a greater mathematical sophistication. © 2009 Elsevier Inc. All rights reserved.
• Pepper, I. L., Gerba, C. P., & Maier, R. M. (2009). Environmental Sample Collection and Processing. Environmental Microbiology, 137-155.
• Pepper, I. L., Gerba, C. P., Newby, D. T., & Rice, C. W. (2009). Soil: A public health threat or savior?. Critical Reviews in Environmental Science and Technology, 39(5), 416-432.
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  Abstract: Soil is the most complicated biomaterial on the planet due to complexsoil architecture and billions of soil microbes with extremebiotic diversity. Soil is potentially a source of human pathogens, which can be defined as geo-indigenous, geo-transportable, or geotreatable. Such pathogens cumulatively can and do result in multiplehuman fatalities annually. A striking example is Helminths, with current infections worldwide estimated to be around two billion. However, soil can also be a source of antibiotics and other natural products that enhance human health. Soilborne antibioticsare used to treat human infections, but can also result inantibiotic-resistant bacteria. Natural products isolated from soil resulted in 60% of new cancer drugs between the period 1983-1994.Soils are also crucial to human health through their impact on human nutrition. Finally, from a global perspective, soils are vitalto the future well-being of nations through their impact on climate change and global warming. A critical review of soil with respectto public health leads to the conclusion that overall soil is a public health savior. The value of soil using a systems approach is estimatedto be $20 trillion, and is by far the most valuable eco systemin the world. Copyright © Taylor & Francis Group, LLC.
• Pepper, I., Mitteldorf, J., & Pepper, I. L. (2009). Senescence as an adaptation to limit the spread of disease. Journal of theoretical biology, 260(2).
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  Aging has the hallmarks of an evolved adaptation. It is controlled by genes that have been conserved over vast evolutionary distances, and most organisms are able to forestall aging in the most challenging of environments. But fundamental theoretical considerations imply that there can be no direct selection for aging. Senescence reduces individual fitness, and any group benefits are weak and widely dispersed over non-relatives. We offer a resolution to this paradox, suggesting a general mechanism by which senescence might have evolved as an adaptation. The proposed benefit is that senescence protects against infectious epidemics by controlling population density and increasing diversity of the host population. This mechanism is, in fact, already well-accepted in another context: it is the Red Queen Hypothesis for the evolution of sex. We illustrate the hypothesis using a spatially explicit agent-based model in which disease transmission is sensitive to population density as well as homogeneity. We find that individual senescence provides crucial population-level advantages, helping to control both these risk factors. Strong population-level advantages to individual senescence can overcome the within-population disadvantage of senescence. We conclude that frequent local extinctions provide a mechanism by which senescence may be selected as a population-level adaptation in its own right, without assuming pleiotropic benefits to the individual.
• Pierson, L. S., Maier, R. M., & Pepper, I. L. (2009). Microbial Communication: Bacteria-Bacteria and Bacteria-Host. Environmental Microbiology, 335-346.
• Roane, T. M., Pepper, I. L., & Maier, R. M. (2009). Microscopic Techniques. Environmental Microbiology, 157-172.
• Roane, T. M., Rensing, C., Pepper, I. L., & Maier, R. M. (2009). Microorganisms and Metal Pollutants. Environmental Microbiology, 421-441.
• Roane, T. M., Reynolds, K. A., Maier, R. M., & Pepper, I. L. (2009). Microorganisms. Environmental Microbiology, 9-36.
• Rodríguez, R. A., Pepper, I. L., & Gerba, C. P. (2009). Application of PCR-based methods to assess the infectivity of enteric viruses in environmental samples. Applied and Environmental Microbiology, 75(2), 297-307.
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  PMID: 19011062;PMCID: PMC2620694;Abstract: The advent of the PCR has greatly enhanced our ability to detect human enteric viral pathogens in the environment, including water, municipal wastes, sewage, food, air, and fomites (2, 3, 59, 69, 79). This is especially true for those viruses which do not grow in cell culture. Despite great sensitivity, PCR methods do have some serious limitations for environmental viral analysis, including small sample volumes, the presence of PCR-inhibitory substances, and an inability to differentiate between infective and noninfective viruses (66). The ability of PCR to assess infectivity would greatly enhance its application for the monitoring of water and food quality and for treatment processes (e.g., disinfection). This review focuses on approaches to overcome these limitations. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
• Gerba, C. P., Campo, N. C., Brooks, J. P., & Pepper, I. L. (2008). Exposure and risk assessment of Salmonella in recycled residuals. Water Science and Technology, 57(7), 1061-1065.
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  PMID: 18441433;Abstract: The potential health effects of Salmonella found in wastewater residuals is dependent on the exposure of individuals to the organism. This paper provides a risk assessment for human infection from Salmonella due to direct contact with Class B biosolids, and from contact with Class A biosolids following regrowth of Salmonella. In addition, a risk assessment is provided for infection via airborne transport of bioaerosols from Class B biosolids and biosolids in which regrowth had occurred, to off-site communities. Results of the risk characterization imply that the risk of human infection from direct contact with Class B land applied residuals and subsequent ingestion is low. In contrast, the risk from direct contact with Class A residuals following regrowth is greater. Risks from airborne transport of Salmonella via bioaerosols away from a Class B land application site are also low. However, once again the risk from aerosols resulting from biosolids in which regrowth had occurred was greater. Based on these analyses, we conclude that it is highly unlikely that Salmonella infections will occur from land applied Class A or B residuals. However, risks become significant if Class A biosolids are stored anaerobically i.e. saturated, prior to land application. © IWA Publishing 2008.
• Pepper, I. L., Zerzghi, H., Brooks, J. P., & Gerba, C. P. (2008). Sustainability of land application of Class B biosolids. Journal of Environmental Quality, 37(SUPPL. 5), S58-S67.
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  PMID: 18765778;Abstract: Land application of Class B biosolids is routinely undertaken in the United States. However, due to public concern over potential hazards, the long-term sustainability of land application has been questioned. Thus, the objective of this review article was to evaluate the sustainability of land application of Class B biosolids. To do this we evaluated (i) the fate and transport of potential biological and chemical hazards within biosolids, and (ii) the influence of long-term land application on the microbial and chemical properties of the soil. Direct risks to human health posed by pathogens in biosolids have been shown to be low. Risks from indirect exposure such as aerosolized pathogens or microbially contaminated ground water are also low. A long-term land application study showed enhanced microbial activity and no adverse toxicity effects on the soil microbial community. Long-term land application also increased soil macronutrients including C, N, and, in particular, P. In fact, care should be taken to avoid contamination of surface waters with high phosphate soils. Available soil metal concentrations remained low over the 20-yr land application period due to the low metal content of the biosolids and a high soil pH. Soil salinity increases were not detected due to the low salt content of biosolids and irrigation rates in excess of consumptive use rates for cotton. Our conclusion, based on these studies, is that long-term land application of Class B biosolids is sustainable. Copyright © 2008 by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. All rights reserved.
• Pepper, I., Gerba, C. P., Castro-del Campo, N., Brooks, J. P., & Pepper, I. L. (2008). Exposure and risk assessment of Salmonella in recycled residuals. Water science and technology : a journal of the International Association on Water Pollution Research, 57(7).
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  The potential health effects of Salmonella found in wastewater residuals is dependent on the exposure of individuals to the organism. This paper provides a risk assessment for human infection from Salmonella due to direct contact with Class B biosolids, and from contact with Class A biosolids following regrowth of Salmonella. In addition, a risk assessment is provided for infection via airborne transport of bioaerosols from Class B biosolids and biosolids in which regrowth had occurred, to off-site communities. Results of the risk characterization imply that the risk of human infection from direct contact with Class B land applied residuals and subsequent ingestion is low. In contrast, the risk from direct contact with Class A residuals following regrowth is greater. Risks from airborne transport of Salmonella via bioaerosols away from a Class B land application site are also low. However, once again the risk from aerosols resulting from biosolids in which regrowth had occurred was greater. Based on these analyses, we conclude that it is highly unlikely that Salmonella infections will occur from land applied Class A or B residuals. However, risks become significant if Class A biosolids are stored anaerobically i.e. saturated, prior to land application.
• Tanner, B. D., Brooks, J. P., Gerba, C. P., Haas, C. N., Josephson, K. L., & Pepper, I. L. (2008). Estimated occupational risk from bioaerosols generated during land application of class B biosolids. Journal of Environmental Quality, 37(6), 2311-2321.
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  PMID: 18948485;Abstract: Some speculate that bioaerosols from land application of biosolids pose occupational risks, but few studies have assessed aerosolization of microorganisms from biosolids or estimated occupational risks of infection. This study investigated levels of microorganisms in air immediately downwind of land application operations and estimated occupational risks from aerosolized microorganisms. In all, more than 300 air samples were collected downwind of biosolids application sites at various locations within the United States. Coliform bacteria, coliphages, and heterotrophic plate count (HPC) bacteria were enumerated from air and biosolids at each site. Concentrations of coliforms relative to Salmonella and concentrations of coliphage relative to enteroviruses in biosolids were used, in conjunction with levels of coliforms and coliphages measured in air during this study, to estimate exposure to Salmonella and enteroviruses in air. The HPC bacteria were ubiquitous in air near land application sites whether or not biosolids were being applied, and concentrations were positively correlated to windspeed. Coliform bacteria were detected only when biosolids were being applied to land or loaded into land applicators. Coliphages were detected in few air samples, and only when biosolids were being loaded into land applicators. In general, environmental parameters had little impact on concentrations of microorganisms in air immediately downwind of land application. The method of land application was most correlated to aerosolization. From this large body of data, the occupational risk of infection from bioaerosols was estimated to be 0.78 to 2.1%/yr. Extraordinary exposure scenarios carried an estimated annual risk of infection of up to 34%, with viruses posing the greatest threat. Risks from aerosolized microorganisms at biosolids land application sites appear to be lower than those at wastewater treatment plants, based on previously reported literature. Copyright © 2008 by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. All rights reserved.
• Brooks, J. P., Gerba, C. P., & Pepper, I. L. (2007). Diversity of aerosolized bacteria during land application of biosolids. Journal of Applied Microbiology, 103(5), 1779-1790.
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  PMID: 17953588;Abstract: Aims: The purpose of this study was to determine the diversity of bacterial communities associated with bioaerosols generated during land application of biosolids using 16S ribosomal RNA (16S rRNA) PCR. Methods and Results: Anaerobically digested Class B biosolids were land applied to an agricultural site located in South Central Arizona. Aerosol samples were collected downwind of the biosolids operations and were collected via the use of SKC Biosamplers and subsequently extracted for the presence of bacterial community DNA. All DNA was amplified using 16S rRNA primers, cloned and sequenced. All sequences were aligned and phylogenetic trees were developed to generate community profiles. The majority of aerosolized bacterial clone sequences belonged to the Actinobacteria and alpha- and beta-proteobacterial taxa. Aerosol samples collected downwind of soil aerosolization produced similar profiles. These profiles differed from upwind and background samples. Conclusions: No one clone sequence isolated from the aerosol samples could be solely attributed to biosolids; on the contrary, the majority appeared to have arisen from soil. Significance and Impact of the Study: This study demonstrates that in dry, arid climates the majority of aerosols associated with biosolids land application appear to be associated with the onsite soil.
• Brooks, J. P., Maxwell, S. L., Rensing, C., Gerba, C. P., & Pepper, I. L. (2007). Occurrence of antibiotic-resistant bacteria and endotoxin associated with the land application of biosolids. Canadian Journal of Microbiology, 53(5), 616-622.
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  PMID: 17668020;Abstract: The purpose of this study was to determine the prevalence of antibiotic-resistant bacteria and endotoxin in soil after land application of biosolids. Soil was collected over a 15 month period following land application of biosolids, and antibiotic resistance was ascertained using clinically relevant antibiotic concentrations. Ampicillin, cephalothin, ciprofloxacin, and tetracycline resistance were all monitored separately for any changes throughout the 15 month period. Endotoxin soil concentrations were monitored using commercially available endotoxin analysis reagents. Overall, land application of biosolids did not increase the percentage of antibiotic-resistant culturable bacteria above background soil levels. Likewise, land application of biosolids did not significantly increase the concentration of endotoxin in soil. This study determined and established a baseline understanding of the overall effect that land application of biosolids had on the land-applied field with respect to antibiotic-resistant bacterial and endotoxin soil densities. © 2007 NRC.
• Campo, N. C., Pepper, I. L., & Gerba, C. P. (2007). Assessment of Salmonella typhimurium growth in class a biosolids and soil/biosolid mixtures. Journal of Residuals Science and Technology, 4(2), 83-88.
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  Abstract: The potential of Salmonella typhimurlum regrowth in Class A biosolid pellets and compost after land application was assessed. Mixtures of soil, soil plus biosolids, and biosolids were inoculated with two different concentrations and monitored during a period of 20 days. No Salmonella growth occurred in any of the soil/biosolid mixtures regardless of inoculum size or moisture content. No growth occurred in any of the biosolids with a moisture content of 20% except the pellets from Texas when inoculated with 10,000 colony forming units/g. Growth of Salmonella did occur in all of the Class A products under saturated conditions. Under all moisture conditions indigenous microflora increased In numbers in the biosolids, soil and biosolid/soll mixtures. In conclusion, these results suggest that while regrowth of Salmonella in biosolids may occur under saturated conditions it does not occur after Class A biosolids land application at typical agronomic rates. © 2007 DEStech Publications, Inc.
• Cheng, L., Chetochine, A. S., Pepper, I. L., & Brusseau, M. L. (2007). Influence of DOC on MS-2 bacteriophage transport in a sandy soil. Water, Air, and Soil Pollution, 178(1-4), 315-322.
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  Abstract: The purpose of this study was to investigate the influence of dissolved organic carbon (DOC) on MS-2 bacteriophage transport in a sandy soil. Miscible-displacement experiments were conducted to examine the retention and transport of MS-2, at two influent concentrations, in the absence and presence of DOC. Effluent recoveries of MS-2 were similar for both sets of experiments. The results of the experiments were analyzed with a mathematical model that incorporated inactivation and rate-limited attachment/detachment. The optimized attachment rate coefficients were similar for all experiments. These results indicate that DOC had no significant influence on the transport of MS-2 in this soil. A mass-balance analysis based on the mathematical-modeling results revealed that attachment was significant during the early stages of the experiments, and that the majority of attached MS-2 became inactivated by the time the experiments were completed. © 2006 Springer Science + Business Media B.V.
• Pepper, I., Brooks, J. P., Maxwell, S. L., Rensing, C., Gerba, C. P., & Pepper, I. L. (2007). Occurrence of antibiotic-resistant bacteria and endotoxin associated with the land application of biosolids. Canadian journal of microbiology, 53(5).
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  The purpose of this study was to determine the prevalence of antibiotic-resistant bacteria and endotoxin in soil after land application of biosolids. Soil was collected over a 15 month period following land application of biosolids, and antibiotic resistance was ascertained using clinically relevant antibiotic concentrations. Ampicillin, cephalothin, ciprofloxacin, and tetracycline resistance were all monitored separately for any changes throughout the 15 month period. Endotoxin soil concentrations were monitored using commercially available endotoxin analysis reagents. Overall, land application of biosolids did not increase the percentage of antibiotic-resistant culturable bacteria above background soil levels. Likewise, land application of biosolids did not significantly increase the concentration of endotoxin in soil. This study determined and established a baseline understanding of the overall effect that land application of biosolids had on the land-applied field with respect to antibiotic-resistant bacterial and endotoxin soil densities.
• Brooks, J. P., Tanner, B. D., Gerba, C. P., & Pepper, I. L. (2006). The measurement of aerosolized endotoxin from land application of Class B biosolids in Southeast Arizona. Canadian Journal of Microbiology, 52(2), 150-156.
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  PMID: 16541151;Abstract: The purpose of this study was to determine aerosolized endotoxin concentrations downwind of a biosolids land application site. Aerosol samples were collected from biosolids land application sites, tractor operation, and an aeration basin located within an open-air wastewater treatment plant. Aerosolized endotoxin above background concentrations was detected from all sites, at levels ranging from below detection up to 1800 EU m-3 of air. Biosolids loading operations resulted in the greatest concentrations of endotoxin (mean 344 EU m-3). As downwind (perpendicular to wind vector) distance increased from sources (2-200 m). levels of endotoxin decreased to near background (without biosolids application) concentrations. Overall, the detected levels of aerosolized endotoxin were within past proposed aerosolized endotoxin limits (250-2000 EU m-3) by other occupational exposure studies. Occasionally, peak concentrations were found to be above these limits. Sites in which soil was being aerosolized resulted in greater concentrations of endotoxin with or without biosolids. which suggested that the majority of endotoxin may in fact be of soil origin. This study evaluated the presence of aerosolized endotoxin from the land application of biosolids and showed that these levels were within ranges for concern suggested by other studies and that this area of research needs further investigation. © 2006 NRC Canada.
• Chetochine, A. S., Brusseau, M. L., Gerba, C. P., & Pepper, I. L. (2006). Leaching of phage from class B biosolids and potential transport through soil. Applied and Environmental Microbiology, 72(1), 665-671.
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  PMID: 16391105;PMCID: PMC1352177;Abstract: The objective of this study was to investigate leaching and transport of viruses, specifically those of an indigenous coliphage host specific to Escherichia coli ATTC 15597 (i.e., MS-2), from a biosolid-soil matrix. Serial extractions of 2% and 7% (solids) class B biosolid matrices were performed to determine the number of phage present in the biosolids and to evaluate their general leaching potential. Significant concentrations of coliphage were removed from the biosolids for each sequential extraction, indicating that many phage remained associated with the solid phase. The fact that phage was associated with or attached to solid particles appeared to influence the potential for release and subsequent transport of phage under saturated-flow conditions, which was examined in a series of column experiments. The results indicated that less than 8% of the indigenous coliphage initially present in the biosolids leached out of the biosolid-soil matrix. A fraction of this was subsequently transported through the sandy porous medium with minimal retention. The minimal retention observed for the indigenous phage, once released from the biosolids, was consistent with the results of control experiments conducted to examine MS-2 transport through the porous medium. Copyright © 2006, American Society for Microbiology. All Rights Reserved.
• Fairley, D. J., Wang, G., Rensing, C., Pepper, I. L., & Larkin, M. J. (2006). Expression of gentisate 1,2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera. Applied Microbiology and Biotechnology, 73(3), 691-695.
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  PMID: 16802151;Abstract: Gentisate-1,2-dioxygenase genes (gdoA), with homology to a number of bacterial dioxygenases, and genes encoding a putative coenzyme A (CoA)-synthetase subunit (acdB) and a CoA-thioesterase (tieA) were identified in two haloarchaeal isolates. In Haloarcula sp. D1, gdoA was expressed during growth on 4-hydroxybenzoate but not benzoate, and acdB and tieA were not expressed during growth on any of the aromatic substrates tested. In contrast, gdoA was expressed in Haloferax sp. D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate and phenylpropionate, and both acdB and tieA were expressed during growth on benzoate, cinnamate and phenylpropionate, but not on 3-hydroxybenzoate. This pattern of induction is consistent with these genes encoding steps in a CoA-mediated benzoate pathway in this strain. © 2006 Springer-Verlag.
• Krutz, L. J., Gentry, T. J., Senseman, S. A., Pepper, I. L., & Tierney, D. P. (2006). Mineralisation of atrazine, metolachlor and their respective metabolites in vegetated filter strip and cultivated soil. Pest Management Science, 62(6), 505-514.
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  PMID: 16612813;Abstract: In vegetated filter strips (VFS) the presence of perennial vegetation, rhizodeposition of labile organic substrates and the accumulation of an organic residue thatch layer may enhance microbial numbers and activity, thereby increasing the potential for mineralisation of herbicides and herbicide metabolites retained during run-off events. The objective of this laboratory experiment was to compare the mineralisation of atrazine and metolachlor with that of their respective metabolites in VFS and cultivated soil. With the exception of total bacteria, propagule density of the microbial groups, endogenous soil enzymes and microbial diversity were higher in the VFS soil. This correlated with increased mineralisation of metolachlor and its metabolites in the VFS soil and indicates potential for VFS to curtail the subsequent transport of these compounds. In contrast, the mineralisation of atrazine and the majority of its metabolites was substantially reduced in VFS soil relative to cultivated soil. Consequently, the potential for subsequent transport of atrazine and many of its metabolites may be greater in VFS soil than in cultivated soil if reduced mineralisation is not offset by increased sorption in the VFS. © 2006 Society of Chemical Industry.
• Pepper, I. L., Brooks, J. P., & Gerba, C. P. (2006). Pathogens in Biosolids. Advances in Agronomy, 90, 1-41.
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  Abstract: The world population of 6.8 billion people all produce sewage. In the developed world most of this is treated by the activated sludge process, which results in large volumes of sludge or biosolids being produced (NRC, 2002). This results in millions of tons of biosolids produced each year in the United States, which must either be disposed of or recycled in some manner. Land application has been seen as the most economical and beneficial way of handling biosolids. Biosolids that result from municipal wastewater treatment processes contain organic matter and nutrients that, when properly treated and applied to farmland, can improve the productivity of soils or enhance revegetation of disturbed ecosystems. However, besides the documented benefits of land application, there are also potential hazards, which have caused the public response to the practice to be mixed. Here we review one of the potential hazards associated with biosolids and its land application, namely human pathogens associated with biosolids. © 2006 Elsevier Inc. All rights reserved.
• Pepper, I., Brooks, J. P., Tanner, B. D., Gerba, C. P., & Pepper, I. L. (2006). The measurement of aerosolized endotoxin from land application of Class B biosolids in Southeast Arizona. Canadian journal of microbiology, 52(2).
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  The purpose of this study was to determine aerosolized endotoxin concentrations downwind of a biosolids land application site. Aerosol samples were collected from biosolids land application sites, tractor operation, and an aeration basin located within an open-air wastewater treatment plant. Aerosolized endotoxin above background concentrations was detected from all sites, at levels ranging from below detection up to 1800 EU m-3 of air. Biosolids loading operations resulted in the greatest concentrations of endotoxin (mean 344 EU m-3). As downwind (perpendicular to wind vector) distance increased from sources (2-200 m), levels of endotoxin decreased to near background (without biosolids application) concentrations. Overall, the detected levels of aerosolized endotoxin were within past proposed aerosolized endotoxin limits (250-2000 EU m-3) by other occupational exposure studies. Occasionally, peak concentrations were found to be above these limits. Sites in which soil was being aerosolized resulted in greater concentrations of endotoxin with or without biosolids, which suggested that the majority of endotoxin may in fact be of soil origin. This study evaluated the presence of aerosolized endotoxin from the land application of biosolids and showed that these levels were within ranges for concern suggested by other studies and that this area of research needs further investigation.
• Pepper, I., Chetochine, A. S., Brusseau, M. L., Gerba, C. P., & Pepper, I. L. (2006). Leaching of phage from Class B biosolids and potential transport through soil. Applied and environmental microbiology, 72(1).
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  The objective of this study was to investigate leaching and transport of viruses, specifically those of an indigenous coliphage host specific to Escherichia coli ATTC 15597 (i.e., MS-2), from a biosolid-soil matrix. Serial extractions of 2% and 7% (solids) class B biosolid matrices were performed to determine the number of phage present in the biosolids and to evaluate their general leaching potential. Significant concentrations of coliphage were removed from the biosolids for each sequential extraction, indicating that many phage remained associated with the solid phase. The fact that phage was associated with or attached to solid particles appeared to influence the potential for release and subsequent transport of phage under saturated-flow conditions, which was examined in a series of column experiments. The results indicated that less than 8% of the indigenous coliphage initially present in the biosolids leached out of the biosolid-soil matrix. A fraction of this was subsequently transported through the sandy porous medium with minimal retention. The minimal retention observed for the indigenous phage, once released from the biosolids, was consistent with the results of control experiments conducted to examine MS-2 transport through the porous medium.
• Brooks, J. P., Tanner, B. D., Gerba, C. P., Haas, C. N., & Pepper, I. L. (2005). Estimation of bioaerosol risk of infection to residents adjacent to a land applied biosolids site using an empirically derived transport model. Journal of Applied Microbiology, 98(2), 397-405.
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  PMID: 15659194;Abstract: Aim: The purpose of this study was to develop an empirically derived transport model, which could be used to predict downwind concentrations of viruses and bacteria during land application of liquid biosolids and subsequently assess microbial risk associated with this practice. Methods and Results: To develop the model, coliphage MS-2 and Escherichia coli were aerosolized after addition to water within a biosolids spray application truck, and bioaerosols were collected at discrete downwind distances ranging from 2 to 70 m. Although coliphage were routinely detected, E. coli did not frequently survive aerosolization. Data on aerosolized coliphage was then used to generate a virus transport model. Risks of infection were calculated for various ranges of human virus concentrations that could be found in biosolids. Conclusions: A conservative estimate at 30-5 m (assumed to be nearest adjacent residences) downwind, resulted in risks of infection of 1: 100 000, to the more realistic 1: 10 000 000 per exposure. Conservative annual risks were calculated to be no more than 7: 100 000 where as a more realistic risk was no greater than 7: 10 000 000. Overall, the viral risk to residences adjacent to land application sites appears to be low, both for one time and annual probabilities of infection. Significance and Impact of the Study: This study demonstrated a simple approach towards modelling viral pathogens aerosolized from land applied liquid biosolids, and offers insight into the associated viral risk.
• Brooks, J. P., Tanner, B. D., Josephson, K. L., Gerba, C. P., Haas, C. N., & Pepper, I. L. (2005). A national study on the residential impact of biological aerosols from the land application of biosolids. Journal of Applied Microbiology, 99(2), 310-322.
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  PMID: 16033462;Abstract: Aims: The purpose of this study was to evaluate the community risk of infection from bioaerosols to residents living near biosolids land application sites. Methods and Results: Approximately 350 aerosol samples from 10 sites located throughout the USA were collected via the use of six SKC Biosamplers®. Downwind aerosol samples from biosolids loading, unloading, land application and background operations were collected from all sites. All samples were analysed for the presence of HPC bacteria, total coliform bacteria, Escherichia coli, Clostridium perfringens, coliphage, enteroviruses, hepatitis A virus and norovirus. Total coliforms, E. coli, C. perfringens and coliphage were not detected with great frequency from any sites, however, biosolids loading operations resulted in the largest concentrations of these aerosolized microbial indicators. Microbial risk analyses were conducted on loading and land application operations and their subsequent residential exposures determined. Conclusions: The greatest annual risks of infection occurred during loading operations, and resulted in a 4 × 10-4 chance of infection from inhalation of coxsackievirus A21. Land application of biosolids resulted in risks that were
• Pepper, I., Brooks, J. P., Tanner, B. D., Gerba, C. P., Haas, C. N., & Pepper, I. L. (2005). Estimation of bioaerosol risk of infection to residents adjacent to a land applied biosolids site using an empirically derived transport model. Journal of applied microbiology, 98(2).
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  The purpose of this study was to develop an empirically derived transport model, which could be used to predict downwind concentrations of viruses and bacteria during land application of liquid biosolids and subsequently assess microbial risk associated with this practice.
• Pepper, I., Brooks, J. P., Tanner, B. D., Josephson, K. L., Gerba, C. P., Haas, C. N., & Pepper, I. L. (2005). A national study on the residential impact of biological aerosols from the land application of biosolids. Journal of applied microbiology, 99(2).
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  The purpose of this study was to evaluate the community risk of infection from bioaerosols to residents living near biosolids land application sites.
• Pepper, I., Tanner, B. D., Brooks, J. P., Haas, C. N., Gerba, C. P., & Pepper, I. L. (2005). Bioaerosol emission rate and plume characteristics during land application of liquid class B biosolids. Environmental science & technology, 39(6).
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  This study investigated bioaerosol emission rates and plume characteristics of bioaerosols generated during land application of liquid Class B biosolids. In addition, it compared the rate of aerosolization of coliphages and total coliform bacteria during land application of liquid Class B biosolids to the rate of aerosolization during land application of groundwater inoculated with similar concentrations of Escherichia coli and coliphage MS2. Air samples were taken immediately downwind of a spray applicator as it applied liquid (approximately 8% solids) biosolids to farmland near Tucson, Arizona. Air samples were also collected immediately downwind of groundwater seeded with MS2 and E. coli applied to land in an identical manner. Air samples, collected with liquid impingers, were taken in horizontal and vertical alignment with respect to the passing spray applicator. Vertical and horizontal sample arrays made it possible to calculate the flux of microorganisms through a virtual plane of air samplers, located 2 m downwind of the passing spray applicator. Neither coliphages nor coliform bacteria were detected in air downwind of spray application of liquid Class B biosolids. Based on limits of detection for the methodology, the rate of aerosolization during land application of liquid biosolids was calculated to be less than 33 plaque forming units (PFU) of coliphage and 10 colony forming units (CFU) of coliform bacteria per meter traveled by the spray applicator. The rate of aerosolization during land application of seeded groundwater was found to be, on average, 2.02 x 10(3) CFU E. coli and 3.86 x 10(3) PFU MS2 aerosolized per meter traveled by the spray applicator. This is greater aerosolization than was observed during land application of biosolids. Because concentrations of coliphages and coliforms were similar in the liquid biosolids and the seeded water, itwas concluded that some property of biosolids reduces aerosolization of microorganisms relative to groundwater. Additional experiments utilizing a novel air sampling protocol showed that the duration of bioaerosol exposure immediately (2 m) downwind of biosolids spray application is brief and the plume of bioaerosols generated is discrete. Additional air samples showed that aerosolization of coliphages and coliform bacteria after liquid biosolids have been applied to land does not occur at detectable levels.
• Pepper, I., Zaleski, K. J., Josephson, K. L., Gerba, C. P., & Pepper, I. L. (2005). Potential regrowth and recolonization of salmonellae and indicators in biosolids and biosolid-amended soil. Applied and environmental microbiology, 71(7).
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  This study evaluated the potential for conversion of Class B to Class A biosolids with respect to salmonellae and fecal coliforms during solar drying in concrete lined drying beds. Anaerobically (8% solids) and aerobically (2% solids) digested Class B biosolids were pumped into field-scale drying beds, and microbial populations and environmental conditions were monitored. Numbers of fecal coliforms and salmonellae decreased as temperature and rate of desiccation increased. After 3 to 4 weeks, Class A requirements were achieved in both biosolids for the pathogens and the indicators. However, following rainfall events, significant increase in numbers was observed for both fecal coliforms and salmonellae. In laboratory studies, regrowth of fecal coliforms was observed in both biosolids and biosolid-amended soil, but the regrowth of salmonellae observed in the concrete-lined drying beds did not occur. These laboratory studies demonstrated that pathogens decreased in numbers when soil was amended with biosolids. Based on serotyping, the increased numbers of salmonellae seen in the concrete lined drying beds following rainfall events was most likely due to recolonization due to contamination from fecal matter introduced by animals and not from regrowth of salmonellae indigenous to biosolids. Overall, we conclude that the use of concrete-lined beds created a situation in which moisture added as rainfall accumulated in the beds, promoting the growth of fecal coliforms and salmonellae added from external sources.
• Stine, S. W., Pepper, I. L., & Gerba, C. P. (2005). Contribution of drinking water to the weekly intake of heterotrophic bacteria from diet in the United States. Water Research, 39(1), 257-263.
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  PMID: 15607184;Abstract: The goal of this study was to assess the relative contribution of heterotrophic bacteria from various sources in the normal diet of an average person in the United States, due to concerns regarding the potential health implications of such bacteria in household tapwater. A literature search was conducted to determine the concentration of heterotrophic plate count (HPC) bacteria in drinking water, as well as foods common to the American diet. Food items were also obtained in Tucson, AZ to further evaluate the consumption of HPC and total coliform bacteria. This was compared to a recent study on HPC bacteria in tapwater with and without POU devices mounted on the tap in Tucson, AZ households. It was determined that only 0.048-4.5% of the average consumer's total heterotrophic bacteria intake is derived from drinking water. Thus, HPC bacteria in drinking water do not represent a significant exposure of total HPC bacteria in the average diet of consumers in the United States. © 2004 Elsevier Ltd. All rights reserved.
• Stine, S. W., Vladich, F. D., Pepper, I. L., & Gerba, C. P. (2005). Development of a method for the concentration and recovery of microsporidia from tap water. Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and Environmental Engineering, 40(5), 913-925.
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  PMID: 15887563;Abstract: Microsporidia are obligate intracellular parasites. Microsporidian spores infect a wide variety of hosts, including humans. The spores may be found in infected hosts' urine and feces, thus waterborne transmission is possible. This study details method development for the detection of microsporidia in tap water. In this study, filtration, centrifugation, purification, and detection parameters were optimized for the detection of microsporidia. The Pall-Gelman Envirocheck sampling capsule (Pall Gelman, Ann Arbor, MI) was chosen as the filter element. Optimal centrifugal force for spore recovery was 1500 × g. Additionally, it was determined that eluting microsporidia spores in a detergent elution buffer solution had a detrimental effect on spore recovery. A direct examination of the concentrate resulted in a greater recovery with less variability than subjecting the sample concentrate to a Percoll-sucrose density gradient purification step. The staining method employed for the detection spores was Calcofluor white (Sigma, St. Louis, MO). Percent recoveries for 10 L tap water samples (n = 5) using the Envirocheck sampling capsule without a density gradient purification step were 26.1 ± 13.4 compared to 25 ± 13.8 for samples subjected to a density gradient purification step.
• Tanner, B. D., Brooks, J. P., Haas, C. N., Gerba, C. P., & Pepper, I. L. (2005). Bioaerosol emission rate and plume characteristics during land application of liquid class B biosolids. Environmental Science and Technology, 39(6), 1584-1590.
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  PMID: 15819213;Abstract: This study investigated bioaerosol emission rates and plume characteristics of bioaerosols generated during land application of liquid Class B biosolids. In addition, it compared the rate of aerosolization of coliphages and total coliform bacteria during land application of liquid Class B biosolids to the rate of aerosolization during land application of groundwater inoculated with similar concentrations of Escherichia co//and coliphage MS2. Air samples were taken immediately downwind of a spray applicator as it applied liquid (∼8% solids) biosolids to farmland near Tucson, Arizona. Air samples were also collected immediately downwind of groundwater seeded with MS2 and E. coli applied to land in an identical manner. Air samples, collected with liquid impingers, were taken in horizontal and vertical alignment with respect to the passing spray applicator. Vertical and horizontal sample arrays made it possible to calculate the flux of microorganisms through a virtual plane of air samplers, located 2 m downwind of the passing spray applicator. Neither coliphages nor coliform bacteria were detected in air downwind of spray application of liquid Class B biosolids. Based on limits of detection for the methodology, the rate of aerosolization during land application of liquid biosolids was calculated to be less than 33 plaque forming units (PFU) of coliphage and 10 colony forming units (CFU) of coliform bacteria per meter traveled by the spray applicator. The rate of aerosolization during land application of seeded groundwater was found to be, on average, 2.02 × 103 CFU E. coli and 3.86 × 103 PFU MS2 aerosolized per meter traveled by the spray applicator. This is greater aerosolization than was observed during land application of biosolids. Because concentrations of coliphages and coliforms were similar in the liquid biosolids and the seeded water, it was concluded that some property of biosolids reduces aerosolization of microorganisms relative to groundwater. Additional experiments utilizing a novel air sampling protocol showed that the duration of bioaerosol exposure immediately (2 m) downwind of biosolids spray application is brief and the plume of bioaerosols generated is discrete. Additional air samples showed that aerosolization of coliphages and coliform bacteria after liquid biosolids have been applied to land does not occur at detectable levels. © 2005 American Chemical Society.
• Zaleski, K. J., Josephson, K. L., Gerba, C. P., & Pepper, I. L. (2005). Potential regrowth and recolonization of salmonellae and indicators in biosolids and biosolid-amended soil. Applied and Environmental Microbiology, 71(7), 3701-3708.
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  PMID: 16000779;PMCID: PMC1169032;Abstract: This study evaluated the potential for conversion of Class B to Class A biosolids with respect to salmonellae and fecal coliforms during solar drying in concrete lined drying beds. Anaerobically (8% solids) and aerobically (2% solids) digested Class B biosolids were pumped into field-scale drying beds, and microbial populations and environmental conditions were monitored. Numbers of fecal coliforms and salmonellae decreased as temperature and rate of desiccation increased. After 3 to 4 weeks, Class A requirements were achieved in both biosolids for the pathogens and the indicators. However, following rainfall events, significant increase in numbers was observed for both fecal coliforms and salmonellae. In laboratory studies, regrowth of fecal coliforms was observed in both biosolids and biosolid-amended soil, but the regrowth of salmonellae observed in the concrete-lined drying beds did not occur. These laboratory studies demonstrated that pathogens decreased in numbers when soil was amended with biosolids. Based on serotyping, the increased numbers of salmonellae seen in the concrete lined drying beds following rainfall events was most likely due to recolonization due to contamination from fecal matter introduced by animals and not from regrowth of salmonellae indigenous to biosolids. Overall, we conclude that the use of concrete-lined beds created a situation in which moisture added as rainfall accumulated in the beds, promoting the growth of fecal coliforms and salmonellae added from external sources. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
• Brooks, J. P., Tanner, B. D., Josephson, K. L., Gerba, C. P., & Pepper, I. L. (2004). Bioaerosols from the land application of biosolids in the desert southwest USA. Water Science and Technology, 50(1), 7-12.
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  PMID: 15318479;Abstract: This study evaluated bioaerosol emissions during land application of Class B biosolids in and around Tucson, Arizona, to aid in developing models of the fate and transport of bioaerosols generated from the land application of biosolids. Samples were collected for 20 min at distances between 2 m and 20 m downwind of point sources, using an SKC BioSampler® impinger. A total of six samples were collected per sampling event, which consisted of a biosolid spray applicator applying liquid biosolids to a cotton field. Each application represented one exposure. Samples were collected in deionised water amended with peptone and antifoam agent. Ambient weather conditions were also monitored every 10 min following initiation of sampling. Concurrently with downwind samples, background (ambient) air samples were collected to compensate for any ambient airborne microorganisms. In addition, biosolids samples were collected for analysis of target indicator and pathogenic organisms. Soil samples were also collected and analysed. Significant numbers of heterotrophic plate count (HPC) bacteria were found in air samples collected during the biosolid application process. These could have arisen from soil particles being aerosolised during the land application process. Aerosolised soil may contribute significantly to the amount of aerosolised microorganisms. Soil particles may be able to more readily aerosolise, due to their low density, small particle size and low mass. Aerosolised HPC bacteria found during biosolids land application were similar to those found during normal tractor operation on non-biosolids applied fields. Coliforms and coliphages were not routinely detected even though they were found to be present in the biosolids at relatively high concentrations, 106 and 104/g (dry weight) of biosolids respectively. This could be due to the die-off rate of aerosolised Gram-negative bacteria or sorption to the solid portion of the biosolids. Low numbers of aerosolised coliphages may likewise be due to sorption phenomena. We theorise that only organisms in the aqueous phase of the biosolids were available to desorb and be aerosolised. Animal viruses, which were not detected in the biosolids, were likewise not detected in the aerosol samples. Clostridium perfringens was detected in only a small percent of aerosol samples although it was detected during all weather conditions; other microorganisms were detected during more favourable environmental conditions (relative humidity >10%). Despite the fact that many of these organisms were present in the biosolids at significant concentrations, their presence in bioaerosols generated during the land application of biosolids was limited to only a small percentage of samples. Bacteria as well as viruses may sorb to biosolids, which contain a high percentage of organic matter, and desorption during land application of biosolids may not readily take place; therefore, these microorganisms may not be readily aerosolised. © IWA Publishing 2004.
• Gentry, T. J., Josephson, K. L., & Pepper, I. L. (2004). Functional establishment of introduced chlorobenzoate degraders following bioaugmentation with newly activated soil: Enhanced contaminant remediation via activated soil bioaugmentation. Biodegradation, 15(1), 67-75.
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  PMID: 14971859;Abstract: Introduced degraders often do not survive when applied to polluted sites; however, the potential for successful bioaugmentation may be increased if newly activated soil (containing indigenous degrader populations recently exposed to the contaminant) or potentially active soil (containing indigenous degrader populations not previously exposed to the contaminant) is used as the inoculant. To investigate this concept, Madera and Oversite soils were amended with 0 or 500 micrograms of 2-, 3-, or 4-chlorobenzoate per gram soil. The Madera degraded 2-chlorobenzoate while the Oversite degraded 3- and 4-chlorobenzoate. After 22 days of incubation, non-active soils that had not degraded chlorobenzoate were bioaugmented with the appropriate activated soil that had been exposed to and degraded chlorobenzoate. Thus, Oversite soil that had not degraded 2-chlorobenzoate was bioaugmented with Madera soil that had degraded 2-chlorobenzoate. Likewise, Madera soil that had not degraded 3- or 4-chlorobenzoate was bioaugmented with the Oversite soil that had degraded 3- or 4-chlorobenzoate. Additionally, the non-active soils were bioaugmented with the corresponding potentially active soils. The Oversite soil amended with activated Madera soil degraded the 2-chlorobenzoate within 3 days of bioaugmentation. The Madera soil amended with activated Oversite soils degraded the 3- and 4-chlorobenzoate within 20 and 6 days, respectively. Large degrader populations developed in microcosms bioaugmented with activated soil, and shifts in the 3- and 4-CB degrader community structures occurred following bioaugmentation. In contrast, bioaugmentation with potentially active soil did not impact degradation. The results indicate the potential for bioaugmentation with newly activated soil to enhance contaminant degradation.
• Gentry, T. J., Rensing, C., & Pepper, I. L. (2004). New approaches for bioaugmentation as a remediation technology. Critical Reviews in Environmental Science and Technology, 34(5), 447-494.
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  Abstract: Bioaugmentation is commonly employed as a remediation technology. However, numerous studies indicate that introduced microorganisms often do not survive in the environment and thus do not increase contaminant remediation. This review details several new approaches that may increase the persistence and activity of exogenous microorganisms and/or genes following introduction into the environment. These techniques include: (1) bioaugmentation with cells encapsulated in a carrier such as alginate; (2) gene bioaugmentation where the goal is for the added inoculant to transfer remediation genes to indigenous microorganisms; (3) rhizosphere bioaugmentation where the microbial inoculant is added to the site along with a plant that serves as a niche for the inoculant's growth; and (4) phytoaugmentation where the remediation genes are engineered directly into a plant for use in remediation without a microbial inoculant. Additionally, the review discusses the generation of genetically engineered microorganisms for use in bioaugmentation along with methods for the control of the engineered microorganisms in the environment, and the potential effects of the release on indigenous organisms. Various methods for the detection of introduced microorganisms such as real-time polymerase chain reaction (PCR) and reporter genes are also addressed. Ultimately, these new approaches may broaden the application of bioaugmentation as a remediation technology.
• Gentry, T. J., Wang, G., Rensing, C., & Pepper, I. L. (2004). Chlorobenzoate-degrading bacteria in similar pristine soils exhibit different community structures and population dynamics in response to anthropogenic 2-, 3-, and 4-chlorobenzoate levels. Microbial Ecology, 48(1), 90-102.
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  PMID: 15085300;Abstract: A study was conducted to determine the diversity of 2-, 3-, and 4-chlorobenzoate (CB) degraders in two pristine soils with similar physical and chemical characteristics. Surface soils were collected from forested sites and amended with 500 μg of 2-, 3-, or 4-CB g-1 soil. The CB levels and degrader numbers were monitored throughout the study. Degraders were isolated, grouped by DNA fingerprints, identified via 16S rDNA sequences, and screened for plasmids. The CB genes in selected degraders were isolated and/or sequenced. In the Madera soil, 2-CB and 4-CB degraded within 11 and 42 d, respectively, but 3-CB did not degrade. In contrast, 3-CB and 4-CB degraded in the Oversite soil within 14 and 28 d, respectively, while 2-CB did not degrade. Approximately 107 CFU g-1 of degraders were detected in the Madera soil with 2-CB, and the Oversite soil with 3- and 4-CB. No degraders were detected in the Madera soil with 4-CB even though the 4-CB degraded. Nearly all of the 2-CB degraders isolated from the Madera soil were identified as a Burkholderia sp. containing chromosomally encoded degradative genes. In contrast, several different 3- and 4-CB degraders were isolated from the Oversite soil, and their populations changed as CB degradation progressed. Most of these 3-CB degraders were identified as Burkholderia spp. while the majority of 4-CB degraders were identified as Bradyrhizobium spp. Several of the 3-CB degraders contained the degradative genes on large plasmids, and there was variation between the plasmids in different isolates. When a fresh sample of Madera soil was amended with 50, 100, or 200 μg 3-CB g-1, 3-CB degradation occurred, suggesting that 500 μg 3-CB g-1 was toxic to the degraders. Also, different 3-CB degraders were isolated from the Madera soil at each of the three lower levels of 3-CB. No 2-CB degradation was detected in the Oversite soil even at lower 2-CB levels. These results indicate that the development of 2-, 3-, and 4-CB degrader populations is site-specific and that 2-, 3-, and 4-CB are degraded by different bacterial populations in pristine soils. These results also imply that the microbial ecology of two soils that develop under similar biotic and abiotic environments can be quite different.
• Pepper, I. L., Rusin, P., Quintanar, D. R., Haney, C., Josephson, K. L., & Gerba, C. P. (2004). Tracking the concentration of heterotrophic plate count bacteria from the source to the consumer's tap. International Journal of Food Microbiology, 92(3), 289-295.
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  PMID: 15145587;Abstract: The goal of this project was to quantify the concentration of heterotrophic plate count (HPC) bacteria within water reaching consumer's taps, and from the sources used by a major utility serving the City of Tucson, AZ. With this information, the amounts and sources of HPC bacteria consumed at the tap could be determined. Samples of water were collected on a monthly basis from two well fields, the CAVSARP recovery well field and Southern Avra Valley well field which serves as one of the groundwater sources for Tucson, AZ, and the distribution system which serves the same homes from which tap water was also tested. The average concentration of HPC in source waters within Southern Avra Valley Wells was 56 CFU/ml (range 1-1995/ml). From the CAVSARP recovery well field, corresponding values were 38 CFU/ml (1 to 502 CFU/ml). Unblended groundwater in the chlorinated distribution system averaged 22 CFU/ml (range 1-794). Blended water at the chlorinated distribution site averaged 47 CFU/ml (range 10-158). There was a major shift in the percentage of gram negative to gram-positive bacteria from the wells to the distribution system, to the tap. In the surface CAP source water, 76% of the bacteria were gram-negative compared to 27% gram-negative in the CAVSARP recovery wells. In contrast, Avra Valley wells contained 17% gram-negative bacteria. In both the Tucson groundwater distribution sites and blended distribution sites, the corresponding number of gram negative bacteria was 12%. Finally at the tap, only 0.2% of the bacteria were gram-negative. The average number of bacteria in household taps averaged 3072 HPC/ml and was equal or greater than 500 ml 68% of the time. This study shows that the number of HPC bacteria increases dramatically from the distribution system to the consumers tap. Thus, the major source of bacteria ingested by the average consumer in Tucson originates from bacteria within the household distribution system or the household tap, rather than from source waters or the distribution system. It is also clear that consumers' regularly consume more than 500 HPC/ml from drinking water taken from the household tap. © 2003 Elsevier B.V. All rights reserved.
• Pepper, I., Brooks, J. P., Tanner, B. D., Josephson, K. L., Gerba, C. P., & Pepper, I. L. (2004). Bioaerosols from the land application of biosolids in the desert southwest USA. Water science and technology : a journal of the International Association on Water Pollution Research, 50(1).
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  This study evaluated bioaerosol emissions during land application of Class B biosolids in and around Tucson, Arizona, to aid in developing models of the fate and transport of bioaerosols generated from the land application of biosolids. Samples were collected for 20 min at distances between 2 m and 20 m downwind of point sources, using an SKC BioSampler impinger. A total of six samples were collected per sampling event, which consisted of a biosolid spray applicator applying liquid biosolids to a cotton field. Each application represented one exposure. Samples were collected in deionised water amended with peptone and antifoam agent. Ambient weather conditions were also monitored every 10 min following initiation of sampling. Concurrently with downwind samples, background (ambient) air samples were collected to compensate for any ambient airborne microorganisms. In addition, biosolids samples were collected for analysis of target indicator and pathogenic organisms. Soil samples were also collected and analysed. Significant numbers of heterotrophic plate count (HPC) bacteria were found in air samples collected during the biosolid application process. These could have arisen from soil particles being aerosolised during the land application process. Aerosolised soil may contribute significantly to the amount of aerosolised microorganisms. Soil particles may be able to more readily aerosolise, due to their low density, small particle size and low mass. Aerosolised HPC bacteria found during biosolids land application were similar to those found during normal tractor operation on non-biosolids applied fields. Coliforms and coliphages were not routinely detected even though they were found to be present in the biosolids at relatively high concentrations, 10(6) and 10(4)/g (dry weight) of biosolids respectively. This could be due to the die-off rate of aerosolised Gram-negative bacteria or sorption to the solid portion of the biosolids. Low numbers of aerosolised coliphages may likewise be due to sorption phenomena. We theorise that only organisms in the aqueous phase of the biosolids were available to desorb and be aerosolised. Animal viruses, which were not detected in the biosolids, were likewise not detected in the aerosol samples. Clostridium perfringens was detected in only a small percent of aerosol samples although it was detected during all weather conditions; other microorganisms were detected during more favourable environmental conditions (relative humidity >10%). Despite the fact that many of these organisms were present in the biosolids at significant concentrations, their presence in bioaerosols generated during the land application of biosolids was limited to only a small percentage of samples. Bacteria as well as viruses may sorb to biosolids, which contain a high percentage of organic matter, and desorption during land application of biosolids may not readily take place; therefore, these microorganisms may not be readily aerosolised.
• Pepper, I., Gentry, T. J., Josephson, K. L., & Pepper, I. L. (2004). Functional establishment of introduced chlorobenzoate degraders following bioaugmentation with newly activated soil. Enhanced contaminant remediation via activated soil bioaugmentation. Biodegradation, 15(1).
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  Introduced degraders often do not survive when applied to polluted sites; however, the potential for successful bioaugmentation may be increased if newly activated soil (containing indigenous degrader populations recently exposed to the contaminant) or potentially active soil (containing indigenous degrader populations not previously exposed to the contaminant) is used as the inoculant. To investigate this concept, Madera and Oversite soils were amended with 0 or 500 micrograms of 2-, 3-, or 4-chlorobenzoate per gram soil. The Madera degraded 2-chlorobenzoate while the Oversite degraded 3- and 4-chlorobenzoate. After 22 days of incubation, non-active soils that had not degraded chlorobenzoate were bioaugmented with the appropriate activated soil that had been exposed to and degraded chlorobenzoate. Thus, Oversite soil that had not degraded 2-chlorobenzoate was bioaugmented with Madera soil that had degraded 2-chlorobenzoate. Likewise, Madera soil that had not degraded 3- or 4-chlorobenzoate was bioaugmented with the Oversite soil that had degraded 3- or 4-chlorobenzoate. Additionally, the non-active soils were bioaugmented with the corresponding potentially active soils. The Oversite soil amended with activated Madera soil degraded the 2-chlorobenzoate within 3 days of bioaugmentation. The Madera soil amended with activated Oversite soils degraded the 3- and 4-chlorobenzoate within 20 and 6 days, respectively. Large degrader populations developed in microcosms bioaugmented with activated soil, and shifts in the 3- and 4-CB degrader community structures occurred following bioaugmentation. In contrast, bioaugmentation with potentially active soil did not impact degradation. The results indicate the potential for bioaugmentation with newly activated soil to enhance contaminant degradation.
• Pepper, I., Gentry, T. J., Wang, G., Rensing, C., & Pepper, I. L. (2004). Chlorobenzoate-degrading bacteria in similar pristine soils exhibit different community structures and population dynamics in response to anthropogenic 2-, 3-, and 4-chlorobenzoate levels. Microbial ecology, 48(1).
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  A study was conducted to determine the diversity of 2-, 3-, and 4-chlorobenzoate (CB) degraders in two pristine soils with similar physical and chemical characteristics. Surface soils were collected from forested sites and amended with 500 microg of 2-, 3-, or 4-CB g(-1) soil. The CB levels and degrader numbers were monitored throughout the study. Degraders were isolated, grouped by DNA fingerprints, identified via 16S rDNA sequences, and screened for plasmids. The CB genes in selected degraders were isolated and/or sequenced. In the Madera soil, 2-CB and 4-CB degraded within 11 and 42 d, respectively, but 3-CB did not degrade. In contrast, 3-CB and 4-CB degraded in the Oversite soil within 14 and 28 d, respectively, while 2-CB did not degrade. Approximately 10(7) CFU g(-1) of degraders were detected in the Madera soil with 2-CB, and the Oversite soil with 3- and 4-CB. No degraders were detected in the Madera soil with 4-CB even though the 4-CB degraded. Nearly all of the 2-CB degraders isolated from the Madera soil were identified as a Burkholderia sp. containing chromosomally encoded degradative genes. In contrast, several different 3- and 4-CB degraders were isolated from the Oversite soil, and their populations changed as CB degradation progressed. Most of these 3-CB degraders were identified as Burkholderia spp. while the majority of 4-CB degraders were identified as Bradyrhizobium spp. Several of the 3-CB degraders contained the degradative genes on large plasmids, and there was variation between the plasmids in different isolates. When a fresh sample of Madera soil was amended with 50, 100, or 200 microg 3-CB g(-1), 3-CB degradation occurred, suggesting that 500 microg 3-CB g(-1) was toxic to the degraders. Also, different 3-CB degraders were isolated from the Madera soil at each of the three lower levels of 3-CB. No 2-CB degradation was detected in the Oversite soil even at lower 2-CB levels. These results indicate that the development of 2-, 3-, and 4-CB degrader populations is site-specific and that 2-, 3-, and 4-CB are degraded by different bacterial populations in pristine soils. These results also imply that the microbial ecology of two soils that develop under similar biotic and abiotic environments can be quite different.
• Pepper, I., Wang, G., Gentry, T. J., Grass, G., Josephson, K., Rensing, C., & Pepper, I. L. (2004). Real-time PCR quantification of a green fluorescent protein-labeled, genetically engineered Pseudomonas putida strain during 2-chlorobenzoate degradation in soil. FEMS microbiology letters, 233(2).
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  The potential for real-time PCR (RTm-PCR) detection of the genetically engineered strain Pseudomonas putida GN2 was studied during 2-chlorobenzoate (2-CB) degradation in three different soils. The strain contained the constructed plasmid pGN2 which encoded genes for 2-CB oxidation (cbdA) and the green fluorescent protein (gfp). P. putida GN2 numbers were assessed by plating onto 2-CB minimal media and also by RTm-PCR detection of cbdA and gfp. Addition of P. putida GN2 decreased the time required to degrade 2-CB in all tested soils by more than 7 days. The RTm-PCR estimations of P. putida GN2 numbers strongly correlated with those obtained from plate count methods during active 2-CB degradation. However, after 2-CB degradation in the soils had ceased, RTm-PCR estimations of cbdA and gfp genes were generally one order of magnitude lower than those from plate counts. These results indicate the potential for RTm-PCR to rapidly determine degrader numbers in soil following bioaugmentation but also the need to exercise caution when attempting to determine cell numbers of degraders from the RTm-PCR quantification of plasmid encoded genes after substrate is depleted.
• Wang, G., Gentry, T. J., Grass, G., Josephson, K., Rensing, C., & Pepper, I. L. (2004). Real-time PCR quantification of a green fluorescent protein-labeled, genetically engineered Pseudomonas putida strain during 2-chlorobenzoate degradation in soil. FEMS Microbiology Letters, 233(2), 307-314.
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  PMID: 15063501;Abstract: The potential for real-time PCR (RTm-PCR) detection of the genetically engineered strain Pseudomonas putida GN2 was studied during 2-chlorobenzoate (2-CB) degradation in three different soils. The strain contained the constructed plasmid pGN2 which encoded genes for 2-CB oxidation (cbdA) and the green fluorescent protein (gfp). P. putida GN2 numbers were assessed by plating onto 2-CB minimal media and also by RTm-PCR detection of cbdA and gfp. Addition of P. putida GN2 decreased the time required to degrade 2-CB in all tested soils by more than 7 days. The RTm-PCR estimations of P. putida GN2 numbers strongly correlated with those obtained from plate count methods during active 2-CB degradation. However, after 2-CB degradation in the soils had ceased, RTm-PCR estimations of cbdA and gfp genes were generally one order of magnitude lower than those from plate counts. These results indicate the potential for RTm-PCR to rapidly determine degrader numbers in soil following bioaugmentation but also the need to exercise caution when attempting to determine cell numbers of degraders from the RTm-PCR quantification of plasmid encoded genes after substrate is depleted. © 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
• Dowd, S. E., John, D., Eliopolus, J., Gerba, C. P., Naranjo, J., Klein, R., López, B., Mejía, M. d., Mendoza, C. E., & Pepper, I. L. (2003). Confirmed detection of Cyclospora cayetanesis, Encephalitozoon intestinalis and Cryptosporidium parvum in water used for drinking.. J Water Health, 1(3), 117-123.
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  PMID: 15384722;Abstract: Human enteropathogenic microsporidia (HEM), Cryptosporidium parvum, Cyclospora cayetanesis, and Giardia lamblia are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of HEM (Encephalitozoon intestinalis and Enterocytozoon bieneusi) and Cyclospora cayetanesis have not been fully elucidated due to lack of sensitive and specific environmental screening methods. The present study was undertaken with recently developed methods, to screen various water sources used for public consumption in rural areas around the city of Guatemala. Water concentrates collected in these areas were subjected to community DNA extraction followed by PCR amplification, PCR sequencing and computer database homology comparison (CDHC). All water samples screened in this study had been previously confirmed positive for Giardia spp. by immunofluorescent assay (IFA). Of the 12 water concentrates screened, 6 showed amplification of microsporidial SSU-rDNA and were subsequently confirmed to be Encephalitozoon intestinalis. Five of the samples allowed for amplification of Cyclospora 18S-rDNA; three of these were confirmed to be Cyclospora cayetanesis while two could not be identified because of inadequate sequence information. Thus, this study represents the first confirmed identification of Cyclospora cayetanesis and Encephalitozoon intestinalis in source water used for consumption. The fact that the waters tested may be used for human consumption indicates that these emerging protozoa may be transmitted by ingestion of contaminated water.
• John, D. E., Nwachuku, N., Pepper, I. L., & Gerba, C. P. (2003). Development and optimization of a quantitative cell culture infectivity assay for the microsporidium Encephalitozoon intestinalis and application to ultraviolet light inactivation. Journal of Microbiological Methods, 52(2), 183-196.
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  PMID: 12459239;Abstract: Microsporidia are unique parasites recognized as a major cause of intestinal illness among immunocompromised patients and occasionally in otherwise healthy hosts. These organisms have been detected in water and are likely transmitted by the fecal-oral route. The most common human pathogenic microsporidia for which cell culture methods have been established is Encephalitozoon intestinalis. This study describes the development of a quantitative cell culture infectivity assay for E. intestinalis and its application to assess inactivation by ultraviolet (UV) light irradiation. The method described here employs calcofluor white, a fluorescent brightener that targets the chitin spore wall, to visualize groups of developing spores in order to confirm infectivity. Serial dilutions of the spore suspension were seeded into tissue culture well slides containing RK-13 cells. Slides were then rinsed, fixed in methanol and stained with calcofluor white and examined microscopically. Large masses of developing spores were easily visible on infected cell monolayers. Positive and negative wells at each dilution step were used to quantify the number of infectious spores in the original suspension using a most-probable-number (MPN) statistical analysis. This assay was used to evaluate the disinfecting potential of ultraviolet light on E. intestinalis spores in water. The ultraviolet dose required for a 3-log10 or 99.9% reduction in the number of infective spores was determined to be 8.43 mW s/cm2. © 2003 Elsevier Science B.V. All rights reserved.
• Lewis, D. L., Gattie, D. K., Rusin, P. A., Maxwell, S. L., Brooks, J. P., Gerba, C. P., & Pepper, I. L. (2003). Comment on "Evidence for the Absence of Staphylococcus aureus in the Land Applied Biosolids" [1] (multiple letters). Environmental Science and Technology, 37(24), 5835-5836.
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  PMID: 14717203;
• Pepper, I., Rusin, P. A., Maxwell, S. L., Brooks, J. P., Gerba, C. P., & Pepper, I. L. (2003). Evidence for the absence of Staphylococcus aureus in land applied biosolids. Environmental science & technology, 37(18).
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  Staphylococcus aureus is an important human pathogen both within the hospital setting and as a community-acquired infection. Recently there has been concern that land applied biosolids may transmit S. aureus. However, no scientific data are available to document whether biosolids are a source of S. aureus. To determine if S. aureus is present in biosolids, we collected samples from 15 sites across the United States. Samples analyzed were as follows: 3 raw untreated sewage samples and 2 undigested primary sewage sludge samples; 23 different biosolid samples; and 27 aerosols obtained during biosolid land application (biosolid aerosols). Although S. aureus were detected in raw sewage samples, none were found in any of the treated biosolids nor in any biosolid aerosol samples. These results suggest that biosolids are not a likely source of S. aureus human exposure or infection.
• Rusin, P. A., Maxwell, S. L., Brooks, J. P., Gerba, C. P., & Pepper, I. L. (2003). Evidence for the absence of Staphylococcus aureus in land applied biosolids. Environmental Science and Technology, 37(18), 4027-4030.
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  PMID: 14524431;Abstract: Staphylococcus aureus is an important human pathogen both within the hospital setting and as a community-acquired infection. Recently there has been concern that land applied biosolids may transmit S. aureus. However, no scientific data are available to document whether biosolids are a source of S. aureus. To determine if S. aureus is present in biosolids, we collected samples from 15 sites across the United States. Samples analyzed were as follows: 3 raw untreated sewage samples and 2 undigested primary sewage sludge samples; 23 different biosolid samples; and 27 aerosols obtained during biosolid land application (biosolid aerosols). Although S. aureus were detected in raw sewage samples, none were found in any of the treated biosolids nor in any biosolid aerosol samples. These results suggest that biosolids are not a likely source of S. aureus human exposure or infection.
• Gerba, C. P., Pepper, I. L., & III, L. W. (2002). A risk assessment of emerging pathogens of concern in the land application of biosolids. Water Science and Technology, 46(10), 225-230.
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  PMID: 12479475;Abstract: Since the development of the United States Environmental Protection Agency's 503 biosolids Rule, which includes treatment requirements to reduce the threat of pathogen transmission, many new pathogens have been recognized which could be transmitted by biosolids. A risk analysis was performed to assess which emerging pathogens would be most likely to survive treatments required for Class B biosolids before land application. The literature was reviewed on the resistance of emerging pathogens to temperature and other environmental factors to assess their probability of surviving various biosolids treatment processes. In addition existing information on occurrence in biosolids and dose response models for each pathogen was reviewed. It was concluded that adenoviruses and hepatitis A virus are the most thermally resistant viruses and can survive for prolonged periods in the environment. The protozoan parasites microsporidia and Cyclospora were unlikely to survive the temperatures achieved in anaerobic digestion and do not survive well under low moisture conditions. A risk model was used to assess the risk of infection and illness from enteric viruses after application of class B biosolids.
• Marlowe, E. M., Wang, J., Pepper, I. L., & Maier, R. M. (2002). Application of a reverse transcription-PCR assay to monitor regulation of the catabolic nahAc gene during phenanthrene degradation. Biodegradation, 13(4), 251-260.
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  PMID: 12521289;Abstract: Biodegradation of polycyclic aromatic hydrocarbons (PAH), such as phenanthrene, in environmental samples is often limited by low bioavailability which results from a combination of low aqueous solubility and/or high sorption. The purpose of this study was to investigate the influence of agents that increase PAH bioavailability on expression of the PAH catabolic gene nahAc. Phenanthrene was used as a model PAH and Pseudomonas putida PpG7, which contains the NAH7 plasmid that encodes the genes responsible for naphthalene and phenanthrene degradation, was used as a model degrader. PAH bioavailability was altered by the addition of two biosurfactants, rhamnolipid and hydroxypropyl-β-cyclodextrin (HPCD). Gene expression was determined by extraction of bacterial mRNA followed by RT-PCR amplification of two transcripts; nahAc, a naphthalene dioxygenase gene, and rpoD, a housekeeping gene. Results indicate that the lag period preceding nahAc gene induction decreased from 312 to 48 h in the presence of biosurfactants. Expression of the nahAc gene, as measured by RT-PCR, in the presence of surfactants was bimodal on a temporal basis, indicating that induction stopped briefly during biodegradation. Cessation of induction could have resulted from the up-regulation of alternate pathways or the accumulation of toxic intermediates. In contrast, expression of the rpoD gene was maintained throughout the duration of each experiment. This research demonstrates that the use of a gene expression assay to monitor the impact of substrate bioavailability on substrate utilization provides unique information concerning the biodegradation process that cannot be obtained from more traditional biodegradation assays such as cell growth or substrate disappearance. Gene expression assays also have the potential for use in assessing the impact of other environmental factors on biodegradation.
• Newby, D. T., & Pepper, I. L. (2002). Dispersal of plasmid pJP4 in unsaturated and saturated 2,4-dichlorophenoxyacetic acid contaminated soil. FEMS Microbiology Ecology, 39(2), 157-164.
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  PMID: 19709195;Abstract: Little is known regarding plasmid fate within contaminated soils. Column studies were used to evaluate dissemination of plasmid pJP4 under unsaturated or saturated flow conditions in a 2,4-dichlorophenoxyacetic acid (2,4-D) contaminated soil. Columns were destructively sampled following 1 week of percolation to assess the vertical distribution of donors, transconjugants, and 2,4-D concentrations within the soil. In unsaturated soil, pJP4 was detected in both culturable donor and transconjugant cells within soil 10.5 cm from the inoculated end of the column. In saturated soil, no transconjugants were detected; however, donors were found throughout the entire length of the column (30.5 cm). These results suggest that donor transport, particularly in conjunction with plasmid transfer to indigenous recipients, allows for significant dispersal of introduced genes through contaminated soil. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
• Pepper, I. L., & Gentry, T. J. (2002). Incidence of Bacillus anthracis in soil. Soil Science, 167(10), 627-635.
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  Abstract: Interest in anthrax has increased recently due to its use in bioterrorism attacks. Bacillus anthracis, the causative agent of anthrax, is genetically similar to other Bacillus spp. that occur in the environment, and is known to persist in soil for years in the form of spores. In many soils, naturally occurring anthrax infections tend to occur during dry periods following a wet period. The "incubator theory" suggests that spores are concentrated in low-lying areas during rainfall events and animals are subsequently exposed to contaminated soil during foraging in dry periods. It is currently believed that B. anthracis spores require a host for germination, and thus do not undergo proliferation cycles in the soil. There is a potential for B. anthracis spores to be transported as an aerosol, but human infection due to inhalation of spores is unlikely given the high minimum infectious dose required to cause disease. Historically, naturally occurring anthrax infections in humans have most commonly occurred due to contact with diseased animals or animal products. However, B. anthracis has been developed as a biological weapon with accidental and intentional releases resulting in human death. With the development of an anthrax vaccine, anthrax outbreaks have generally been controlled in developed countries; however, anthrax is still a major problem in many parts of the world. The advent of molecular techniques has enhanced the detection of B. anthracis spores, which is now possible in less than 1 h. However, due to the persistence of spores, it is difficult to eliminate B. anthracis contamination from the environment. There remains a need for additional research on anthrax in several areas including evaluation of the conditions that favor B. anthracis survival in soil, determination of whether B. anthracis undergoes a growth cycle in soil, and determination of the potential for transfer of B. anthracis virulence genes to other soil microorganisms.
• Pepper, I. L., Gentry, T. J., Newby, D. T., Roane, T. M., & Josephson, K. L. (2002). The role of cell bioaugmentation and gene bioaugmentation in the remediation of co-contaminated soils. Environmental Health Perspectives, 110(SUPPL. 6), 943-946.
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  PMID: 12634123;PMCID: PMC1241276;Abstract: Soils co-contaminated with metals and organics present special problems for remediation. Metal contamination can delay or inhibit microbial of organic pollutants such that for effective in situ biodegradation, bioaugmentation is necessary. We monitored the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) or 3-chlorobenzoate (3-CB) in two different soils with and without cadmium (Cd) contamination. Additionally, we evaluated the ability of bioaugmentation to enhance organic degradation in these co-contaminated soils. Finally, we determined whether enhanced degradation was due to survival of the introduced organism (cell bioaugmentation) or plasmid transfer to indigenous microbial populations (gene bioaugmentation). In Brazito soil, dual inoculation with a Cd-resistant bacterium plus a known 2,4-D-degrading bacterium, Ralstonia eutropha JM134, enhanced 2,4-D-degradation. Escherichia coli D11, which lacks chromosomal genes necessary for complete 2,4-D mineralization, was used for gene bioaugmentation in Madera soil. Significant gene transfer of the plasmid to the indigenous populations was observed, and the rate of 2,4-D degradation was enhanced relative to that of controls. Cell bioaugmentation mentation was further demonstrated when Comamonas testosteroni was used to enhance biodegradation of 3-CB in Madera soil. In this case no transfer of plasmid pBRC60 to indigenous soil recipients was observed. For the Madera soil, nonbioaugmented samples ultimately showed complete 2,4-D degradation. In contrast, nonbioaugmented Brazito soils showed incomplete 2,4-D degradation. These studies are unique in showing that both cell bioaugmentation and gene bioaugmentation can be effective in enhancing organic degradation in co-contaminated soils. Ultimately, the bioaugmentation strategy may depend on the degree of contamination and the time frame available for remediation.
• Pepper, I., Newby, D. T., & Pepper, I. L. (2002). Dispersal of plasmid pJP4 in unsaturated and saturated 2,4-dichlorophenoxyacetic acid contaminated soil. FEMS microbiology ecology, 39(2).
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  Little is known regarding plasmid fate within contaminated soils. Column studies were used to evaluate dissemination of plasmid pJP4 under unsaturated or saturated flow conditions in a 2,4-dichlorophenoxyacetic acid (2,4-D) contaminated soil. Columns were destructively sampled following 1 week of percolation to assess the vertical distribution of donors, transconjugants, and 2,4-D concentrations within the soil. In unsaturated soil, pJP4 was detected in both culturable donor and transconjugant cells within soil 10.5 cm from the inoculated end of the column. In saturated soil, no transconjugants were detected; however, donors were found throughout the entire length of the column (30.5 cm). These results suggest that donor transport, particularly in conjunction with plasmid transfer to indigenous recipients, allows for significant dispersal of introduced genes through contaminated soil.
• Rensing, C., Newby, D. T., & Pepper, I. L. (2002). The role of selective pressure and selfish DNA in horizontal gene transfer and soil microbial community adaptation. Soil Biology and Biochemistry, 34(3), 285-296.
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  Abstract: Recent advances in genome sequencing and horizontal gene transfer in soil have led to new insights on soil microbial community adaptation. In this review, we document and evaluate the role of selective pressure and selfish DNA in propagating horizontal gene transfer in soil through the use of a model system involving the organic pesticide 2,4-dichlorophenoxyacetic acid and the metal cadmium. This review provides a theoretical framework for microbial adaptation, wherein it is the selfish nature of DNA that provides the initial stimulus for adaptation rather than the host cells themselves. Subsequent to selfish DNA transfer, if useful to host cells, the transferred DNA may become integrated into the host chromosome. Following these events, ultimately the growth of more fit individuals within the newly created ecological niche allows for adaptation of the soil microbial community. © 2002 Elsevier Science Ltd. All rights reserved.
• Thurston-Enriquez, J., Watt, P., Dowd, S. E., Enriquez, R., Pepper, I. L., & Gerba, C. P. (2002). Detection of protozoan parasites and microsporidia in irrigation waters used for crop production. Journal of Food Protection, 65(2), 378-382.
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  PMID: 11848571;Abstract: The occurrence of human pathogenic parasites in irrigation waters used for food crops traditionally eaten raw was investigated. The polymerase chain reaction was used to detect human pathogenic microsporidia in irrigation waters from the United States and several Central American countries. In addition, the occurrence of both Cryptosporidium oocysts and Giardia cysts was determined by immunofluorescent techniques. Twenty-eight percent of the irrigation water samples tested positive for microsporidia, 60% tested positive for Giardia cysts, and 36% tested positive for Cryptosporidium oocysts. The average concentrations in samples from Central America containing Giardia cysts and Cryptosporidium oocysts were 559 cysts and 227 oocysts per 100 liters. In samples from the United States, averages of 25 Giardia cysts per 100 liters and
• Gentry, T. J., Newby, D. T., Josephson, K. L., & Pepper, I. L. (2001). Soil microbial population dynamics following bioaugmentation with a 3-chlorobenzoate-degrading bacterial culture: Bioaugmentation effects on soil microorganisms. Biodegradation, 12(5), 349-357.
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  PMID: 11998824;Abstract: Changes in microbial populations were evaluated following inoculation of contaminated soil with a 3-chlorobenzoate degrader. Madera sandy loam was amended with 0, 500, or 1000 μg 3-chlorobenzoate g-1 dry soil. Selected microcosms were inoculated with the degrader Comamonas testosteroni BR60. Culturable bacterial degraders were enumerated on minimal salts media containing 3-chlorobenzoate. Culturable heterotrophic bacteria were enumerated on R2A. Isolated degraders were grouped by enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction fingerprints and identified based on 16S ribosomal-DNA sequences. Bioaugmentation increased the rate of degradation at both levels of 3-chlorobenzoate. In both the 500 and 1000 μg 3-chlorobenzoate g-1 dry soil inoculated microcosms, degraders increased from the initial inoculum and decreased following degradation of 3-CB. Inoculation delayed the development of indigenous 3-chlorobenzoate degrading populations. It is unclear if inoculation altered the composition of indigenous degrader populations. In the uninoculated soil, degraders increased from undetectable levels to 6.6 × 107 colony-forming-units g-1 dry soil in the 500 μg 3-chlorobenzoate g-1 dry soil microcosms, but none were detected in the 1000 μg 3-chlorobenzoate g-1 dry soil microcosms. Degraders isolated from uninoculated soil were identified as one of two distinct Burkholderia species. In the uninoculated soil, numbers of culturable heterotrophic bacteria initially decreased following addition of 1000 μg 3-chlorobenzoate g-1 dry soil. Inoculation with C. testosteroni reduced this negative impact on culturable bacterial numbers. The results indicate that bioaugmentation may not only increase the rate of 3-chlorobenzoate degradation but also reduce the deleterious effects of 3-chlorbenzoate on indigenous soil microbial populations.
• Pepper, I., Gentry, T. J., Newby, D. T., Josephson, K. L., & Pepper, I. L. (2001). Soil microbial population dynamics following bioaugmentation with a 3-chlorobenzoate-degrading bacterial culture. Bioaugmentation effects on soil microorganisms. Biodegradation, 12(5).
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  Changes in microbial populations were evaluated following inoculation of contaminated soil with a 3-chlorobenzoate degrader. Madera sandy loam was amended with 0, 500, or 1,000 microg 3-chlorobenzoate g(-1) dry soil. Selected microcosms were inoculated with the degrader Comamonas testosteroni BR60. Culturable bacterial degraders were enumerated on minimal salts media containing 3-chlorobenzoate. Culturable heterotrophic bacteria were enumerated on R2A. Isolated degraders were grouped by enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction fingerprints and identified based on 16S ribosomal-DNA sequences. Bioaugmentation increased the rate of degradation at both levels of 3-chlorobenzoate. In both the 500 and 1,000 microg 3-chlorobenzoate g(-1) dry soil inoculated microcosms, degraders increased from the initial inoculum and decreased following degradation of 3-CB. Inoculation delayed the development of indigenous 3-chlorobenzoate degrading populations. It is unclear if inoculation altered the composition of indigenous degrader populations. In the uninoculated soil, degraders increased from undetectable levels to 6.6 x 10(7) colony-forming-units g(-1) dry soil in the 500 microg 3-chlorobenzoate g(-1) dry soil microcosms, but none were detected in the 1,000 microg 3-chlorobenzoate g(-1) dry soil microcosms. Degraders isolated from uninoculated soil were identified as one of two distinct Burkholderia species. In the uninoculated soil, numbers of culturable heterotrophic bacteria initially decreased following addition of 1,000 microg 3-chlorobenzoate g(-1) dry soil. Inoculation with C. testosteroni reduced this negative impact on culturable bacterial numbers. The results indicate that bioaugmentation may not only increase the rate of 3-chlorobenzoate degradation but also reduce the deleterious effects of 3-chlorbenzoate on indigenous soil microbial populations.
• Pepper, I., Reynolds, K. A., Gerba, C. P., Abbaszadegan, M., & Pepper, I. L. (2001). ICC/PCR detection of enteroviruses and hepatitis A virus in environmental samples. Canadian journal of microbiology, 47(2).
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  This study applied the integrated cell culture/polymerase chain reaction methodology (ICC/PCR) for rapid and specific detection of both cytopathogenic and noncytopathogenic viruses. Results of this study showed that the use of direct RT-PCR or conventional cell culture alone may yield erroneous results with the analysis of environmental samples. The purpose of this study was to compare cultural, molecular, and combined assays for the most effective method of virus detection in variable environmental samples. Using ICC/PCR, stock enterovirus inocula of > or =10 PFU were PCR positive in at least 4/5 replicate flasks after only 5 h of incubation in cell culture, and in all flasks after > or =10 h. An inoculum of one PFU was detected by PCR after 20 h of cell culture incubation while for concentrations of virus below one PFU, 25 h of incubation was sufficient. Similarly, hepatitis A virus (HAV) inocula of 100 MPN/flask, produced indeterminate CPE in cell culture, but were clearly detected by ICC/PCR following 48 h of incubation. Lower levels of HAV, 1 and 10 MPN, were detected by ICC/PCR after 96 to 72 h of incubation, respectively. Cell culture lysates from 11 environmental sample concentrates of sewage, marine water, and surface drinking water sources, were positive for enteroviruses by ICC/PCR compared to 3 positive by direct RT-PCR alone. Results from ICC/PCR eventually agreed with cell culture but required < or =48 h of incubation, compared to as long as 3 weeks for CPE following incubation with BGM and FRhK cells.
• Pepper, I., Roane, T. M., Josephson, K. L., & Pepper, I. L. (2001). Dual-bioaugmentation strategy to enhance remediation of cocontaminated soil. Applied and environmental microbiology, 67(7).
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  Although metals are thought to inhibit the ability of microorganisms to degrade organic pollutants, several microbial mechanisms of resistance to metal are known to exist. This study examined the potential of cadmium-resistant microorganisms to reduce soluble cadmium levels to enhance degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) under conditions of cocontamination. Four cadmium-resistant soil microorganisms were examined in this study. Resistant up to a cadmium concentration of 275 microg ml(-1), these isolates represented the common soil genera Arthrobacter, Bacillus, and Pseudomonas. Isolates Pseudomonas sp. strain H1 and Bacillus sp. strain H9 had a plasmid-dependent intracellular mechanism of cadmium detoxification, reducing soluble cadmium levels by 36%. Isolates Arthrobacter strain D9 and Pseudomonas strain I1a both produced an extracellular polymer layer that bound and reduced soluble cadmium levels by 22 and 11%, respectively. Although none of the cadmium-resistant isolates could degrade 2,4-D, results of dual-bioaugmentation studies conducted with both pure culture and laboratory soil microcosms showed that each of four cadmium-resistant isolates supported the degradation of 500-microg ml(-1) 2,4-D by the cadmium-sensitive 2,4-D degrader Ralstonia eutropha JMP134. Degradation occurred in the presence of up to 24 microg of cadmium ml(-1) in pure culture and up to 60 microg of cadmium g(-1) in amended soil microcosms. In a pilot field study conducted with 5-gallon soil bioreactors, the dual-bioaugmentation strategy was again evaluated. Here, the cadmium-resistant isolate Pseudomonas strain H1 enhanced degradation of 2,4-D in reactors inoculated with R. eutropha JMP134 in the presence of 60 microg of cadmium g(-1). Overall, dual bioaugmentation appears to be a viable approach in the remediation of cocontaminated soils.
• Reynolds, K. A., Gerba, C. P., Abbaszadegan, M., & Pepper, I. L. (2001). ICC/PCR detection of enteroviruses and hepatitis A virus in environmental samples. Canadian Journal of Microbiology, 47(2), 153-157.
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  PMID: 11261495;Abstract: This study applied the integrated cell culture/polymerase chain reaction methodology (ICC/PCR) for rapid and specific detection of both cytopathogenic and noncytopathogenic viruses. Results of this study showed that the use of direct RT-PCR or conventional cell culture alone may yield erroneous results with the analysis of environmental samples. The purpose of this study was to compare cultural, molecular, and combined assays for the most effective method of virus detection in variable environmental samples. Using ICC/PCR, stock enterovirus inocula of ≥10 PFU were PCR positive in at least 4/5 replicate flasks after only 5 h of incubation in cell culture, and in all flasks after ≥10 h. An inoculum of one PFU was detected by PCR after 20 h of cell culture incubation while for concentrations of virus below one PFU, 25 h of incubation was sufficient. Similarly, hepatitis A virus (HAV) inocula of 100 MPN/flask, produced indeterminate CPE in cell culture, but were clearly detected by ICC/PCR following 48 h of incubation. Lower levels of HAV, 1 and 10 MPN, were detected by ICC/PCR after 96 to 72 h of incubation, respectively. Cell culture lysates from 11 environmental sample concentrates of sewage, marine water, and surface drinking water sources, were positive for enteroviruses by ICC/PCR compared to 3 positive by direct RT-PCR alone. Results from ICC/PCR eventually agreed with cell culture but required ≤ 48 h of incubation, compared to as long as 3 weeks for CPE following incubation with BGM and FRhK cells.
• Roane, T. M., Josephson, K. L., & Pepper, I. L. (2001). Dual-Bioaugmentation Strategy to Enhance Remediation of Cocontaminated Soil. Applied and Environmental Microbiology, 67(7), 3208-3215.
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  PMID: 11425743;PMCID: PMC93002;Abstract: Although metals are thought to inhibit the ability of microorganisms to degrade organic pollutants, several microbial mechanisms of resistance to metal are known to exist. This study examined the potential of cadmium-resistant microorganisms to reduce soluble cadmium levels to enhance degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) under conditions of cocontamination. Four cadmium-resistant soil microorganisms were examined in this study. Resistant up to a cadmium concentration of 275 μg ml-1, these isolates represented the common soil genera Arthrobacter, Bacillus, and Pseudomonas. Isolates Pseudomonas sp. strain H1 and Bacillus sp. strain H9 had a plasmid-dependent intracellular mechanism of cadmium detoxification, reducing soluble cadmium levels by 36%. Isolates Arthrobacter strain D9 and Pseudomonas strain I1a both produced an extracellular polymer layer that bound and reduced soluble cadmium levels by 22 and 11%, respectively. Although none of the cadmium-resistant isolates could degrade 2,4-D, results of dual-bioaugmentation studies conducted with both pure culture and laboratory soil microcosms showed that each of four cadmium-resistant isolates supported the degradation of 500-μg ml-1 2,4-D by the cadmium-sensitive 2,4-D degrader Ralstonia eutropha JMP134. Degradation occurred in the presence of up to 24 μg of cadmium ml-1 in pure culture and up to 60 μg of cadmium g-1 in amended soil microcosms. In a pilot field study conducted with 5-gallon soil bioreactors, the dual-bioaugmentation strategy was again evaluated. Here, the cadmium-resistant isolate Pseudomonas strain H1 enhanced degradation of 2,4-D in reactors inoculated with R. eutropha JMP134 in the presence of 60 μg of cadmium g-1. Overall, dual bioaugmentation appears to be a viable approach in the remediation of cocontaminated soils.
• Blackmer, F., Reynolds, K. A., Gerba, C. P., & Pepper, I. L. (2000). Use of integrated cell culture-PCR to evaluate the effectiveness of poliovirus inactivation by chlorine. Applied and Environmental Microbiology, 66(5), 2267-2268.
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  PMID: 10788415;PMCID: PMC101488;Abstract: Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.
• Dowd, S. E., Gerba, C. P., Pepper, I. L., & Pillai, S. D. (2000). Bioaerosol transport modeling and risk assessment in relation to biosolid placement. Journal of Environmental Quality, 29(1), 343-348.
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  Abstract: A field study was performed in which bioaerosols were sampled at a field site undergoing land placement of anaerobically digested, de-watered biosolid material. The data from these field studies were then used to generate microbial release rates from the biosolids for use in modeling bioaerosol transport. Continuous-point sources represented by large biosolid piles (temporary storage before placement) in the field, and continuous-area sources represented by large fields upon which biosolids were placed by spraying, were modeled using microbial transport models; and downwind microbial concentrations were generated. These quantified transport data were then entered into microbial dose-response models in an attempt to characterize the risk of pathogenic bacteria and viruses infecting workers and nearby population centers. The risk of viral and bacterial infection to workers at biosolid land application sites is 3:100 and 2:100, respectively, under 2-m/s wind conditions and 1 hr of exposure. The route of exposure proposed in this model is the transport, inhalation, deposition, and swallowing of bacterial or viral pathogens. Note that these risk models by nature would tend to overestimate the actual risk to populations (wastewater workers) consisting primarily of immunocompetent individuals. Under these low-wind conditions, nearby population centers where such immunocompetent populations may exist (here considered to be 10 000 m from the land application sites) are predicted to be at little risk (1.95 x 10-2:100) of infection from aerosolized bacteria and at no risk from aerosolized viruses.
• Newby, D. T., Gentry, T. J., & Pepper, I. L. (2000). Comparison of 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in soil resulting from bioaugmentation with two different pJP4 donors. Applied and Environmental Microbiology, 66(8), 3399-3407.
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  PMID: 10919798;PMCID: PMC92162;Abstract: A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection, Escherichia coli D11. The R. eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E. coli strain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of the Burkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the E. coli D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful.
• Newby, D. T., Josephson, K. L., & Pepper, I. L. (2000). Detection and characterization of plasmid pJP4 transfer to indigenous soil bacteria. Applied and Environmental Microbiology, 66(1), 290-296.
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  PMID: 10618238;PMCID: PMC91820;Abstract: Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism, Escherichia coli ATCC 15224, by plate mating with Ralstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2,4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 107 and 108 transconjugants g of dry soil-1 for samples supplemented with 500 and 1,000 μg of 2,4-D g of dry soil-1, respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genus Burkholderia or the genus Pseudomonas. No mercury-resistant, 2,4- D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.
• Pepper, I. L. (2000). Environmental science: A new opportunity for soil science. Soil Science, 165(1), 41-46.
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  Abstract: During the golden era of soil science - from the 1950s to the 1980s - the main focus of this discipline was on the role of soil in production agriculture. More recently, renewed interest in the area of environmental science has offered new opportunities to soil scientists. Thus, many soil scientists are now working in areas such as bioremediation, waste recycling, and/or contaminant transport. Environmental science has, therefore, not only changed the traditional research role of soil scientists at land grant institutions but has also influenced student enrollment, the traditional soil science curriculum, and faculty recruitment. These changes require a new breed of soil scientist, one with a background not only in soil science but also in other areas of environmental science as well.
• Pepper, I., Blackmer, F., Reynolds, K. A., Gerba, C. P., & Pepper, I. L. (2000). Use of integrated cell culture-PCR to evaluate the effectiveness of poliovirus inactivation by chlorine. Applied and environmental microbiology, 66(5).
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  Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.
• Pepper, I., Newby, D. T., Gentry, T. J., & Pepper, I. L. (2000). Comparison of 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in soil resulting from bioaugmentation with two different pJP4 donors. Applied and environmental microbiology, 66(8).
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  A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection, Escherichia coli D11. The R. eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E. coli strain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of the Burkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the E. coli D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful.
• Pepper, I., Newby, D. T., Josephson, K. L., & Pepper, I. L. (2000). Detection and characterization of plasmid pJP4 transfer to indigenous soil bacteria. Applied and environmental microbiology, 66(1).
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  Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism, Escherichia coli ATCC 15224, by plate mating with Ralstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2, 4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 10(7) and 10(8) transconjugants g of dry soil(-1) for samples supplemented with 500 and 1,000 microg of 2,4-D g of dry soil(-1), respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genus Burkholderia or the genus Pseudomonas. No mercury-resistant, 2, 4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.
• Dowd, S. E., Gerba, C. P., Kamper, M., & Pepper, I. L. (1999). Evaluation of methodologies including immunofluorescent assay (IFA) and the polymerase chain reaction (PCR) for detection of human pathogenic microsporidia in water. Journal of Microbiological Methods, 35(1), 43-52.
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  PMID: 10076630;Abstract: Microsporidia is a term used to describe a group of emerging protozoan pathogens whose environmental occurrence has only recently been documented due to lack of detection methodologies. This study evaluates and describes current methods for detection of microsporidia in water. Standard methods, for the collection and processing of large volumes of water to detect protozoa, showed only a 4.8% recovery, of microsporidia spores, from 100 l volumes of tap. Immunofluorescent assay (IFA) analysis was assessed using two different antibodies specific for human pathogenic microsporidia. Results indicated that the use of IFA for routine screening of water for microsporidia was not an acceptable approach. The antibodies tested for the IFA resulted in false positives and false negatives and did not react with Enterocytozoon bieneusi, which is an important human pathogenic microsporidia. Finally, the small sizes of the human pathogenic microsporidia prevent confirmation and species determination by light microscopic methods. Two methods for isolating microsporidia DNA from water for use in polymerase chain reaction (PCR) amplification of microsporidia target sequences were assessed. Both of these DNA isolation methods when combined with the PCR showed the ability to detect less than ten spores in purified water concentrates. Thus, this study represents the first documentation and evaluation of current methods for the detection of human pathogenic microsporidia in water.
• Pepper, I., Dowd, S. E., Gerba, C. P., Kamper, M., & Pepper, I. L. (1999). Evaluation of methodologies including immunofluorescent assay (IFA) and the polymerase chain reaction (PCR) for detection of human pathogenic microsporidia in water. Journal of microbiological methods, 35(1).
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  Microsporidia is a term used to describe a group of emerging protozoan pathogens whose environmental occurrence has only recently been documented due to lack of detection methodologies. This study evaluates and describes current methods for detection of microsporidia in water. Standard methods, for the collection and processing of large volumes of water to detect protozoa, showed only a 4.8% recovery, of microsporidia spores, from 100 l volumes of tap. Immunofluorescent assay (IFA) analysis was assessed using two different antibodies specific for human pathogenic microsporidia. Results indicated that the use of IFA for routine screening of water for microsporidia was not an acceptable approach. The antibodies tested for the IFA resulted in false positives and false negatives and did not react with Enterocytozoon bieneusi, which is an important human pathogenic microsporidia. Finally, the small sizes of the human pathogenic microsporidia prevent confirmation and species determination by light microscopic methods. Two methods for isolating microsporidia DNA from water for use in polymerase chain reaction (PCR) amplification of microsporidia target sequences were assessed. Both of these DNA isolation methods when combined with the PCR showed the ability to detect less than ten spores in purified water concentrates. Thus, this study represents the first documentation and evaluation of current methods for the detection of human pathogenic microsporidia in water.
• Roane, T. M., & Pepper, I. L. (1999). Microbial responses to environmentally toxic cadmium. Microbial Ecology, 38(4), 358-364.
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  Abstract: We analyzed the soil microbial communities from one uncontaminated and two metal-impacted soils and found that while cadmium adversely affected the numbers of culturable bacteria in all soils, cadmium-resistant isolates were found from each of the soils. With exposure to 24 and 48 μg ml-1 soluble cadmium, the metal-contaminated soil communities were more resistant than the uncontaminated soil community. In addition, in one metal-stressed soil, the resistant population became more resistant with increased cadmium levels. Ribosomal 16S DNA sequencing identified the isolates as Arthrobacter, Bacillus, or Pseudomonas spp. Further characterization demonstrated that two of the isolates were highly resistant to soluble cadmium with maximum resistance at 275 μg ml-1 cadmium. These isolates were also resistant to a variety of antibiotics, namely ampicillin, gentamicin, penicillin, and streptomycin, but no overall correlation was found between enhanced antibiotic resistance and cadmium resistance. One Pseudomonas isolate H1 did become more resistant with increasing cadmium levels, suggesting a different resistance mechanism at high cadmium concentrations.
• Burr, M. D., Josephson, K. L., & Pepper, I. L. (1998). An evaluation of DNA-based methodologies for subtyping Salmonella. Critical Reviews in Environmental Science and Technology, 28(3), 283-323.
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  Abstract: Bacteria of the genus Salmonella are major human and animal pathogens worldwide, with over 2000 serotypes. Although most authorities agree that there are only one or two true species of Salmonella, serotypes are accepted as pseudo-species. In addition, although serotype identification is important, without additional subtyping, serotyping has limited usefulness for epidemiology as three serotypes (S. typhimurium, S. enteriditis, and S. heidelberg) account for 50% of human infections worldwide. The purpose of this review is to evaluate different methods of DNA typing and fingerprinting of Salmonella strains and to report the most important epidemiological and phylogenetic discoveries that have been made using these methods. These include plasmid analysis (plasmid profiles and plasmid fingerprints, restriction fragment length polymorphism (RFLP) analysis, and polymerase chain reaction (PCR) fingerprinting). Plasmids are common but not universal in Salmonella isolates and can be used to discriminate isolates through plasmid profiles or restriction digests of plasmid DNA. They have been used with some success to subtype some serotypes. The correlation, however, is not always perfect. RFLP fingerprinting has also been used to subtype serotypes. Specifically, the IS200 insertion sequence has been used to probe restriction digests of chromosomal DNA. Probes derived from 16S sequences have been used similarly in 'ribotyping' studies but with less success. RFLP fingerprinting has been used in both phylogenetic and epidemiology studies of Salmonella and is considered to be a more stable and reliable method than plasmid analysis. PCR fingerprinting techniques, including arbitrarily primed PCR (AP PCR), repetitive sequence (REP) PCR and restriction digests of PCR-amplified 16S rDNA sequences have also been used to discriminate between Salmonella serotypes. The success of PCR methods is variable and depends on specific primers utilized. Overall, many fingerprinting techniques can discriminate between isolates but are often not successful in subtyping serotypes.
• Burr, M. D., Josephson, K. L., & Pepper, I. L. (1998). An evaluation of ERIC PCR and AP PCR fingerprinting for discriminating Salmonella serotypes. Letters in Applied Microbiology, 27(1), 24-30.
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  PMID: 9722993;Abstract: PCR fingerprints of 89 Salmonella isolates belonging to 22 serotypes were obtained using ERIC PCR (enterobacterial repetitive intergenic consensus PCR) and AP PCR (arbitrarily primed PCR) to evaluate the ability of different fingerprinting methods to differentiate or identify serotypes and subtypes. Fingerprints were scored and comparisons were made using a computer program. ERIC PCR produced a unique, complex fingerprint for almost every isolate, but these fingerprints did not identify serotypes. One AP CR primer also produced complex fingerprints that discriminated among isolates, but again did not identify serotypes. A second AP PCR primer produced simple patterns, including one pattern shared by 35 isolates from 12 different serotypes. In general, the three sets of PCR fingerprints distinguished isolates, but were not correlated with serotypes. Matching fingerprints from different gels by computer was difficult, since similarities were based on both intense and faint bands. In addition, this study suggests that dendrograms created from PCR fingerprints should be viewed with caution.
• Dowd, S. E., Gerba, C. P., & Pepper, I. L. (1998). Confirmation of the human-pathogenic microsporidia Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Vittaforma corneae in water. Applied and Environmental Microbiology, 64(9), 3332-3335.
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  PMID: 9726879;PMCID: PMC106729;Abstract: Microsporidia, as a group, cause a wide range of infections, though two species of microsporidia in particular, Enterocytozoon bieneusi and Encephalitozoon intestinalis, are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of microsporidia have not been elucidated due to lack of sensitive and specific screening methods. The present study was undertaken with recently developed methods to screen several significant water sources. Water concentrates were subjected to community DNA extraction followed by microsporidium-specific PCR amplification, PCR sequencing, and database homology comparison. A total of 14 water concentrates were screened; 7 of these contained human-pathogenic microsporidia. The presence of Encephalitozoon intestinalis was confirmed in tertiary sewage effluent, surface water, and groundwater; the presence of Enterocytozoon bieneusi was confirmed in surface water; and the presence of Vittaforma corneae was confirmed in tertiary effluent. Thus, this study represents the first confirmation, to the species level, of human-pathogenic microsporidia in water, indicating that these human-pathogenic microsporidia may be waterborne pathogens.
• Dowd, S. E., Gerba, C. P., Enriquez, F. J., & Pepper, I. L. (1998). PCR amplification and species determination of microsporidia in formalin-fixed feces after immunomagnetic separation. Applied and Environmental Microbiology, 64(1), 333-336.
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  PMID: 9435086;PMCID: PMC124713;Abstract: The term microsporidia is used to describe several species of opportunistic protozoan parasites. Encephalitozoon intestinalis and Enterocytozoon bieneusi have been found in stools of more than 40% of AIDS patients with diarrhea. Diagnosis of infection with these small protozoans has been difficult, and until recently their occurrence has not been well documented. Formalin is widely used to preserve clinical specimens, but due to the nature of the fixation process, subsequent analysis, especially analysis by the PCR, is difficult. This study evaluated methods used to prepare formalin-fixed fecal specimens for PCR amplification of microsporidial DNA. Two methods were devised to allow PCR detection and subsequent identification of microsporidia in formalin-fixed fecal specimens to the species level. One method involved immunomagnetic separation to concentrate microsporidial spores from fecal specimens. In the second method Chelex resin (Bio-Rad, Hercules, Calif.) was used to remove inhibitory substances, followed by a DNA concentration step. Both methods resulted in reproducible, confirmed detection of microsporidia in formalinized fecal specimens and subsequent species determination by PCR sequencing. The detection sensitivity was two in vitro culture-derived spores (Encephalitozoon intestinalis) for the direct PCR. The reproducible detection sensitivity for DNA amplification from formalin-fixed fecal samples was 200 spores for either the Chelex method or the immunomagnetic bead separation method. Thus, we developed two methods for rapid, inexpensive detection of microsporidial spores in formalin-fixed fecal specimens.
• Pepper, I., Burr, M. D., Josephson, K. L., & Pepper, I. L. (1998). An evaluation of ERIC PCR and AP PCR fingerprinting for discriminating Salmonella serotypes. Letters in applied microbiology, 27(1).
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  PCR fingerprints of 89 Salmonella isolates belonging to 22 serotypes were obtained using ERIC PCR (enterobacterial repetitive intergenic consensus PCR) and AP PCR (arbitrarily primed PCR) to evaluate the ability of different fingerprinting methods to differentiate or identify serotypes and subtypes. Fingerprints were scored and comparisons were made using a computer program. ERIC PCR produced a unique, complex fingerprint for almost every isolate, but these fingerprints did not identify serotypes. One AP PCR primer also produced complex fingerprints that discriminated among isolates, but again did not identify serotypes. A second AP PCR primer produced simple patterns, including one pattern shared by 35 isolates from 12 different serotypes. In general, the three sets of PCR fingerprints distinguished isolates, but were not correlated with serotypes. Matching fingerprints from different gels by computer was difficult, since similarities were based on both intense and faint bands. In addition, this study suggests that dendrograms created from PCR fingerprints should be viewed with caution.
• Pepper, I., Dowd, S. E., Gerba, C. P., & Pepper, I. L. (1998). Confirmation of the human-pathogenic microsporidia Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Vittaforma corneae in water. Applied and environmental microbiology, 64(9).
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  Microsporidia, as a group, cause a wide range of infections, though two species of microsporidia in particular, Enterocytozoon bieneusi and Encephalitozoon intestinalis, are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of microsporidia have not been elucidated due to lack of sensitive and specific screening methods. The present study was undertaken with recently developed methods to screen several significant water sources. Water concentrates were subjected to community DNA extraction followed by microsporidium-specific PCR amplification, PCR sequencing, and database homology comparison. A total of 14 water concentrates were screened; 7 of these contained human-pathogenic microsporidia. The presence of Encephalitozoon intestinalis was confirmed in tertiary sewage effluent, surface water, and groundwater; the presence of Enterocytozoon bieneusi was confirmed in surface water; and the presence of Vittaforma corneae was confirmed in tertiary effluent. Thus, this study represents the first confirmation, to the species level, of human-pathogenic microsporidia in water, indicating that these human-pathogenic microsporidia may be waterborne pathogens.
• Pepper, I., Dowd, S. E., Gerba, C. P., Enriquez, F. J., & Pepper, I. L. (1998). PCR amplification and species determination of microsporidia in formalin-fixed feces after immunomagnetic separation. Applied and environmental microbiology, 64(1).
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  The term microsporidia is used to describe several species of opportunistic protozoan parasites. Encephalitozoon intestinalis and Enterocytozoon bieneusi have been found in stools of more than 40% of AIDS patients with diarrhea. Diagnosis of infection with these small protozoans has been difficult, and until recently their occurrence has not been well documented. Formalin is widely used to preserve clinical specimens, but due to the nature of the fixation process, subsequent analysis, especially analysis by the PCR, is difficult. This study evaluated methods used to prepare formalin-fixed fecal specimens for PCR amplification of microsporidial DNA. Two methods were devised to allow PCR detection and subsequent identification of microsporidia in formalin-fixed fecal specimens to the species level. One method involved immunomagnetic separation to concentrate microsporidial spores from fecal specimens. In the second method Chelex resin (Bio-Rad, Hercules, Calif.) was used to remove inhibitory substances, followed by a DNA concentration step. Both methods resulted in reproducible, confirmed detection of microsporidia in formalinized fecal specimens and subsequent species determination by PCR sequencing. The detection sensitivity was two in vitro culture-derived spores (Encephalitozoon intestinalis) for the direct PCR. The reproducible detection sensitivity for DNA amplification from formalin-fixed fecal samples was 200 spores for either the Chelex method or the immunomagnetic bead separation method. Thus, we developed two methods for rapid, inexpensive detection of microsporidial spores in formalin-fixed fecal specimens.
• Pepper, I., Reynolds, K. A., Roll, K., Fujioka, R. S., Gerba, C. P., & Pepper, I. L. (1998). Incidence of enteroviruses in Mamala Bay, Hawaii using cell culture and direct polymerase chain reaction methodologies. Canadian journal of microbiology, 44(6).
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  The consequence of point and nonpoint pollution sources, discharged into marine waters, on public recreational beaches in Mamala Bay, Hawaii was evaluated using virus cell culture and direct reverse transcriptase-polymerase chain reaction (RT-PCR). Twelve sites, nine marine, two freshwater (one stream and one canal), and one sewage, were assessed either quarterly or monthly for 1 year to detect the presence of human enteric viruses. Water samples were concentrated from initial volumes of 400 L to final volumes of 30 mL using Filterite electronegative cartridge filters and a modified beef extract elution procedure. Cell culture was applied using the Buffalo Green Monkey kidney cell line to analyze samples for enteroviruses. Positive samples were also evaluated by RT-PCR, using enterovirus-specific primers. Levels of RT-PCR inhibition varied with each concentrated sample. Resin column purification increased PCR detection sensitivity by at least one order of magnitude in a variety of sewage outfall and recreational marine water samples but not in the freshwater canal samples. Using cell culture, viable enteroviruses were found in 50 and 17% of all outfall and canal samples, respectively. Samples were positive at beaches 8% of the time. These data illustrate the potential public health hazard associated with recreational waters. Using direct PCR, viruses were detected at the outfall but were not found in any beach or canal samples, in part, owing to substances that inhibit PCR. Therefore, conventional cell culture is the most effective means of detecting low levels of infectious enteroviruses in environmental waters, whereas direct RT-PCR is rendered less effective by inhibitory compounds and low equivalent reaction volumes.
• Reynolds, K. A., Roll, K., Fujioka, R. S., Gerba, C. P., & Pepper, I. L. (1998). Incidence of enteroviruses in Mamala Bay, Hawaii using cell culture and direct polymerase chain reaction methodologies. Canadian Journal of Microbiology, 44(6), 598-604.
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  PMID: 9734309;Abstract: The consequence of point and nonpoint pollution sources, discharged into marine waters, on public recreational beaches in Mamala Bay, Hawaii was evaluated using virus cell culture and direct reverse transcriptase - polymerase chain reaction (RT-PCR). Twelve sites, nine marine,two freshwater (one stream and one canal), and one sewage, were assessed either quarterly or monthly for 1 year to detect the presence of human enteric viruses. Water samples were concentrated from initial volumes of 400 L to final volumes of 30 mL using Filterite electronegative cartridge filters and a modified beef extract elution procedure. Cell culture was applied using the Buffalo Green Monkey kidney cell line to analyze samples for enteroviruses. Positive samples were also evaluated by RT-PCR, using enterovirus-specific primers. Levels of RT-PCR inhibition varied with each concentrated sample. Resin column purification increased PCR detection sensitivity by at least one order of magnitude in a variety of sewage outfall and recreational marine water samples but not in the freshwater canal samples. Using cell culture, viable enteroviruses were found in 50 and 17% of all outfall and canal samples, respectively. Samples were positive at beaches 8% of the time. These data illustrate the potential public health hazard associated with recreational waters. Using direct PCR, viruses were detected at the outfall but were not found in any beach or canal samples, in part,owing to substances that inhibit PCR. Therefore, conventional cell culture is the most effective means of detecting low levels of infectious enteroviruses in environmental waters, whereas direct RT-PCR is rendered less effective by inhibitory compounds and low equivalent reaction volumes.
• Abbaszadegan, M., Huber, M. S., Gerba, C. P., & Pepper, I. L. (1997). Detection of viable Giardia cysts by amplification of heat shock- induced mRNA. Applied and Environmental Microbiology, 63(1), 324-328.
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  PMID: 8979360;PMCID: PMC168324;Abstract: Primers obtained from gene sequences coding for heat shock proteins (HSP) were used to specifically detect enteric protozoans of the genus Giardia. The HSP primers amplified Giardia DNA or the corresponding RNA sequences obtained from lysed cysts and gave a 163-bp product. Since the presence of the product did not indicate whether the cysts were viable, these amplifications are a presence/absence test only. In contrast, amplification of heat shock-induced mRNA utilizing the same HSP primers was indicative of viable Giardia cysts. The limit of sensitivity of the presence/absence test was 1 cyst, whereas for the viability test it was 10 cysts. Thus, viable Giardia cysts can be rapidly and specifically detected with great sensitivity through the use of PCR amplifications.
• Burr, M. D., & Pepper, I. L. (1997). Variability in presence-absence scoring of AP PCR fingerprints affects computer matching of bacterial isolates. Journal of Microbiological Methods, 29(1), 63-68.
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  Abstract: Sources of variation in scoring bands in arbitrarily primed PCR (AP PCR) fingerprints of bacterial isolates were identified, and their effect on computer matching of fingerprints was determined. E. coli and five Salmonella serotypes were fingerprinted. PCR reactions and gel electrophoresis analysis of PCR products were replicated, including comparisons in the same gel and in different gels. Bands in the images were assigned by two different people on a presence-absence basis. Variations in scoring the images occurred at all levels, and prevented correct identification of isolates. E. coli was distinguished from Salmonella, but discrimination among different Salmonella serotypes and between two isolates of the same serotype was poor. Our results suggest that computer analysis of AP PCR fingerprints scored on a presence absence basis may not correctly match isolates. Side-by-side visual comparison of isolates is recommended.
• Day Jr., W. A., Pepper, I. L., & Joens, L. A. (1997). Use of an arbitrarily primed PCR product in the development of a Campylobacter jejuni-specific PCR. Applied and Environmental Microbiology, 63(3), 1019-1023.
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  PMID: 9055418;PMCID: PMC168393;Abstract: Development of a PCR assay for Campylobacter jejuni is based on the isolation of species-specific DNA. An arbitrarily primed PCR incorporating 10-mer primers was used to generate fingerprints of C. jejuni M129 genomic DNA. Fingerprint products were then screened individually for their species specificity in dot blot hybridizations with 6 C. jejuni isolates, 4 Campylobacter species other than C. jejuni, and 27 enteric bacterial species other than Campylobacter spp. A 486-bp fingerprint product hybridized specifically to C. jejuni DNA under stringent conditions; no binding to Campylobacter DNA other than that of C. jejuni or to DNA from enteric bacteria was detected. The 486-bp fingerprint product was sequenced, and primers corresponding to three overlapping regions of the DNA probe were synthesized. Evaluation of the three primer pairs for specificity to C. jejuni DNA identified an oligonucleotide primer pair which amplified a 265- bp product from six C. jejuni isolates only. In sensitivity studies using a crude M129 lysate as the template, the C. jejuni-specific PCR amplified the 265-bp product in a lysate with as few as 100 bacteria.
• Johnson, D. C., Enriquez, C. E., Pepper, I. L., Davis, T. L., Gerba, C. P., & Rose, J. B. (1997). Survival of Giardia, Cryptosporidium, poliovirus and Salmonella in marine waters. Water Science and Technology, 35(11-12), 261-268.
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  Abstract: Discharge of sewage into the ocean is still a common method of disposal worldwide. Both treated and untreated sewage may contain significant concentrations of waterborne pathogens, such as Giardia, Cryptosporidium, poliovirus and Salmonella. Limited studies exist on the survival of poliovirus and Salmonella in marine waters; however, almost no information exists on the survival of protozoan parasites in marine waters. This study examined the survival of Giardia muris cysts, Cryptosporidium parvum oocysts, poliovirus-1 and Salmonella typhimurium in marine waters. The survival of the microorganisms varied according to the presence of light, salinity and water quality (as determined by quantity of enterococci). All microorganisms survived longer in the dark than in sunlight, the order of survival in sunlight being: Cryptosporidium > poliovirus > Giardia > Salmonella.
• Josephson, K. L., Rubino, J. R., & Pepper, I. L. (1997). Characterization and quantification of bacterial pathogens and indicator organisms in household kitchens with and without the use of a disinfectant cleaner. Journal of Applied Microbiology, 83(6), 737-750.
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  PMID: 9449812;Abstract: This two year study evaluated the prevalence of indicator bacteria and specific pathogens in 10 'normal' kitchens in the United States. In Phase I, none of the kitchens was cleaned with an antimicrobial cleaner or disinfectant. Eight locations within the kitchens were monitored for: total heterotrophs, staphylococci, Pseudomonas, total coliforms and faecal coliforms. Almost all locations at all households exhibited contamination, with the sink and sponge samples exhibiting large bacterial concentrations. The faecal coliform concentrations in sink and sponge samples were very high, with 63 and 67% of all samples being positive, respectively. Escherichia coli was detected in 16·7% of all sink surfaces and 33·3% of all sponges. Salmonella was detected once and Campylobacter, on two occasions. In a second phase, households were provided with an antimicrobial disinfectant cleaner which families were encouraged to use but not forced to do so; in some cases, the product was used infrequently or not at all. This regimen did not demonstrate any consistent reduction in the incidence of bacterial contamination. By contrast, in the final phase of the study where disinfectant use was targeted for surfaces soon after contamination with foods or hands, the incidence of contamination decreased dramatically. These data show that normal kitchens can easily be contaminated with a variety of bacterial contaminants including faecal coliforms, E. coli, Salmonella and Campylobacter. Irregular use, or not using antimicrobial agents, is unlikely to reduce the risk of these infectious agents. By contrast, targeted use is likely to reduce the incidence of bacterial contaminants.
• Jutras, E. M., Smart, C. M., Rupert, R., Pepper, I. L., & Miller, R. M. (1997). Field-scale biofiltration of gasoline vapors extracted from beneath a leaking underground storage tank. Biodegradation, 8(1), 31-42.
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  Abstract: Approximately 15000 L of unleaded gasoline were released into the surrounding vadose zone from a leaking underground storage tank. Initial remediation was by soil vapor extraction and combustion which soon became cost prohibitive, as added propane was required to reach the combustion limit of the extracted vapors. As a cost effective alternative, a field-scale compost based biofilter was used in conjunction with soil vapor extraction to remediate the vadose zone. The biofilter was constructed on site using 4:1 diatomaceous earth:composted horse manure. Results of a five month study showed that the biofilter removed approximately 90% of total petroleum hydrocarbons (TPH) and >90% of the BTEX compounds (benzene, toluene, ethylbenzene, xylene), achieving the stringent permit requirements set at either 90% TPH reduction or less than 1.36 kg per day of volatile organic compounds (VOC's) released to the atmosphere. The biofilter showed the capacity to readily adapt to changing environmental conditions such as increased contaminant loading, and variations in temperature and moisture. The bacterial population in the biofilter was uniformly diverse throughout the biofilter, suggesting that a consortium of bacteria was needed for efficient biodegradation. The cost of biofilter set up and operation saved 90% in the first year alone of the operating expenses incurred by soil vapor extraction and combustion.
• Marlowe, E. M., Josephson, K. L., Miller, R. M., & Pepper, I. L. (1997). A method for the detection and quantitation of PCR template in environmental samples by high performance liquid chromatography. Journal of Microbiological Methods, 28(1), 45-53.
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  Abstract: This study describes methodology for the detection and quantitation of PCR amplified DNA. Specifically we report the estimation of the prevalence of E. coli in marine waters and other environmental samples from Mamala Bay, Hawaii. High performance liquid chromatography (HPLC) was used to quantitate PCR products containing between 1 and 250 ng DNA. PCR was used to amplify E. coli DNA through the use of lamB primers. A standard curve was generated that related initial cell template concentrations to amplified product DNA concentrations within a template range of 520 to 5.2 x 107 cells. The standard curve was used to determine initial template concentrations of the lamB gene sequence present within 11 different environmental samples. Quantified PCR analyses were most useful when samples contained only low numbers of target organisms, and when environmental samples contained few PCR inhibitory substances, as for example in marine water samples. Quantitation of amplified DNA and comparison with culture data also suggested the presence of viable but nonculturable organisms in some environmental samples. Overall these data are unique in that they indicate the successful use of HPLC to quantitate PCR amplifications with concomitant estimation of PCR template within environmental samples.
• Pepper, I., Josephson, K. L., Rubino, J. R., & Pepper, I. L. (1997). Characterization and quantification of bacterial pathogens and indicator organisms in household kitchens with and without the use of a disinfectant cleaner. Journal of applied microbiology, 83(6).
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  This two year study evaluated the prevalence of indicator bacteria and specific pathogens in 10 'normal' kitchens in the United States. In Phase I, none of the kitchens was cleaned with an antimicrobial cleaner or disinfectant. Eight locations within the kitchens were monitored for: total heterotrophs, staphylococci, Pseudomonas, total coliforms and faecal coliforms. Almost all locations at all households exhibited contamination, with the sink and sponge samples exhibiting large bacterial concentrations. The faecal coliform concentrations in sink and sponge samples were very high, with 63 and 67% of all samples being positive, respectively. Escherichia coli was detected in 16.7% of all sink surfaces and 33.3% of all sponges. Salmonella was detected once and Campylobacter, on two occasions. In a second phase, households were provided with an antimicrobial disinfectant cleaner which families were encouraged to use but not forced to do so; in some cases, the product was used infrequently or not at all. This regimen did not demonstrate any consistent reduction in the incidence of bacterial contamination. By contrast, in the final phase of the study where disinfectant use was targeted for surfaces soon after contamination with foods or hands, the incidence of contamination decreased dramatically. These data show that normal kitchens can easily be contaminated with a variety of bacterial contaminants including faecal coliforms, E. coli, Salmonella and Campylobacter. Irregular use, or not using antimicrobial agents, is unlikely to reduce the risk of these infectious agents. By contrast, targeted use is likely to reduce the incidence of bacterial contaminants.
• Reynolds, K. S., Gerba, C. P., & Pepper, I. L. (1997). Rapid PCR-based monitoring of infectious enteroviruses in drinking water. Water Science and Technology, 35(11-12), 423-427.
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  Abstract: Currently, the standard method for the detection of enteroviruses and hepatitis A virus in water involves cell culture assay which is expensive and time consuming. Direct RT-PCR offers a rapid and sensitive alternative to virus detection but sensivity is often reduced by PCR inhibitory substances and the requirement for small reaction volumes. Rapid methods for detection of infectious enteroviruses in PCR inhibitory environmental samples are being developed utilising an integrated cell culture/PCR approach (ICC/PCR). With this approach, 300-400l of water were concentrated using charged filters followed by a modified 11, 1.5% BEV/glycine elution and organic flocculation reconcentration. Water concentrates were analysed by direct RT-PCR, conventional cell culture and ICC/PCR. For ICC/PCR, sample concentrates were incubated with BGM or FRhK cells for 24-48h. The cell culture lysates were collected following freeze-thaw cycles, centrifuged, resin column purified and PCR amplified. In this study viruses known to be present by cell culture analysis could not be detected by direct PCR. Using the integrated method, virus concentrations as low as 0.001 MPN/l of original water were detected in samples which were previously inhibitory to direct PCR. In addition, confirmed enterovirus results were achieved as soon as 48 h against 5-16 d with cell culture alone. Therefore, the integrated approach overcame some of the traditional problems associated with conventional cell culture and direct RT-PCR by allowing rapid, confirmed detection of low levels of enteroviruses in PCR inhibitory samples.
• DiGiovanni, G. D., Neilson, J. W., Pepper, I. L., & Sinclair, N. A. (1996). Gene transfer of Alcaligenes eutrophus JMP134 plasmid pJP4 to indigenous soil recipients. Applied and Environmental Microbiology, 62(7), 2521-2526.
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  PMID: 8779592;PMCID: PMC168035;Abstract: This study evaluated the potential for gene transfer of a large catabolic plasmid from an introduced organism to indigenous soil recipients. The donor organism Alcaligenes eutrophus JMP134 contained the 80-kb plasmid pJP4, which contains genes that code for mercury resistance. Genes on this plasmid plus chromosomal genes also allow degradation of 2,4- dichlorophenoxyacetic acid (2,4-D). When JMP134 was inoculated into a nonsterile soil microcosm amended with 1,000 μg of 2,4.D g-1, significant (106 g of soil-1) populations of indigenous recipients or transconjugants arose. These transconjugants all contained an 80-kb plasmid similar in size to pJP4, and all degraded 2,4-D. In addition, all transconjugants were resistant to mercury and contained the tfdB gene of pJP4 as detected by PCR. No mercury-resistant, 2,4-D-degrading organisms with large plasmids or the tfdB gene were found in the 2,4-D-amended but uninoculated control microcosm. These data clearly show that the plasmid pJP4 was transferred to indigenous soil recipients. Even more striking is the fact that not only did the indigenous transconjugant population survive and proliferate but also enhanced rates of 2,4-D degradation occurred relative to microcosms in which no such gene transfer occurred. Overall, these data indicate that gene transfer from introduced organisms is an effective means of bioaugmentation and that survival of the introduced organism is not a prerequisite for biodegradation that utilizes introduced biodegradative genes.
• Giovanni, G. D., Neilson, J. W., Pepper, I. L., & Sinclair, N. A. (1996). Plasmid diversity within a 2,4-dichlorophenoxyacetic acid-degrading Variovorax paradoxus population isolated from a contaminated soil. Journal of Environmental Science and Health - Part A Environmental Science and Engineering and Toxic and Hazardous Substance Control, 31(5), 963-976.
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  Abstract: Thirty two 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading {Tfd+) isolates of Variovorax paradoxus were obtained from a sample of contaminated soil by enrichment culture and were divided into six groups based on the size and number of plasmids they contained. Isolates within each of the six groups had identical and unique plasmid profiles consisting of multiple plasmids of diverse size. Curing of plasmids resulted in loss of ability to degrade 2,4- D. EcoR1 restriction patterns of plasmids contained by Tfd+ and Tfd- clones suggested that the plasmids did not derive from a common origin, since patterns revealed unique differences as well as similarities in restriction fragment size. In addition, the data suggest that in situ gene transfer and recombination events occurred frequently within this population.
• Pepper, I., Reynolds, K. A., Gerba, C. P., & Pepper, I. L. (1996). Detection of infectious enteroviruses by an integrated cell culture-PCR procedure. Applied and environmental microbiology, 62(4).
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  Rapid detection of infectious enteroviruses in environmental samples was made possible by utilizing an integrated cell culture-reverse transcriptase PCR approach. By this method, the presence of infectious enterovirus was confirmed within 24 h, compared with > or = 3 days by cell culture alone. The combined methodology eliminated typical problems normally associated with direct reverse transcriptase PCR by increasing the equivalent volume of environmental sample examined and reducing the effects of inhibitory compounds.
• Reynolds, K. A., Gerba, C. P., & Pepper, I. L. (1996). Detection of infectious enteroviruses by an integrated cell culture-PCR procedure. Applied and Environmental Microbiology, 62(4), 1424-1427.
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  PMID: 8919804;PMCID: PMC167909;Abstract: Rapid detection of infectious enteroviruses in environmental samples was made possible by utilizing an integrated cell culture-reverse transcriptase PCR approach. By this method, the presence of infectious enterovirus was confirmed within 24 h, compared with ≥3 days by cell culture alone. The combined methodology eliminated typical problems normally associated with direct reverse transcriptase PCR by increasing the equivalent volume of environmental sample examined and reducing the effects of inhibitory compounds.
• Johnson, D. C., Reynolds, K. A., Gerba, C. P., Pepper, I. L., & Rose, J. B. (1995). Detection of Giardia and Cryptosporidium in marine waters. Water Science and Technology, 31(5-6), 439-442.
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  Abstract: Raw sewage disposal in marine waters is a common practice in many countries. This practice raises health risk concerns of possible transmission of Giardia and Cryptosporidium. Both of these protozoa have been shown to be transmitted by recreational swimming. To date no studies have determined the efficiency of their detection and concentration in marine waters. This study evaluated the efficiency of their detection in tap water and from marine waters in Hawaii with two different filter types. This study compared a polypropylene fiber cartridge filter, DPPPY (1.0 μm nominal porosity) (Cuno, Meriden, CT) which is typically used for parasite detection and the Filterite negatively charged filter (0.45μm) (Filtemp Sales, Inc., Phoenix, AZ). The latter would allow for both viruses and parasites to be concentrated simultaneously. The organisms were removed from the filter by passing the eluent through the filters in the opposite direction of collection and detected by indirect immunofluorescence antibody staining specific for Giardia and Cryptosporidium. Processing was simpler and faster with the Filterite filter and the overall efficiency for both Giardia and Cryptosporidium detection was greater. These methods are currently being used for the detection of the oocysts aid cysts at bathing beaches in Hawaii impacted by marine sewage discharge.
• Jutras, E. M., Miller, R. M., & Pepper, I. L. (1995). Optimization of arbitrarily primed PCR for the identification of bacterial isolates. Journal of Microbiological Methods, 24(1), 55-63.
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  Abstract: Arbitrarily primed polymerase chain reaction (AP-PCR) has been used extensively for genetic mapping, and the identification of bacterial isolates. To ensure that the results will be reproducible and due to true genetic variations, the AP-PCR reaction conditions must be optimized. In this study, three cultured bacterial isolates were screened with 100 arbitrary primers. Of these, five were chosen for the optimization study. The parameters optimized included: the operating conditions of the thermal cycler, the agarose gel concentration, the annealing temperature, and the concentrations of Taq polymerase enzyme, magnesium chloride, primer, and template. The final optimized PCR reaction conditions were 1 × buffer (3.5 mM MgCl2, 10 mM Tris-HCl, 50 mM KCl and 0.1 mg ml-1 gelatin), 200 μM dNTP, 0.4 μM primer, 2.5 U AmpliTaq® (Perkin-Elmer Cetus) polymerase enzyme, and 5 μl of template (at least 106 lysed bacterial cells). The Perkin-Elmer Gene-Amp™ 9600 PCR System was used with the following cycling conditions; a 94°C 15 s denaturation step, a 45°C 15 s annealing step, and 72°C 30 s extension step for a total of 35 cycles. Reproducible, unique fingerprints were generated for the three isolates using each of the five arbitrary primers. © 1995.
• Ma, J., Gerba, C. P., & Pepper, I. L. (1995). Increased sensitivity of poliovirus detection in tap water concentrates by reverse transcriptase-polymerase chain reaction. Journal of Virological Methods, 55(3), 295-302.
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  PMID: 8609195;Abstract: This study developed a methodology to increase the sensitivity of enteric virus detection in tap water concentrates. Polymerase chain reaction (PCR) detection of virus in reduced volumes of virus-containing water concentrates was successful following removal of PCR inhibitory substances. Poliovirus 1 and coxsackievirus B3 were seeded into 378 1 of tap water, concentrated with 1MDS filters, and reconcentrated by organic flocculation. The volume of concentrates was successfully reduced from 25 to 5 ml without loss of virus recovery. PCR detection of virus after treatment of a water concentrate (1.1 × 105-fold concentration) with a Sephadex G-100 plus Chelex-100 column, or Sephadex G-50 plus Chelex-100 column, followed by heat treatment to release viral RNA, was compared with direct phenol-chloroform-isoamyl alcohol (PCI) extraction of viral RNA. The Sephadex G-50 plus Chelex-100 column did not remove inhibitory substances efficiently. The Sephadex G-100 plus Chelex-100 column could remove inhibitory substances, however, 99% of the viruses were also removed by the column. PCI extraction was found to be sufficient to remove inhibitory substances for reverse transcriptase (RT)-seminested PCR with a sensitivity of 0.2 plaque-forming units/10 μl (0.2 PFU/l tap water). © 1995.
• Pepper, I., Ma, J. F., Gerba, C. P., & Pepper, I. L. (1995). Increased sensitivity of poliovirus detection in tap water concentrates by reverse transcriptase-polymerase chain reaction. Journal of virological methods, 55(3).
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  This study developed a methodology to increase the sensitivity of enteric virus detection in tap water concentrates. Polymerase chain reaction (PCR) detection of virus in reduced volumes of virus-containing water concentrates was successful following removal of PCR inhibitory substances. Poliovirus 1 and coxsackievirus B3 were seeded into 378 l of tap water, concentrated with 1MDS filters, and reconcentrated by organic flocculation. The volume of concentrates was successfully reduced from 25 to 5 ml without loss of virus recovery. PCR detection of virus after treatment of a water concentrate (1.1 x 10(5)-fold concentration) with a Sephadex G-100 plus Chelex-100 column, or Sephadex G-50 plus Chelex-100 column, followed by heat treatment to release viral RNA, was compared with direct phenol-chloroform-isoamyl alcohol (PCI) extraction of viral RNA. The Sephadex G-50 plus Chelex-100 column did not remove inhibitory substances efficiently. The Sephadex G-100 plus Chelex-100 column could remove inhibitory substances, however, 99% of the viruses were also removed by the column. PCI extraction was found to be sufficient to remove inhibitory substances for reverse transcriptase (RT)-seminested PCR with a sensitivity of 0.2 plaque-forming units/10 microliters (0.2 PFU/l tap water).
• Reynolds, K. A., Gerba, C. P., & Pepper, I. L. (1995). Detection of enteroviruses in marine waters by direct RT-PCR and cell culture. Water Science and Technology, 31(5-6), 323-328.
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  Abstract: Sewage outfalls and storm water runoff introduces pathogenic human enteric viruses into marine coastal waters, which may pose a potential public health risk. Although members of the enterovirus group have been suggested as possible indicators of sewage pollution in marine waters, the lack of rapid, sensitive and cost effective methods have prevented routine monitoring in the United States. This study compared traditional cell culture and direct RT-PCR (reverse transcriptase-polymerase chain reaction) amplification for detection of an enterovirus. Poliovirus could be recovered from 100 L of artificial seawater with an average efficiency of 77%, using adsorption and elution from electronegative filters. Viruses were eluted from the filters with 1.5% beef extract for viruses (BEV) adjusted to pH 9.5 and reconcentrated by organic flocculation to a volume of 30 mL. Substances which interfered with detection by RT-PCR were removed by treatment of the concentrates with sephadex and chelex resins. Direct RT-PCR could detect 2.5 and 0.025 PFU (plaque forming units) for single (25 cycles) and double PCR (2 X 25 cycles) in 10 μL of pure culture poliovirus samples, respectively. These methods are currently being applied to assess the occurrence of enteroviruses at marine bathing beaches influenced by sewage discharges.
• Straub, T. M., Pepper, I. L., & Gerba, C. P. (1995). Comparison of PCR and cell culture for detection of enteroviruses in sludge-amended field soils and determination of their transport. Applied and Environmental Microbiology, 61(5), 2066-2068.
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  PMID: 7646051;PMCID: PMC167478;Abstract: PCR and cell culture assays for enteroviruses were conducted on soil samples collected from an experimental farm that had received mesophilic anaerobically digested sludge for the past 7 years. Of 24 samples assayed, 21 samples were positive by PCR, implying that at least some viral nucleic acid sequences remained intact. However, these viral particles were unable to infect the Buffalo Green Monkey cell line used in subsequent cell culture assays. It is significant that positive PCR detection of nucleic acid sequences occurred even though the most recent sludge application was 3 months prior to soil sampling. Viral nucleic acid sequences were detected by PCR at points vertically and laterally displaced from sludge injections, illustrating significant transport of viruses. Rainfall and irrigation events may have contributed to viral transport.
• Straub, T. M., Pepper, I. L., & Gerba, C. P. (1995). Removal of PCR inhibiting substances in sewage sludge amended soil. Water Science and Technology, 31(5-6), 311-315.
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  Abstract: Current methods for the detection of enteric viruses in soil involve elution of viruses from soil colloids using beef extract or other proteins. These eluates are then assayed in cell culture and observed daily for cytopathic effects (CPE). While this method is suitable for detection of enteric viruses by cell culture, these eluates contain humic acids and heavy metals that interfere with polymerase chain reaction (PCR) detection. Using beef extract eluates prepared from sludge amended soil, 10 different methods of eluate purification were evaluated for their ability to remove PCR inhibition and maximize sensitivity. The treatment method providing the greatest sensitivity of poliovirus detection by PCR was the combination of Sephadex G-50 and Chelex-100. Using this method 2 plaque forming units (PFU) could be detected after reverse transcription and 30 cycles of PCR. Thirty (30) cycles of seminested PCR were performed on these samples to verify nucleic acid sequences and increase sensitivity after the first 30 cycles of PCR. Using seminested PCR sensitivity of detection using the Sephadex G-50 and Chelex-100 treatment method to 0.2 PFU. In addition to providing excellent sensitivity for viruses in sludge amended soils, this treatment method is relatively simple compared to other methods.
• Ma, J. -., Straub, T. M., Pepper, I. L., & Gerba, C. P. (1994). Cell culture and PCR determination of poliovirus inactivation by disinfectants. Applied and Environmental Microbiology, 60(11), 4203-4206.
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  PMID: 7993102;PMCID: PMC201963;Abstract: Inactivation of poliovirus type 1 by 1 N HCl, 1 N NaOH, 0.5 and 1.0 mg of free chlorine per liter, and UV light was compared by using cell culture and seminested PCR (30 cycles of reverse transcriptase-PCR plus 30 cycles of seminested PCR). A minimum contact time of 45 min with HCl, 3 min with NaOH, 3 and 6 min with 1.0 and 0.5 mg of free chlorine per liter, respectively, was required to render 1.64 x 102 PFU of poliovirus type 1 per ml undetectable by seminested PCR. In cell culture, a minimum contact time of 5 min to HCl, 30 s to NaOH, and 1 min to either chlorine concentration was required to render the viruses undetectable by the plaque assay method. No correlation was observed between results by PCR and cell culture when viruses were exposed to UV light. These data suggest that inactivated virus with intact nucleic acid sequences can be detected by PCR.
• Pepper, I. L., Brendecke, J. W., & Axelson, R. D. (1994). Metal contamination on soil microorganisms. Soil Biology and Biochemistry, 26(8), 1099-.
• Pepper, I. L., Josephson, K. L., Bailey, R. L., Burr, M. D., Pillai, S. D., Tolliver, D. L., & Pulido, S. (1994). Measuring bacterial contaminants in ultrapure water. A rapid analytical method. Microcontamination, 12(10).
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  Abstract: The availability of ultrapure water is one of the basic requirements of semiconductor manufacturing. The methods currently used to detect microbial contaminants in UP water can significantly underestimate the actual levels of contamination. This article describes the use of a specialized molecular biology technique known as the polymerase chain reaction that provides rapid and sensitive detection of microbial contaminants. By employing this technique on UP-water samples collected over an 11-month period, we demonstrated that microbial contamination was episodic in nature and that 44.1% of the samples tested showed a positive indication of bacterial contamination of at least 1 CFU/L. The attractive features of this technique are its sensitivity, cost-effectiveness, and speed of detection.
• Soares, A. C., Straub, T. M., Pepper, I. L., & Gerba, C. P. (1994). Effect of anaerobic digestion on the occurrence of enteroviruses and Giardia cysts in sewage sludge. Journal of Environmental Science and Health - Part A Environmental Science and Engineering, 29(9), 1887-1897.
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  Abstract: The disposal of anaerobically digested sewage sludge onto farmland has created the necessity to evaluate the fate of pathogens which are often present. The occurrence of enteroviruses and Giardia cysts in mesophilic anaerobically digested sludge before and after treatment, was monitored for a period of 14 months. The concentration of enteroviruses in sludge determined by Most Probable Number (MPN) ranged from 4.36 x 103 to 7.00 x 105 MPN/Kg before anaerobic digestion and from < 6.25 to 2.52 x 105 MPN/Kg after treatment. The concentration of Giardia cysts as determined by immunofluorescence ranged from 7.33 x 104 to 3.30 x 106 cysts/Kg of undigested sludge and 1.00 x 105 to 4.14 x 106 cysts/Kg of anaerobically digested sludge. The percentage virus removal after anaerobic digestion varied from 68% to > 99.94%. The levels of intact Giardia cysts did not show any significant removal after sludge treatment. However, cyst viability could not be assessed by the method used for detection. Results of this study suggest that significant concentrations of both groups of pathogens can be present in anaerobically digested sludge.
• Straub, T. M., Pepper, I. L., & Gerba, C. P. (1994). Detection of naturally occurring enteroviruses and hepatitis A virus in undigested and anaerobically digested sludge using the polymerase chain reaction. Canadian Journal of Microbiology, 40(10), 884-888.
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  PMID: 7528092;Abstract: Four undigested and four anaerobically digested sewage sludge samples were analyzed for enteroviruses and hepatitis A virus using seminested and double polymerase chain reaction (PCR), respectively. For enteroviruses, all eight samples were positive when detection was by seminested PCR. Using cell culture all samples except two digested sludge samples were positive. For hepatitis A virus, seven out of eight samples were positive by PCR detection. In all samples, PCR inhibitory substances were removed by passage through Sephadex G-50 and Chelex 100 columns. Overall the PCR methodology was highly successful in identifying the presence of both viruses; however, with this methodology, there was no indication as to whether en enteroviruses or hepatitis A viruses not confirmed in cell culture were infectious.
• Straub, T. M., Pepper, I. L., Abbaszadegan, M., & Gerba, C. P. (1994). A method to detect enteroviruses in sewage sludge-amended soil using the PCR. Applied and Environmental Microbiology, 60(3), 1014-1017.
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  PMID: 8161168;PMCID: PMC201425;Abstract: PCR detection of seeded poliovirus type 1 in sludge-amended soil was made possible by utilizing Sephadex G-50 and Chelex-100 resins to remove compounds present in sludge-amended soil that may inhibit PCR. With this method, enteroviruses indigenous to an anerobically digested sludge were detected by PCR in 10 different soils amended with this sludge.
• Abbaszadegan, M., Huber, M. S., Gerba, C. P., & Pepper, I. L. (1993). Detection of enteroviruses in groundwater with the polymerase chain reaction. Applied and Environmental Microbiology, 59(5), 1318-1324.
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  PMID: 7685998;PMCID: PMC182083;Abstract: Standard methods for the detection of enteroviruses in environmental samples involve the use of cell culture, which is expensive and time- consuming. The polymerase chain reaction (PCR) is an attractive method for the detection of enteroviruses in water because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target DNAs and RNAs usually found in environmental samples. However, environmental samples often contain substances that inhibit PCR amplification of target DNA and RNA. Procedures that remove substances that interfere with the amplification process need to be developed if PCR is to be successfully applied to environmental samples. An RNA-PCR assay for the detection of enteroviruses in water was developed and used to test a variety of groundwater concentrates and humic acid solutions seeded with poliovirus type 1. The groundwater samples and humic acid solutions were treated with Sephadex G-50, Sephadex G-100, Sephadex G-200, Chelex-100 resin, and a mixed bed resin to remove PCR-inhibitory material from the samples. Sephadex G-100 in combination with Chelex-100 was found to be very effective in removing inhibitory factors for the detection of enteroviruses in groundwater concentrates by PCR. Viruses were detected in two of the groundwater concentrates by the RNA-PCR assay after treatment with Sephadex G-100 plus Chelex-100. This was confirmed by tissue culture, suggesting that the treatment protocol and, subsequently, the RNA-PCR assay are applicable for the detection of enteroviruses in environmental samples.
• Brendecke, J. W., Axelson, R. D., & Pepper, I. L. (1993). Soil microbial activity as an indicator of soil fertility: Long-term effects of municipal sewage sludge on an arid soil. Soil Biology and Biochemistry, 25(6), 751-758.
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  Abstract: The utility of a selection of soil microbial assays for predicting the effects of land application of municipal sewage sludge on long-term soil fertility was evaluated in a 4 yr study with one unfertilized control plot, and plots with anaerobically digested sewage sludge applied at the optimal rate (based on N requirements) for cotton growth and three times the optimal rate. These loading rates were 8.0 and 24tha-1 yr-1 (dry wt) during 4 yr. The soil was a Pima clay loam (Typic Torrifluvent) which was planted to Upland cotton (Gossypium hirsutum L.). After 4 yr of annual sludge application and annual growth of cotton crops, it was found that sewage sludge application had had no significant effect on various measured soil physical and chemical properties other than an increase in available PO4-P. Total heavy metal contents were not affected by sludge application, but some DTPA-TEA-extractable metals (Zn, Cu, Pb and Ni) increased significantly with sludge treatment. Results also indicated that 4 yr of sewage sludge application had had no significant adverse effect on soil microbial populations or activity. Soil microbial activity was measured by viable heterotrophic plate counts for bacteria, actinomycetes and fungi; acridine orange direct counts for bacteria; the dehydrogenase assay and CO2 evolution. In some cases, there was a significant elevation of a few of the measured variables (dehydrogenase activity and CO2 evolution). Cotton lint yields in year 4 of the study were not significantly affected by treatment, but plant stand was significantly decreased with higher sludge application. As there was no significant association (Pearson product-moment correlation coefficient or the Kendall ρ b, as applicable) between measurements of microbial activity and cotton plant growth, this study lends no support to using soil microbial activity as measured here as a predictive index of soil fertility as affected by land application of sewage sludge. However, this study does illustrate that long-term applications of sludge to arid southwestern desert soils does not adversely affect microbial populations or activity. © 1993.
• Enriquez, C. E., Abbaszadegan, M., Pepper, I. L., Richardson, K. J., & Gerba, C. P. (1993). Poliovirus detection in water by cell culture and nucleic acid hybridization. Water Research, 27(7), 1113-1118.
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  Abstract: Nucleic acid hybridization has been used to detect viral nucleic acid in water. This type of assay, in contrast with tissue culture assays, may not distinguish between viable and non-viable viruses. We evaluated, by comparison with tissue culture infectivity assay (plaque forming method), the ability of the gene probe assay to detect viable poliovirus I (LSc) in well water, autoclaved well water, filter-sterilized well water and autoclaved phosphate buffered saline kept at 37 and 15°C for 75 days, and in dechlorinated tap water held at room temperature. A gradual decline in numbers of poliovirus was observed in all of the samples by cell culture assay. With the exception of autoclaved well water and phosphate buffer, a parallel decline in virus detectable by gene probe occurred in all other water samples. These results suggest that detection of poliovirus 1, by gene probe, is influenced by the presence of microorganisms or their products and to a lesser extent by temperature. This suggests that in some natural waters, the detection of poliovirus 1, by gene probe, may be expected to correlate to detection by tissue culture.
• Enriquez, C. E., Abbaszadegan, M., Pepper, I. L., Richardson, K. J., Margolin, A. B., & Gerba, C. P. (1993). Comparison of poliovirus detection in water by cell culture and nucleic acid hybridization. Water Science and Technology, 27(3-4), 315-319.
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  Abstract: The nucleic acid hybridization technique has been used to detect viral nucleic acid in environmental water samples. This type of assay, in contrast with tissue culture assays, may not distinguish between viable and non-viable viruses. We evaluated, by comparison with tissue culture infectivity assay (plaque forming method), the ability of the gene probe assay to detect viable poliovirus 1 (LSc) in well water, autoclaved well water, filter-sterilized well water and autoclaved phosphate buffered saline kept at 37° C and 15° C for 75 days, and in dechlorinated tapwater held at room temperature. A gradual decline in numbers of poliovirus was observed in all of the samples by cell culture assay. With the exception of autoclaved well water and phosphate buffer samples, a parallel decline in virus detectable by gene probe occurred in all other water samples.
• Estrella, M. R., Brusseau, M. L., Maier, R. S., Pepper, I. L., Wierenga, P. J., & Miller, R. M. (1993). Biodegradation, sorption, and transport of 2,4-dichlorophenoxyacetic acid in saturated and unsaturated soils. Applied and Environmental Microbiology, 59(12), 4266-4273.
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  PMID: 8285717;PMCID: PMC195895;Abstract: The fate of an organic contaminant in soil depends on many factors, including sorption, biodegradation, and transport. The herbicide 2,4- dichlorophenoxyacetic acid (2,4-D) was used as a model compound to illustrate the impact of these interacting factors on the fate of an organic contaminant. Batch and column experiments performed with a sandy loam soil mixture under saturated and unsaturated conditions were used to determine the effects of sorption and biodegradation on the fate and transport of 2,4-D. Sorption of 2,4-D was found to have a slight but significant effect on transport of 2,4-D under saturated conditions (retardation factor, 1.8) and unsaturated conditions (retardation factor, 3.4). Biodegradation of 2,4-D was extensive under both batch and column conditions and was found to have a significant impact on 2,4-D transport in column experiments. In batch experiments, complete mineralization of 2,4-D (100 mg kg-1) occurred over a 4-day period following a 3-day lag phase under both saturated and unsaturated conditions. The biodegradation rate parameters calculated for batch experiments were found to be significantly different from those estimated for column experiments.
• Josephson, K. L., Gerba, C. P., & Pepper, I. L. (1993). Polymerase chain reaction detection of nonviable bacterial pathogens. Applied and Environmental Microbiology, 59(10), 3513-3515.
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  PMID: 8250575;PMCID: PMC182487;Abstract: Polymerase chain reaction (PCR) methodologies for detection of pathogens in environmental samples are currently available. However, positive amplification products for any set of primers only signal that the appropriate target nucleic acid sequences were present in the sample. The presence of the amplification products does not imply that the target organisms were viable. Here we show that PCR will detect nonviable cells, as long as intact target nucleic acid sequences are available. In an environmental water sample, nucleic acids degraded quickly and were not detectable by PCR after 3 weeks even when stored at 4°C. However, these data show that there is a window of opportunity for PCR analyses to result in false positives with respect to viable cells. We further show that care must be taken in the way samples are stored for future PCR amplifications and that filter sterilization of media is not acceptable for long-term preservation of samples for PCR.
• Pepper, I. L., Josephson, K. L., Bailey, R. L., Burr, M. D., & Gerba, C. P. (1993). Survival of indicator organisms in Sonoran Desert soil amended with sewage sludge. Journal of Environmental Science and Health - Part A Environmental Science and Engineering, 28(6), 1287-1302.
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  Abstract: Anaerobically digested sewage sludge is currently applied to farmland surrounding Tucson, Arizona to supply nutrients for the growth of cotton. Bacterial pathogens introduced into the environment by this practice may survive or move through the soil profile thus posing health risks to animals and humans. We have conducted both laboratory and field studies to monitor the survival and transport of bacterial pathogens added to soil via sludge. In laboratory studies, sewage sludge was added to soil and incubated at constant moisture and temperature conditions. Populations of fecal streptococci, fecal coliforms and total coliforms were monitored at weekly intervals using the Most Probable Number (MPN) method. Two soils and 3 temperatures were evaluated. Field studies determined the survival of indicator organisms in the surface horizon, and survival and transport of fecal coliforms up to a depth of 300 cm. We found that soil moisture, texture and temperature all affected the persistence of bacterial pathogens in sludge amended soil. Survival of organisms increased with increasing soil moisture and clay content, and with decreased soil temperatures. In the field, when soil moisture content increased after rainfall events, regrowth of indicator organisms occurred. Soil acted as an efficient filter negating bacterial transport, and thus fecal coliforms did not appear to migrate through the soil profile under non-irrigated conditions.
• Pepper, I. L., Josephson, K. L., Bailey, R. L., Burr, M. D., Pillai, S. D., Tolliver, D., & Pulido, S. (1993). Rapid and systematic analytical method for measuring bacterial contaminants in ultrapure water. Conference Proceedings - Annual Semiconductor Pure Water and Chemicals Conference, 50-62.
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  Abstract: In this paper, we describe the use of a specialized molecular biology technique known as polymerase chain reaction (PCR) to allow rapid and systematic determination of bacterial contaminants in ultrapure water. PCR is a rapid enzymatic process involving repeated amplification of DNA sequences found within microbial cells. This amplification leads to easy identification of contaminating microbes or molecules.
• Pepper, I., Abbaszadegan, M., Huber, M. S., Gerba, C. P., & Pepper, I. L. (1993). Detection of enteroviruses in groundwater with the polymerase chain reaction. Applied and environmental microbiology, 59(5).
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  Standard methods for the detection of enteroviruses in environmental samples involve the use of cell culture, which is expensive and time-consuming. The polymerase chain reaction (PCR) is an attractive method for the detection of enteroviruses in water because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target DNAs and RNAs usually found in environmental samples. However, environmental samples often contain substances that inhibit PCR amplification of target DNA and RNA. Procedures that remove substances that interfere with the amplification process need to be developed if PCR is to be successfully applied to environmental samples. An RNA-PCR assay for the detection of enteroviruses in water was developed and used to test a variety of groundwater concentrates and humic acid solutions seeded with poliovirus type 1. The groundwater samples and humic acid solutions were treated with Sephadex G-50, Sephadex G-100, Sephadex G-200, Chelex-100 resin, and a mixed bed resin to remove PCR-inhibitory material from the samples. Sephadex G-100 in combination with Chelex-100 was found to be very effective in removing inhibitory factors for the detection of enteroviruses in groundwater concentrates by PCR. Viruses were detected in two of the groundwater concentrates by the RNA-PCR assay after treatment with Sephadex G-100 plus Chelex-100. This was confirmed by tissue culture, suggesting that the treatment protocol and, subsequently, the RNA-PCR assay are applicable for the detection of enteroviruses in environmental samples.
• Pepper, I., Josephson, K. L., Gerba, C. P., & Pepper, I. L. (1993). Polymerase chain reaction detection of nonviable bacterial pathogens. Applied and environmental microbiology, 59(10).
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  Polymerase chain reaction (PCR) methodologies for detection of pathogens in environmental samples are currently available. However, positive amplification products for any set of primers only signal that the appropriate target nucleic acid sequences were present in the sample. The presence of the amplification products does not imply that the target organisms were viable. Here we show that PCR will detect nonviable cells, as long as intact target nucleic acid sequences are available. In an environmental water sample, nucleic acids degraded quickly and were not detectable by PCR after 3 weeks even when stored at 4 degrees C. However, these data show that there is a window of opportunity for PCR analyses to result in false positives with respect to viable cells. We further show that care must be taken in the way samples are stored for future PCR amplifications and that filter sterilization of media is not acceptable for long-term preservation of samples for PCR.
• Pepper, I., Way, J. S., Josephson, K. L., Pillai, S. D., Abbaszadegan, M., Gerba, C. P., & Pepper, I. L. (1993). Specific detection of Salmonella spp. by multiplex polymerase chain reaction. Applied and environmental microbiology, 59(5).
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  Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. phoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-1i primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonella species and are not present in Shigella, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of Salmonella species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 10(2) CFU after 25 cycles of PCR and 1 (10(0)) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as Salmonella species by the use of conventional cultural techniques. In addition, positive amplifications resulted from Salmonella species in environmental samples including soil and water.
• Straub, T. M., Pepper, I. L., & Gerba, C. P. (1993). Hazards from pathogenic microorganisms in land-disposed sewage sludge. Reviews of Environmental Contamination and Toxicology, 132, 55-91.
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  PMID: 8346362;
• Straub, T. M., Pepper, I. L., & Gerba, C. P. (1993). Virus survival in sewage sludge amended desert soil. Water Science and Technology, 27(3-4), 421-424.
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  Abstract: Currently Pima County, Arizona, disposes all of its anaerobically digested sewage sludge in liquid form (1.5% solids) on agricultural land used for non-food crop production by subsurface injection or surface spreading. Present in these sludges are human enteric viruses in concentrations as high as 1,000 per liter of sludge. These viruses could potentially contaminate surface and groundwater sources during periods of irrigation or extended rainfall. This study was designed to assess the survival of viruses under field conditions typical of the arid Southwestern United States during the winter and summer months. This study was also conducted in the laboratory to simulate field conditions. Soil samples taken from freshly amended fields were seeded with poliovirus type 1 (stock titer = 106/ml) and bacteriophage MS2 (stock titer = 1010/ml) and thoroughly mixed with the sludged soil. The seeded samples were put into containers and buried 10 cm below the soil surface, and samples were taken at pre-determined time intervals. Average soil temperature (measured at the 10 cm depth) ranged from 15°C in the winter to 33°C in the summer. Soil moisture decreased from 25% to 15% in the winter and from 40% to less than 5% in the summer. During the winter study, no inactivation of poliovirus was observed after 7 days, while greater than a 90% reduction was observed for MS-2. During the summer study, no poliovirus was recovered after 7 days, and no MS-2 was recovered after 3 days. The results of this study suggest that high soil temperature and rapid loss of moisture limit the survival of viruses in desert soils.
• Way, J. S., Josephson, K. L., Pillai, S. D., Abbaszadegan, M., Gerba, C. P., & Pepper, I. L. (1993). Specific detection of Salmonella spp. by multiplex polymerase chain reaction. Applied and Environmental Microbiology, 59(5), 1473-1479.
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  PMID: 8517741;PMCID: PMC182106;Abstract: Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. phoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-li primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-li flagellin gene, respectively, were used. Both Hin and H- li primers are specific to motile Salmonella species and are not present in Shigella, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of Salmonella species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 102 CFU after 25 cycles of PCR and 1 (100) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as Salmonella species by the use of conventional cultural techniques. In addition, positive amplifications resulted from Salmonella species in environmental samples including soil and water.
• Artiola, J. F., & Pepper, I. L. (1992). Denitrification activity in the root zone of a sludge-amended desert soil. Biology and Fertility of Soils, 13(4), 200-205.
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  Abstract: We evaluated potential NOinf3sup-losses from organic and inorganic N sources applied to improve the growth of cotton (Gossypium hirsutum) on a Pima clay loam soil (Typic Torrifluvent). An initial set of soil cores (April 1989) was collected to a depth of 270 cm from sites in a cotton field previously amended with anaerobically digested sewage sludge or an inorganic N fertilizer. The denitrification potential was estimated in all soil samples by measuring N2O with gas chromatography. Soils amended with a low or high rate of sludge showed increased denitrification activity over soil samples amended with a low rate or inorganic N fertilizer. All amended samples showed greater denitrification activity than control soils. The denitrification decreased with soil depth in all treatments, and was only evident as deep as 90 cm in the soils treated with the high sludge rate. However, when soils collected from depths greater than 90 cm were amended with a C substrate, significant denitrification activity occurred. These date imply that organisms capable of denitrification were present in all soil samples, even those at depths far beneath the root zone. Hence, denitrification was C-substrate limited. A second series of soil cores taken later in the growing season (July 1989) confirmed these data. Denitrification losses (under laboratory conditions) to a soil depth of 270 cm represented 1-4% of total soil N depending on treatment, when the activity was C-substrate limited. With additional C substrate, the denitrification losses increased to 15-22% of the total soil N. © 1992 Springer-Verlag.
• Artiola, J. F., & Pepper, I. L. (1992). Longterm influence of liquid sewage sludge on the organic carbon and nitrogen content of a furrow-irrigated desert soil. Biology and Fertility of Soils, 14(1), 30-36.
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  Abstract: In this study we evaluated the impact of five annual liquid sewage-sludge applications on the organic C and N content of a furrow-irrigated desert soil. Mineralization rates showed that sludge organic matter is mineralized rapidly (65% per year). Resistant residual sludge organic matter accumulation resulted in a theoretical increase in total soil organic C of 0.013% for the single sludge rate or 0.038% for three annual applications. These small additions were not detected in sludged soils at any depth to 270 cm. Similarly, increases in total soil N were not detected at any depth. However, soluble forms of organic C and N did increase in sludged soils relative to the non-sludged soils. In addition, soluble C:N ratios decreased significantly in the sludged soils. Soluble C and N also increased with depth due to leaching. This study therefore shows that applications of liquid sludge onto desert soils could affect the status of underground aquifers with respect to nitrate pollution. © 1992 Springer-Verlag.
• Neilson, J. W., Josephson, K. L., Pillai, S. D., & Pepper, I. L. (1992). Polymerase chain reaction and gene probe detection of the 2,4- dichlorophenoxyacetic acid degradation plasmid, pJP4. Applied and Environmental Microbiology, 58(4), 1271-1275.
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  PMID: 1599246;PMCID: PMC195586;Abstract: Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4- dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.
• Pepper, I., Neilson, J. W., Josephson, K. L., Pillai, S. D., & Pepper, I. L. (1992). Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4. Applied and environmental microbiology, 58(4).
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  Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4-dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.
• Pillai, S. D., Josephson, K. L., Bailey, R. L., & Pepper, I. L. (1992). Specific detection of rhizobia in root nodules and soil using the polymerase chain reaction. Soil Biology and Biochemistry, 24(9), 885-891.
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  Abstract: The polymerase chain reaction (PCR) amplification of specific DNA sequences, allows specific and sensitive detection of bacteria at the genus, species or strain level depending on the design of the oligonucleotide primers. In this study we utilized 20 mer primers that flanked a 300 bp region of the npt II gene of the transposon Tn5 thus allowing for the amplification of this region. Insertion of the Tn 5 element into rhizobia allowed for detection of these cells using PCR amplification. Using the npt II-specific primers and Tn5 insertion mutants of Rhizobium leguminosarum bv. phaseoli we were able to detect these specific rhizobia strains in root nodules of bean plants and in inoculated soils. Utilization of genus-specific gene sequences would allow for estimates of cells of that genus in environmental samples. Conversely, use of gene sequences common to rhizobia, e.g. nif and nod sequences, would give estimates of the population of rhizobia. This paper serves to illustrate the use of PCR, for detecting gene sequences in an environmental sample such as a root nodule. © 1992.
• Soares, A. C., Pepper, I. L., & Gerba, C. P. (1992). Recovery of poliovirus from sludge-amended soils. Journal of Environmental Science and Health - Part A Environmental Science and Engineering, 27(4), 999-1005.
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  Abstract: A method to recover enteroviruses from sludge amended soils was developed, using Poliovirus type 1 (strain LSc) as a model. Recovery efficiencies were evaluated for ratios of 1:1, 1:2.5, 1:5, and 1:10 of sludge amended soil to eluent, using 3% buffered beef extract at pH 9.5. Viral recoveries varied from 1.7% to 98.8%, with a significant increase when larger volumes of the eluent were used. This study also showed that there is a linear correlation (r = 0.92) between the volume of the eluent and the percent recovery of poliovirus.
• Straub, T. M., Pepper, I. L., & Gerba, C. P. (1992). Persistence of viruses in desert soils amended with anaerobically digested sewage sludge. Applied and Environmental Microbiology, 58(2), 636-641.
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  PMID: 16348651;PMCID: PMC195295;Abstract: Pima County, Ariz., is currently investigating the potential benefits of land application of sewage sludge. To assess risks associated with the presence of pathogenic enteric viruses present in the sludge, laboratory studies were conducted to measure the inactivation rate (k = log10 reduction per day) of poliovirus type 1 and bacteriophages MS2 and PRD-1 in two sludge-amended desert agricultural soils (Brazito Sandy Loam and Pima Clay Loam). Under constant moisture (approximately -0.05 x 105 Pa for both soils) and temperatures of 15, 27, and 40°C, the main factors controlling the inactivation of these viruses were soil temperature and texture. As the temperature increased from 15 to 40°C, the inactivation rate increased significantly for poliovirus and MS2, whereas, for PRD-1, a significant increase in the inactivation rate was observed only at 40°C. Clay loam soils afforded more protection to all three viruses than sandy soils. At 15°C, the inactivation rate for MS2 ranged from 0.366 to 0.394 log10 reduction per day in clay loam and sandy loam soils, respectively. At 27°C, this rate increased to 0.629 log10 reduction per day in clay loam soil and to 0.652 in sandy loam soil. A similar trend was observed for poliovirus at 15°C (k = 0.064 log10 reduction per day, clay loam; k = 0.095 log10 reduction per day, sandy loam) and 27°C (k = 0.133 log10 reduction per day, clay loam; k = 0.154 log10 reduction per day, sandy loam). Neither MS2 nor poliovirus was recovered after 24 h at 40°C. No reduction of PRD-1 was observed after 28 days at 15°C and after 16 days at 27°C. At 40°C, the inactivation rates were 0.208 log10 reduction per day in amended clay loam soil and 0.282 log10 reduction per day in sandy loam soil. Evaporation to less than 5% soil moisture completely inactivated all three viruses within 7 days at 15°C, within 3 days at 27°C, and within 2 days at 40°C regardless of soil type. This suggests that a combination of high soil temperature and rapid loss of soil moisture will significantly reduce risks caused by viruses in sludge.
• Josephson, K. L., Bourque, D. P., Bliss, F. A., & Pepper, I. L. (1991). Competitiveness of KIM 5 and VIKING 1 bean rhizobia: Strain by cultivar interactions. Soil Biology and Biochemistry, 23(3), 249-253.
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  Abstract: Competitiveness, which in rhizobia refers to the relative ability of a strain to inject a legume and cause nodule formation in the presence of other strains, is critical to biological N2-fixation. The mechanisms for strain competitiveness are unknown, but are probably affected by biotic and abiotic factors. The competitiveness of two strains of rhizobia (Rhizobium leguminosarum biovar phaseoli) KIM 5 and VIKING 1 that are known to be highly competitive were evaluated when added together as inoculant to 12 bean (Phaseolus vulgaris L.) host cultivars grown in Leonard jars in sterilized sand. Strain identification of bacteria from harvested nodules was determined by fluorescent antibodies. KIM 5 was generally more competitive than VIKING 1, but competitiveness of both strains was highly dependent on strain by cultivar interactions. With Tendergreen, no nodules contained VIKING 1 alone, whereas with Jampa, 46.3 of the nodules contained only VIKING 1. Comparisons of parent cultivar and sib progeny lines showed that even small differences in the host genotype affected the competitiveness of both strains. All symbioses were effective in fixing N2. Based on these data, studies evaluating the competitiveness of rhizobia should utilize several host genotypes to avoid misinterpretations of the competitiveness of a given strain. © 1991.
• Josephson, K. L., Pillai, S. D., Way, J., Gerba, C. P., & Pepper, I. L. (1991). Fecal coliforms in soil detected by polymerase chain reaction and DNA-DNA hybridizations. Soil Science Society of America Journal, 55(5), 1326-1332.
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  Abstract: The application of sewage sludge on agricultural lands is a common practice in the USA. The survival of pathogens and indicator organisms added to soil via sludge needs to be determined. A rapid method utilizing polymerase chain reaction (PCR) and a gene-specific probe was used to detect low numbers of fecal coliforms in soil. Two 23-base primers were designed from a region of the lamB gene that codes for an outer-membrane protein present specifically in fecal coliforms. Use of these primers for PCR amplification of a 179-bp fragment of lamB allowed specific detection of fecal-coliform indicator organisms. Sensivity of amplification from whole-cell lysates of pure cultures of Escherichia coli using detection via ethidium bromide was 104 colony-forming units (CFUs). When detection of these cell lysates was via a lamB gene-specific probe, a total of 4 × 102 CFUs could be detected from these pure cultures. When genomic deoxyribonucleic acid (DNA) preparations were made from these pure cultures, 100 ag (10-18) of DNA was detectable. Following the inoculation of E. coli into soil, cells were extracted with CaCl2, and concentrated and purified by sucrose density centrifugation. The DNA obtained by lysing the cells was amplified using a new 'double PCR' 50-cycle protocol. Escherichia coli were detected in Brazito sandy loam (mixed, thermic Typic Torripsamment) and Pima silty clay loam (fine-silty, mixed (calcareous), thermic Typic Torrifluvent). Using a 5′ end-labeled lamB-specific probe to detect PCR products, one CFU of E. coli was detectable in 1 g of soil. One PCR-amplificable CFU g-1 would be equivalent to many copies of amplificable target DNA, due to the presence of dead and lysed cells along with cells containing more than one copy of the genome. Sample processing and PCR amplification can be completed within 7 to 8 h. Thus, the use of PCR for sensitive detection of introduced bacteria in environmental soil samples is possible.
• Pepper, I. L., & Upchurch, R. P. (1991). Nitrogen fixation by desert legumes associated with Rhizobia. Semiarid lands and deserts, 443-467.
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  Abstract: Legumes can fix nitrogen, and Rhizobium can assist, together they offer excellent opportunities for semi-arid and arid land management, restoration and development. -C.J.Barrow
• Pepper, I., Pillai, S. D., Josephson, K. L., Bailey, R. L., Gerba, C. P., & Pepper, I. L. (1991). Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences. Applied and environmental microbiology, 57(8).
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  Bacterial cells can be differentially separated from soil colloids on the basis of their buoyant densities. By using this principle, a modified sucrose gradient centrifugation protocol has been developed for separating bacterial cells from most of the soil colloids. Since the bacterial cell suspension still contained some colloidal soil particles, which inhibited polymerase chain reaction amplification, a new "double" polymerase chain reaction method of analysis was adopted for amplification of Tn5-specific gene sequences. This new protocol allowed rapid detection of small numbers (1 to 10 CFU/g) of bacterial cells present in soil samples.
• Pillai, S. D., & Pepper, I. L. (1991). Transposon Tn 5 as an identifiable marker in rhizobia: Survival and genetic stability of Tn 5 mutant bean rhizobia under temperature stressed conditions in desert soils. Microbial Ecology, 21(1), 21-33.
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  PMID: 24194199;Abstract: Five transposon Tn 5 insertion mutants of a bean Rhizobium strain (Rhizobium leguminosarum b. v. phaseoli) were used in an ecological study to evaluate the extent to which transposon Tn 5 was stable to serve as an identifiable marker in rhizobia under a high temperature stress condition in two Sonoran Desert soils. All the mutants possessed single chromosomal insertions of the transposon. In both soils, under the temperature stress conditions that were employed (40°C), both wild type and mutant populations possessing functional transposable elements declined rapidly. After 12 days, mutant cells, when screened using the Tn 5 coded antibiotic resistance markers, were significantly less in number than when they were screened using only their intrinsic antibiotic resistance markers. There were no significant differences in numbers between the mutant cell population and the wild type when the mutant cells were screened using only the intrinsic antibiotic resistance markers. DNA-DNA hybridizations using a probe indicated neither deletion nor transposition of the transposable element. The results indicate that transposon DNA sequences are present within cells under high temperature stress conditions, but kanamycin/neomycin resistance is not expressed by some of these cells, suggesting that Tn 5 undergoes a possible functional inactivation under these conditions. The possible implications of these findings are discussed. © 1991 Springer-Verlag New York Inc.
• Pillai, S. D., Josephson, K. L., Bailey, R. L., Gerba, C. P., & Pepper, I. L. (1991). Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences. Applied and Environmental Microbiology, 57(8), 2283-2286.
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  PMID: 1662931;PMCID: PMC183564;Abstract: Bacterial cells can be differentially separated from soil colloids on the basis of their buoyant densities. By using this principle, a modified sucrose gradient centrifugation protocol has been developed for separating bacterial cells from most of the soil colloids. Since the bacterial cell suspension still contained some colloidal soil particles, which inhibited polymerase chain reaction amplification, a new 'double' polymerase chain reaction method of analysis was adopted for amplification of Tn5-specific gene sequences. This new protocol allowed rapid detection of small numbers (1 to 10 CFU/g) of bacterial cells present in soil samples.
• Day, A. D., Ottman, M. J., Taylor, B. B., Pepper, I. L., & Swingle, R. S. (1990). Wheat responds to sewage sludge as fertilizer in an arid environment. Journal of Arid Environments, 18(2), 239-244.
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  Abstract: Triticum durum was grown on a sandy loam soil and fertilized with recommended rates of inorganic N and recommended rates of plant-available N from anaerobically digested liquid sewage sludge. Fertilization with sludge increased number of days from planting to heading, plant height, and tillering. The low sludge rate and the inorganic N treatment produced similar yields of hay, grain, and straw. High sludge rate produced the most hay and straw, but did not result in a significant increase in grain yield. Wheat hay, grain, and straw grown with sewage sludge and inorganic N were similar in livestock feeding qualities. Heavy metal concentrations in wheat hay, grain, and straw were low in all fertilizer treatments. When wheat was grown to maturity, more heavy metals accumulated in grain than in straw. -from Authors
• Neilson, J. W., & Pepper, I. L. (1990). Soil respiration as an index of soil aeration. Soil Science Society of America Journal, 54(2), 428-432.
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  Abstract: The accurate determination of available soil O2 is problematic due to the dynamic interaction of physical and biological soil properties. This study was conducted to evaluate combined effects of soil bulk density, water content, and organic and inorganic amendments on available soil O2 using CO2 evolution from soil respiration as an index. Optimal levels of soil aeration vary with respiratory O2 consumption, which is influenced by soil amendments. Comparisons were made between Pima clay loam [fine-silty, mixed (calcareous), thermic, Typic torrifluvent] amended with inorganic fertilizer or anaerobically digested liquid sewage sludge at equivalent N loading rates. Respiration rates in the inorganic-fertilizer- and sludge-amended soil samples were equivalent.
• Pillai, S. D., & Pepper, I. L. (1990). Survival of Tn5 mutant bean rhizobia in desert soils: Phenotypic expression of Tn5 under moisture stress. Soil Biology and Biochemistry, 22(2), 265-270.
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  Abstract: The survival and phenotypic expression of a transposon Tn5 mutant of bean rhizobia (Rhizobium leguminosarum bv. phaseoli) and the corresponding wildtype under increasing moisture stress conditions was studied in two Sonoran Desert soil types. Survival studies in sterile and nonsterile soil showed that both biotic and abiotic factors influenced the survival of the mutant and the wild type. In the silty clay loam, the Tn5 mutant population averaged ca 105cfu g-1 soil even at a moisture content of 2% (corresponding to a soil water potential of
• Shishido, M., & Pepper, I. L. (1990). Identification of dominant indigenous Rhizobium meliloti by plasmid profiles and intrinsic antibiotic resistance. Soil Biology and Biochemistry, 22(1), 11-16.
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  Abstract: Yields of alfalfa (Medicago sativa L.) in irrigated production agriculture in the southwest U.S.A. varies widely, but is generally higher than in other areas of the U.S.A. Since nitrogen fertilizer is rarely applied, high yields are likely to be due in part to biological N2 fixation in the symbiotic association between alfalfa and Rhizobium meliloti. Dominant types of R. meliloti responsible for N2 fixation were identified from nodule isolates collected from five locations throughout the state of Arizona, which were not known to have been inoculated. The locations were sampled in the winter of 1987 and the summer of 1988. The dominant types (≥20% nodule occupancy at each sampling site) were identified through plasmid profile analysis and intrinsic antibiotic resistance patterns. Four types were found to be dominant throughout the state. A single example of each of these four dominant types and a commercial strain (Nitragin Co., Milwaukee, Wis.) were of equal symbiotic effectiveness as M. sativa cv. Lew. No significant differences were found (P ≥ 0.05) in shoot weight, root weight, nodule weight, acetylene reduction and total plant N content. Several of the selected cultures may have potential as inoculants for use in arid lands due to their effectiveness and their ability to survive in the extreme environmental conditions prevailing in the soils from which they were isolated. © 1990.
• Pepper, I. L., Josephson, K. L., Nautiyal, C. S., & Bourque, D. P. (1989). Strain identification of highly-competitive bean rhizobia isolated from root nodules: Use of fluorescent antibodies, plasmid profiles and gene probes. Soil Biology and Biochemistry, 21(6), 749-753.
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  Abstract: Kim 5 and Viking 1 are highly-competitive N-fixing bean rhizobia (Rhizobium leguminosarum biovar phaseoli). Leonard jar studies were used to determine which of the two isolates was the most competitive. A mixed inoculant of both strains was added to bean seed (Phaseolus vulgar is cultivar Tendergreen) in sterile sand. Single-strain inoculants were used for seed grown in soil containing indigenous rhizobia. Nodule occupancy was determined after harvest. Plasmid profiles of nodule isolates from bean seed inoculated with equal numbers of both strains showed that Kim 5 occupied 72% of the nodules. Plasmid profiles for Kim 5 revealed three distinctive bands as compared to two bands from Viking 1. One band appeared to be common to both strains, whereas Kim 5 had two unique bands. A 32P-labelled gene probe to one of the unique Kim 5 plasmids was used to-detect Kim 5 isolated from nodules by colony hybridization. This technique showed that Kim 5 was found in 76.2% of the nodules. Fluorescent antibodies were also used to distinguish between isolates. When single-strain inoculants were used in non-sterile soil, fluorescent antibodies showed that Kim 5 was recovered in 67.5% of the nodule isolates compared to 17.6% for Viking 1, while the other isolates were indigenous rhizobia. These data demonstrate that Kim 5 is more competitive than Viking 1. © 1989.
• Day, A. D., Ottman, M. J., Taylor, B. B., Pepper, I. L., & Swingle, R. S. (1988). Liquid sludge as fertilizer for wheat. BioCycle, 29(10), 60-61.
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  Abstract: Application of liquid sewage sludge on agricultural lands has been practiced since 1984 in Pima County, Arizona. Experiments were conducted in Arizona to compare the plant growth, yield, and livestock feeding qualities of wheat hay, grain, and straw when fertilized with liquid sewage sludge and inorganic fertilizers. Anaerobically digested liquid sewage sludge was obtained from the Pima County Wastewater Treatment Plant at Ina Road near Tucson, Arizona. The sewage sludge rates used in this study resulted in plant growth, yield, and quality of wheat hay, grain, and straw that were similar to the plant growth, yield, and quality of wheat fertilized with inorganic N. Cadmium and nickel concentrations in all plant parts tested were below the detectable limit in all fertilizer treatments. Copper, lead, and zinc in wheat hay, grain, and straw showed similar patterns of accumulation for all fertilizer treatments. The foregoing observations, in addition to Pima county's industrial sludge, pre-treatment requirements and state guidelines limiting sludge applications to the N requirements of crops, make sewage sludge from Tucson, Arizona safe for continuous use in commercial agriculture.
• Miller, M., & Pepper, I. L. (1988). Physiological and biochemical characteristics of a fast-growing strain of lupin rhizobia isolated from the sonoran desert. Soil Biology and Biochemistry, 20(3), 319-322.
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  Abstract: An effective, fast-growing strain of lupin rhizobia was isolated from a species of Lupinus native to the Sonoran Desert near San Felipe, Baja, Mexico (generation time, 3.6 h). Bacteria isolated from the roots of lupins are normally slow growing, however. Lupin 43 was fast-growing, possessed multiple flagella and produced acid in a denned medium. In comparison to a slow-growing lupin strain. Nitragin 96AII; Lupin 43 had a low intrinsic resistance to antibiotics and was able to utilize a wider range of C and N-sources. When compared to a fast-growing strain of Rhizobium meliloti, also isolated from the Sonoran Desert, Lupin 43 had a slightly faster growth rate, was less resistant to antibiotics and produced alkaline end products when asparagine was supplied as N-source. Based on these characteristics. Lupin 43 was more similar to other fast-growing rhizobia than the slow growing lupin rhizobia. © 1988.
• Miller, M., & Pepper, I. L. (1988). Survival of a fast-growing strain of lupin rhizobia in sonoran desert soils. Soil Biology and Biochemistry, 20(3), 323-327.
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  Abstract: Bacteria isolated from the roots of lupins are normally slow-growing, but an effective, fast-growing strain (generation rime, 3.6 h) of Rhizobium was isolated from a species of Lupinus native to the Sonoran Desert near San Felipe, Baja, Mexico. This strain (Lupin 43) possesses multiple ffageila and produces acid in a denned medium. Field and incubator studies were conducted to determine if the Actaptation of the characteristics of fast-growing strains enabled Lupin 43 to survive under the desert conditions of the southwestern U.S. In the field, where no moisture was added after initial inoculation, Lupin 43 survived in significantly higher numbers than Lupin 96AI 1, a commercial, slow-growing strain. In laboratory studies, at a constant moisture tension of - kPa, differences in survival between the two strains were mostly dependent on soil texture, pH and temperature. © 1988.
• Ferguson, G. A., & Pepper, I. L. (1987). AMMONIUM RETENTION IN SAND AMENDED WITH CLINOPTILOLITE.. Soil Science Society of America Journal, 51(1), 231-234.
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  Abstract: Clinoptilolite zeolite has a rigid three-dimensional lattice with 10** minus **9 m sized tunnels, and an affinity for NH** plus //4 on its internal exchange sites. These internal tunnels are too small for 10** minus **6 m size microbes to access; hence, clinoptilolite has the potential to physically protect NH** plus //4 from nitrification by microbes, and may reduce volatilization losses. The objective of this study was to evaluate NH** plus //4 retention of sand amended with clinoptilolite. Retention of NH** plus //4 on clinoptilolite amended sands incubated at 20% volumetric water content, was studied in the laboratory. Clinoptilolite reduced NH** plus //4 losses from the soil mix and would allow increased plant N fertilizer-use efficiency. Thus clinoptilolite-amended sands have potential as a growth medium for turfgrasses on golf greens, where N use efficiency is typically low.
• Roskoski, J. P., Pepper, I., & Pardo, E. (1986). Inoculation of leguminous trees with rhizobia and VA mycorrhizal fungi. Forest Ecology and Management, 16(1-4), 57-68.
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  Abstract: Many leguminous trees are tripartite symbioses composed of the plant, Rhizobium bacteria, and vesicular-arbuscular (VA) mycorrhizal fungi. However, few field studies have examined the efficacy of inoculating tree legumes with microsymbionts. A Rhizobium inoculation trial was run with Leucaena leucocephala and Acacia pennatula at two sites in Mexico. Results showed that inoculated plants of both species exhibited greater growth than uninoculated controls in Xalapa, a site with acid soils, but not in LaBalsa which has alkaline soils. Furthermore, the best inoculant strain on A. pennatula in Xalapa was isolated from the very field where the inoculation trial was run. The data suggest that while the inoculation response of leguminous trees may vary with site or species, the benefits possible from the establishment of a highly effective tripartite symbiosis warrant including Rhizobium and VA mycorrhizae inoculation trials in all tree legume programs. © 1986.
• Shoushtari, N. H., & Pepper, I. L. (1985). Mesquite rhizobia isolated from the Sonoran Desert: Competitiveness and survival in soil. Soil Biology and Biochemistry, 17(6), 803-806.
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  Abstract: The competitiveness of a mesquite Rhizobium (AZ-M1) and its ability to survive in desert soils was compared to a selected commercial strain (31A5). In a greenhouse study, the native isolate out-competed strain 31A5 in nodule occupancy, when applied as a mixed inoculant to seed germinated and grown in sand culture, and irrigated with N-free nutrients. A high incidence of nodule double occupancy was found when double strain inoculants were used. The survival rate of the two strains was tested in three desert soils in a controlled laboratory study. The desert strain AZ-M1 grew and survived in all the soils for 1 month. The commercial strain 31A5, did not grow, and the population decreased in 14 days from 108 cells g-1 dry soil to below 104 cells g-1. Both strains survived to a lesser extent in a saline-sodic soil. A significant morphological change from a rod to a coccus was observed 2 days after strain 31A5 had been introduced into the desert soils. © 1985.
• Shoushtari, N. H., & Pepper, I. L. (1985). Mesquite rhizobia isolated from the Sonoran Desert: Physiology and effectiveness. Soil Biology and Biochemistry, 17(6), 797-802.
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  Abstract: A mesquite Rhizobium isolated from the Sonoran Desert (strain AZ-M1) and a commercial mesquite Rhizobium obtained from the Nitragin Company (strain 31A5) were chosen as superior strains from among many evaluated in a screening study of the efficiency of mesquite rhizobia. Both strains were fast-growing and acid-producing in denned media. The desert strain AZ-M1 had the shortest mean generation time of 3.5 h. Strain 31A5 grew better in broth amended with various sugars and amino acids, but generally produced less acid. The desert strain showed greater resistance to various antibiotics than did 31A5. In a greenhouse study N applied at high rates inhibited N2 fixation when either strain was used as inoculant for mesquite seed. At low N rates, AZ-M1 fixed more N than 31A5. Total N, nodule weight, C2H2 reduction, mesquite shoot weight and root weight were all significantly increased when AZ-M1 was the applied inoculant. This study shows that the mesquite Rhizobium AZ-M 1 isolated from the Sonoran Desert is infective and effective on mesquite seedlings. Fast growth rate, acid production and high resistance to antibiotics in laboratory media may indicate the adaptation of this organism to its microbial niche in the Sonoran Desert. © 1985.
• Watson, J. E., Pepper, I. L., Unger, M., & Fuller, W. H. (1985). Yields and leaf elemental composition of cotton grown on sludge-amended soil. Journal of Environmental Quality, 14(2), 174-177.
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  Abstract: A 3-yr field study was conducted to determine the effects of land application of anaerobically digested, air-dried sewage sludge on growth of upland cotton (Gossypium hirsutum L.). Cotton lint yields obtained with sludge rates from 20 to 80 Mg/ha were comparable with those obtained by area farmers employing conventional fertilizer practices. Rates of 80 Mg/ha had no significant effect on lint yields, although lint/seed ratios tended to decrease with increased sludge rate for all years. After 3 yr of sludge application, the leaf and seed concentrations of Cd, Zn, Ni, and Cu were not significantly different from those on the fertilized check plots. Leaf concentrations of Cd were higher than seed concentrations, but the reverse trend was true for Zn, indicating that Zn may not be directly useable as a model for Cd behavior in cotton.
• Babiker, H. M., & Pepper, I. L. (1984). Microbial production of ethylene in desert soils. Soil Biology and Biochemistry, 16(6), 559-564.
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  Abstract: Ethylene production was monitored in 12 desert soils. In all but two soils with high organic matter content, C2H4 production was low. Statistical analysis showed a good correlation between organic matter content and C2H4, production. Minimum levels of C2H4 were observed in saline and sodic soils. Addition of l-methionine to soil significantly increased C2H4 formation, indicating its possible role as a precursor for C2H4. Addition of salt to the high C2H4-producing soils suppressed C2H4 production most probably because of a direct effect on C2H4-producing microorganisms through toxic salt levels, high osmotic pressure, increased pH or a combination of these factors. Leaching of four saline soils and subsequent incubation resulted in significant increases in C2H4 in two soils. Ethylene producers, previously inhibited by salinity, were probably reactivated when the salts were removed. A Fusarium isolated from the high C2H4-producing soil, produced the most C2H4 in pure culture followed by isolates belonging to the genera Aspergillus, Penicillium, Curvularia and Rhizopus. A sterilized saline soil produced significant C2H4 when inoculated with spores of Mucor hiemalis or the Fusarium isolate, indicating an originally low population of C2H4-producing organisms in the saline soil. The two high organic matter soils when sterilized and similarly inoculated produced only a fraction of the C2H4 produced in non-sterilized samples, indicating the involvement of a number of species in the production of C2H4 in these soils. © 1984.
• Josephson, K. L., & Pepper, I. L. (1984). Competitiveness and effectiveness of strains of Rhizobium phaseoli isolated from the sonoran desert. Soil Biology and Biochemistry, 16(6), 651-655.
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  Abstract: Four strains of Rhizobium phaseoli were examined for N2 fixation effectiveness and for competitiveness for nodule occupancy by utilizing strain-specific fluorescent antibodies. Competition studies in Leonard jars held in a growth chamber showed strain KIM-5 (a cool season isolate from Kimberly, Idaho) consistently occupied the majority of nodules on bean plants (Phaseolus vulgaris L.) cv. Kentucky Wonder, when applied as a mixed inoculant with desert strains (K-1, 36 or 90). Competitiveness of KIM-5 was relatively independent of cell numbers as shown by the high recovery of KIM-5 from nodules, even when extensively outnumbered in the inoculant. KIM-5 out-competed the desert strains regardless of whether they were ineffective (strains 36 and 90) or highly effective (K-1). Although KIM-5 was more competitive than K-l, no difference in infectiveness (as shown by nodule mass) or effectiveness (as shown by % N, total plant N, C2H2 reduction and total plant weight) was observed. In YEM broth, strain K-l showed increasing growth rates when the temperature was increased from 27° to 35°C, and was viable at 40°C. These data indicate K-1 to be an unusually heat-tolerant strain. Growth rates of KIM-5 were constant from 27° to 35°C and the organism was not viable at 40°C. Both strains produced acid in a defined broth medium. The effectiveness of KIM-5 and K-l was also evaluated under field conditions using single strain inoculants with two cultivars of pinto beans (P. vulgaris L.) ev. Mexicali 80 and Delicias 71. Inoculation with K-1 resulted in yield increases with both cultivars over uninoculated plants, whereas there was little difference between KIM-5 inoculated and uninoculated plants. © 1984.
• Pepper, I. L., Bezdicek, D. F., Baker, A. S., & Sims, J. M. (1983). Silage corn uptake of sludge-applied zinc and cadmium as affected by soil pH. Journal of Environmental Quality, 12(2), 270-275.
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  Abstract: Liming is often recommended to control metal uptake in plants. In western Washington, where silage corn (Zea mays L.) is ofted fed to dairy cattle, studies were conducted to determine the effect of liming on the uptake of Zn, and especially Cd from land-applied sewage sludge. Two soils, a Sultan silt loam and a Puyallup fine sandy loam were amended annually for 2 y with anaerobically digested sewage sludge, and seeded to silage corn. Half of the plots were limed, which increased soil pH from 4.6 to 6.5 for both soils. In general, liming reduced Zn uptake in most corn tissues, whereas Cd uptake was generally unaffected. Concentration of Zn and Cd in corn leaves from both soils increased significantly with increased sludge rate. Liming significantly lowered leaf Zn, although leaf Cd was not significantly decreased by liming. In fact, at higher sludge application rates, leaf Cd was greater from limed than unlimed plots for the Puyallup soil in 1976. Kernel Zn and Cd concentrations for both soils generally increased with sludge rate. For the Sultan soil, kernel Cd concentration at silage harvest was similar, regardless of lime treatment, whereas Cd values for the limed Puyallup soil increased significantly over those from unlimed soil in 1976. For both soils, Cd and Zn concentrations followed the order: leaves > stover > cobs > kernels. Leaf and DTPA-extractable Zn/Cd ratios from limed plots were lower than those from unlimed plots. These results suggest that liming to pH 6.5, as recommended in a number of sludge application guides, would not reduce Cd uptake by corn in the soils studied.
• Anderson, E. L., Pepper, I. L., & Kneebone, W. R. (1981). Reclamation of wastewater with a soil-turf filter. I. Removal of nitrogen. Journal of the Water Pollution Control Federation, 53(9), 1402-1407.
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  Abstract: This study investigated the maximum rate of wastewater that could be applied to two different soils to achieve suitable water quality in the form of leachate, without harming turfgrass and without excess nitrate concentrations. A sand-turf filter removed less nitrogen than a mix-turf filter, but its lower water-holding capacity resulted in higher recharge percentages. The maximum rate of wastewater application that maintained leachate nitrate concentrations below 10 mg/l was also influenced by season. In the summer, the sand-turf filter could accept up to 12.7 cm/wk yielding 9 cm/wk recharge, compared to 18.7 cm/wk yielding 12.5 cm/wk recharge for the mix-turf filter. Corresponding winter values for the sand-turf filter were 13.6 cm/wk applied with 10.8 cm/wk recharge, and 21.9 cm/wk applied to the mix-turf filter yielding 16 cm/wk recharge. The sand-turf removed 52% of wastewater nitrogen (averaging all application rates over the 42-week study). The sand removed 33% of the nitrogen, with 19% removed by the turf. For the mix-turf filter, 64% of wastewater nitrogen was removed - 37% by soil processes and 27% by turf growth.
• Anderson, E. L., Pepper, I. L., Kneebone, W. R., & Drake, R. J. (1981). Reclamation of wastewater with a soil-turf filter. II. Removal of phosphorus, boron, sodium and chlorine. Journal of the Water Pollution Control Federation, 53(9), 1408-1412.
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  Abstract: Secondarily treated wastewater was applied separately to sandy and siltaceous soil-turf filters to study relative removals of phosphorus, sodium, boron, and chlorine and the associated implications for groundwater recharge. Removal of phosphorus was greater in the siltaceous filter, but in both filters leachate phosphorus concentrations increased with rate of wastewater application and with time; amendment of soils with lime may be necessary to keep leachate phosphorus concentrations low. That neither filter retained boron indicates potential problems may arise if wastewater borate concentrations increase. Sodium adsorption ratios were 3 to 4, regardless of application rate or soil type, and seemed to pose no hazard. Leachate chlorine was undetectable; however, high-pH-induced iron chlorosis could be harmful to turf.
• Borrelli, J., Pochop, L. O., Kneebone, W. R., Pepper, I. L., Danielson, R. E., Hart, W. E., & Youngner, V. B. (1981). BLANEY-CRIDDLE COEFFICIENTS FOR WESTERN TURF GRASSES.. Tetsu-To-Hagane/Journal of the Iron and Steel Institute of Japan, 1, 81-88.
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  Abstract: Evapotranspiration rates were measured for several turf grasses in Arizona, California, Colorado, and Wyoming, and these data were used to calibrate the Blaney-Criddle method for estimating evapotranspiration rates. Warm-season grasses used less water than did cool-season grasses when grown in warm climates. However, Kentucky bluegrass did exhibit heat stress that reduced its evapotranspiration rates below what would be expected when soil temperatures exceed 25 degree C. The data were collected in urban areas and should be representative of city-wide water requirements for turfed areas.
• Vigue, G. T., Pepper, I. L., & Bezdicek, D. F. (1981). The effect of cadmium on nodulation and N₂(C₂H₂)-fixation by dry beans (Phaseolus vulgaris L.). Journal of Environmental Quality, 10(1), 87-90.
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  Abstract: Experiments were conducted to determine the toxicity of Cd on the nodulation, N2(C2H2)-fixation, and growth of dry beans. Plants were grown in the greenhouse with hydroponic solutions to which 0-500 μm Cd/liter had been added. The total N content of shoots, nodule weight, nodule number, and N2(C2H2)-fixation were significantly decreased at 10 μm Cd/liter. Nodulation was completely inhibited at 500 μm Cd/liter. Nodulation and the N2(C2H2)-fixation rate were negatively correlated with solution Cd concentration (r = -0.97). Cadmium uptake and translocation were slow; therefore, Cd levels were highest in the roots, much lower in the nodules, and lowest in the shoot tissue. For example, at 20 μm Cd/liter, respective tissue Cd levels averaged 431, 80, and 35 μg Cd/g, on a dry weight basis. Cadmium levels in roots or shoots from comparable Cd treatments were essentially the same whether the plants obtained their N by fixation or from nutrient solution NO3. Root Cd concentrations were exponentially correlated with nodule number, nodule weight, and acetylene reduction, with correlation coefficients of -0.99, -0.99, and -0.97, respectively. Shoot Cd concentrations were logarithmically correlated with nodule number, nodule weight, and acetylene reduction, with correlation coefficients of -0.89, -0.91, and -0.84, respectively.

Proceedings Publications

• Pepper, I. L. (2018, November). Anammox for sidestream treatmento of wastewater effluent. In 23rd Euaropean Biosolids and Organic Resources Conference.

Presentations

• Pepper, I. L. (2018, April). The Soil Health: Human Health Nexus. 2018 William A. Albrecht Earth Day Lecture. Columbia Missouri, USA.
• Pepper, I. L. (2018, August). In fluence of soil on human health. 21st World Congress of Soil Science. Rio de Janeiro, brazil: World congress of Soil Science.
• Pepper, I. L. (2018, September). Biosolids update : new concepts and technologies. Northwest Biosolids Biofest. Lake chelan Washington, USA: North West Biosolids.
• Pepper, I. L., & IKner, L. (2018, August). A new index for the determination of soil health. 21st World Congresss of Soil Science. Rio de Janeiro, Brazil: World Congress of Soil Science.
• Pepper, I. L. (2012, May). Fate of Chemical and Biological Emerging Contaminants in Biosolids and After Land Application. Residuals and Biosolids 2011. Sacreamento, CA.
• Pepper, I. L. (2012, November). Real-time Security for Potable Water. University of Adelaide. Adelaide, Australia: University of Adelaide.
• Pepper, I. L. (2011, May). Fate of Chemical and Biological Emerging Contaminants in Biosolids and After Land Application. Residuals and Biosolids 2011. Sacreamento, CA.
• Pepper, I. L. (2011, November). Real-time Security for Potable Water. Adelaide, Australia: University of Adelaide.
• Pepper, I. L. (2011, November). Real-time Security for Potable Water. University of Adelaide. Adelaide, Australia: University of Adelaide.

Poster Presentations

• Pepper, I. L. (2012, October). Effects of Residence Time on Reclaimed Water Quality in Storage Tanks. ASA-CSSA-SSSA Annual Meetings. San Antonio, TX.
• Pepper, I. L. (2011, October). Effects of Residence Time on Reclaimed Water Quality in Storage Tanks. ASA-CSSA-SSSA Annual Meetings. San Antonio, TX.
• Pepper, I. L. (2010, Fall). Ensuring Safe Water Through Advanced Oxidation and Real-time Sensors. IWA-WCE 2012. World Congress on Water, Climate, and Energy.