Prevalence and abundance of antibiotic-resistant genes in culturable bacteria inhabiting a non-polar passu glacier, karakorum mountains range, Pakistan.

Natural pristine environments including cold habitats are thought to be the potent reservoirs of antibiotic-resistant genes and have been recurrently reported in polar glaciers' native bacteria, nevertheless, their abundance among the non-polar glaciers' inhabitant bacteria is mostly uncharted. Herein we evaluated antibiotic resistance profile, abundance of antibiotic-resistant genes plus class 1, 2, and 3 integron integrases in 65 culturable bacterial isolates retrieved from a non-polar glacier. The 16S rRNA gene sequencing analysis identified predominantly Gram-negative 43 (66.15%) and Gram-positive 22 (33.84%) isolates. Among the Gram-negative bacteria, Gammaproteobacteria were dominant (62.79%), followed by Betaproteobacteria (18.60%) and Alphaproteobacteria (9.30%), whereas Phyla Actinobacteria (50%) and Firmicutes (40.90%) were predominant among Gram-positive. The Kirby Bauer disc diffusion method evaluated significant antibiotic resistance among the isolates. PCR amplification revealed phylum Proteobacteria predominantly carrying 21 disparate antibiotic-resistant genes like; blaAmpC 6 (100%), blaVIM-1, blaSHV and blaDHA 5 (100%) each, blaOXA-1 1 (100%), blaCMY-4 4 (100%), followed by Actinobacteria 14, Firmicutes 13 and Bacteroidetes 11. Tested isolates were negative for blaKPC, qnrA, vanA, ermA, ermB, intl2, and intl3. Predominant Gram-negative isolates had higher MAR index values, compared to Gram-positive. Alignment of protein homology sequences of antibiotic-resistant genes with references revealed amino acid variations in blaNDM-1, blaOXA-1, blaSHV, mecA, aac(6)-Ib3, tetA, tetB, sul2, qnrB, gyrA, and intI1. Promising antibiotic-resistant bacteria, harbored with numerous antibiotic-resistant genes and class 1 integron integrase with some amino acid variations detected, accentuating the mandatory focus to evaluate the intricate transcriptome analysis of glaciated bacteria conferring antibiotic resistance.

Antibiotic Resistant Bacteria in Municipal Wastes: Is There Reason for Concern?

Recently, there has been increased concern about the presence of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARG), in treated domestic wastewaters, animal manures and municipal biosolids. The concern is whether these additional sources of ARB contribute to antibiotic resistance levels in the environment, that is, "environmental antibiotic resistance." ARB and ARG occur naturally in soil and water, and it remains unclear whether the introduction of ARB in liquid and solid municipal and animal wastes via land application have any significant impact on the background levels of antibiotic resistance in the environment, and whether they affect human exposure to ARB. In this current review, we examine and re-evaluate the incidence of ARB and ARG resulting from land application activities, and offer a new perspective on the threat of antibiotic resistance to public health via exposure from nonclinical environmental sources. Based on inputs of ARBs and ARGs from land application, their fate in soil due to soil microbial ecology principles, and background indigenous levels of ARBs and ARGs already present in soil, we conclude that while antibiotic resistance levels in soil are increased temporally by land application of wastes, their persistence is not guaranteed and is in fact variable, and often contradictory based on application site. Furthermore, the application of wastes may not produce the most direct impact of ARGs and ARB on public health. Further investigation is still warranted in agriculture and public health, including continued scrutiny of antibiotic use in both sectors.

Reduction of Cryptosporidium, Giardia, and Fecal Indicators by Bardenpho Wastewater Treatment.

Increased demand for water reuse and reclamation accentuates the importance for optimal wastewater treatment to limit protozoa in effluents. Two wastewater treatment plants utilizing advanced Bardenpho were investigated over a 12-month period to determine the incidence and reduction of Cryptosporidium, Giardia, Cyclospora, and fecal indicators. Results were compared to facilities that previously operated in the same geographical area. Protozoa (oo)cysts were concentrated using an electronegative filter and subsequently detected by fluorescent microscopy and/or PCR methods. Cryptosporidium and Giardia were frequently detected in raw sewage, but Cyclospora was not detected in any wastewater samples. Facilities with Bardenpho treatment exhibited higher removals of (oo)cysts than facilities utilizing activated sludge or trickling filters. This was likely due to Bardenpho systems having increased solid wasting rates; however, this mechanism cannot be confirmed as sludge samples were not analyzed. Use of dissolved-air-flotation instead of sedimentation tanks did not result in more efficient removal of (oo)cysts. Concentrations of protozoa were compared with each other, Escherichia coli, somatic coliphage, and viruses (pepper mild mottle virus, Aichi virus 1, adenovirus, and polyomaviruses JC and BK). Although significant correlations were rare, somatic coliphage showed the highest potential as an indicator for the abundance of protozoa in wastewaters.

Effects of Subsurface Banding and Broadcast of Poultry Litter and Cover Crop on Soil Microbial Populations.

Agronomic management is aimed at managing the crop environment to maximize crop yield, but soil biology is often ignored. This study aimed to compare the application of poultry litter via broadcast and subsurface banding versus standard inorganic fertilizer to cotton ( L.) and their effects on soil bacterial populations and fecal indicator bacteria. The study comprised a randomized complete block design, with fertilizer and time of application as treatment effects and cover crop as a main effect. Soil cores were collected and analyzed from 2008 to 2014. Fecal indicator bacteria were at detection limits for all treatments, where the integron 1 gene was significantly elevated in litter plots. There were few differences between litter application approaches, but both significantly increased key biogeochemical genes over control plots, whereas a cover crop only increased soil moisture and urease C. Data suggested a positive residual effect of litter application with 16S, phosphatase A, and urease C genes elevated over controls, but similar to standard fertilizer plots. High-throughput 16S ribosomal RNA analysis suggested increased diversity and enrichment indices in litter and standard fertilizer over untreated control plots. Litter and standard fertilizer effects persisted 4 and 2 yr after application, respectively, as evidenced by residual library community structures. This study demonstrated the positive effects of litter application on the soil bacterial community when compared with untreated control plots. Some differences between standard fertilization and litter practices were noted and suggest that there is a positive residual effect on soil microbial populations associated with both practices.

Near Real-Time Detection of E. coli in Reclaimed Water.

Advanced treatment of reclaimed water prior to potable reuse normally results in the inactivation of bacterial populations, however, incremental treatment failure can result in bacteria, including pathogens, remaining viable. Therefore, potential microorganisms need to be detected in real-time to preclude potential adverse human health effects. Real-time detection of microbes presents unique problems which are dependent on the water quality of the test water, including parameters such as particulate content and turbidity, and natural organic matter content. In addition, microbes are unusual in that: (i) viability and culturability are not always synonymous; (ii) viability in water can be reduced by osmotic stress; and (iii) bacteria can invoke repair mechanisms in response to UV disinfection resulting in regrowth of bacterial populations. All these issues related to bacteria affect the efficacy of real-time detection for bacteria. Here we evaluate three different sensors suitable for specific water qualities. The sensor A is an on-line, real-time sensor that allows for the continuous monitoring of particulates (including microbial contaminants) using multi-angle-light scattering (MALS) technology. The sensor B is a microbial detection system that uses optical technique, Mie light scattering, for particle sizing and fluorescence emission for viable bacteria detection. The last sensor C was based on adenosine triphosphate (ATP) production. E. coli was used a model organism and out of all tested sensors, we found the sensor C to be the most accurate. It has a great potential as a surrogate parameter for microbial loads in test waters and be useful for process control in treatment trains.

Virus Reduction during Advanced Bardenpho and Conventional Wastewater Treatment Processes.

The present study investigated wastewater treatment for the removal of 11 different virus types (pepper mild mottle virus; Aichi virus; genogroup I, II, and IV noroviruses; enterovirus; sapovirus; group-A rotavirus; adenovirus; and JC and BK polyomaviruses) by two wastewater treatment facilities utilizing advanced Bardenpho technology and compared the results with conventional treatment processes. To our knowledge, this is the first study comparing full-scale treatment processes that all received sewage influent from the same region. The incidence of viruses in wastewater was assessed with respect to absolute abundance, occurrence, and reduction in monthly samples collected throughout a 12 month period in southern Arizona. Samples were concentrated via an electronegative filter method and quantified using TaqMan-based quantitative polymerase chain reaction (qPCR). Results suggest that Plant D, utilizing an advanced Bardenpho process as secondary treatment, effectively reduced pathogenic viruses better than facilities using conventional processes. However, the absence of cell-culture assays did not allow an accurate assessment of infective viruses. On the basis of these data, the Aichi virus is suggested as a conservative viral marker for adequate wastewater treatment, as it most often showed the best correlation coefficients to viral pathogens, was always detected at higher concentrations, and may overestimate the potential virus risk.

Occurrence and genetic diversity of human cosavirus in influent and effluent of wastewater treatment plants in Arizona, United States.

Human cosavirus (HCoSV) is a novel member of the family Picornaviridae. We investigated the prevalence and genetic diversity of HCoSV in influent and effluent wastewater in Arizona over a 12-month period, from August 2011 to July 2012. HCoSV sequences were identified in six (25%) influent samples and one (4%) effluent sample, with the highest concentration of 3.24 × 10(5) and 1.54 × 10(3) copies/liter in influent and effluent, respectively. The strains were characterized based on their 5' untranslated region and classified into species A and D, demonstrating that genetically heterogeneous HCoSV were circulating with a clear temporal shift of predominant strains in the study area.

Online monitoring of Escherichia coli and Bacillus thuringiensis spore inactivation after advanced oxidation treatment.

Various studies have shown that advanced oxidation processes (AOPs) such as UV light in combination with hydrogen peroxide is an efficient process for the removal of a large variety of emerging contaminants including microorganisms. The mechanism of destruction in the presence of hydrogen peroxide (H2O2) is the enhanced formation of hydroxyl (·OH) radicals, which have a high oxidation potential. The goal of this study was to utilize in-line advanced oxidation to inactivate microbes, and document the inactivation via an in-line, real-time sensor. Escherichia coli cells and Bacillus thuringiensis spores were exposed to UV/H2O2 treatment in DI water, and the online sensor BioSentry(®) was evaluated for its potential to monitor inactivation in real-time. B. thuringiensis was selected as a non-pathogenic surrogate for B. anthracis, the causative agent of anthrax and a proven biological weapon. UV radiation and UV/H2O2 exposure resulted in a >6 log10 reduction of the viable culturable counts of E. coli vegetative cells, and a 3 log10 reduction of B. thuringiensis spores. Scanning electron microscopy of the treated samples revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the morphology of the B. thuringiensis spores. Following AOP exposure, the BioSentry sensor showed an increase in the categories of unknown, rod and spores counts, but overall, did not correspond well with viable count assays. Data from this study show that advanced oxidation processes effectively inactivate E. coli vegetative cells, but not B. thuringiensis spores, which were more resistant to AOP. Further, the BioSentry in-line sensor was not successful in documenting destruction of the microbial cells in real-time.

Inactivation of MS2 coliphage by UV and hydrogen peroxide: comparison by cultural and molecular methodologies.

The use of advanced oxidation processes (AOP) are expected to increase for removal of emerging contaminants and pathogens from drinking water. In this study, the performance of a small community ultraviolet light reactor in combination with hydrogen peroxide (H2O2) for MS2 coliphage inactivation with two different flow rate conditions of 1 gal/min (gpm) and 2 gpm was evaluated. Following UV radiation, MS2 showed a reduction of 5.3-5.8 log10 when quantified with cultural plaque counts, whereas corresponding quantitative polymerase chain reaction (qPCR) data showed only a 1.7-2.8 log10 reduction in viral RNA copy number. When H2O2 was added at either 2.5 or 5 ppm with UV at both flow rate conditions, enhanced MS2 inactivation occurred with a more than 7 log10 reduction observed via plaque counts, indicating that all added MS2 had been inactivated, since no plaques were formed after incubation at 37 °C for 24 h. In contrast, qPCR only showed a corresponding 3-4 log10 reduction in viral RNA copy number. This research also sheds light on the inactivation of MS2 with ultraviolet light and in the presence of hydroxyl radicals and provides a practical use of qPCR to detect MS2 concentration following advanced oxidation relative to traditional plaque methodology; however qPCR detection overestimates the true number of infective virus.

Assessment of virus removal by managed aquifer recharge at three full-scale operations.

Managed aquifer recharge (MAR) systems such as riverbank filtration and soil-aquifer treatment all involve the use of natural subsurface systems to improve the quality of recharged water (i.e. surface water, stormwater, reclaimed water) before reuse. During MAR, water is either infiltrated via basins, subsurface injected or abstracted from wells adjacent to rivers. The goal of this study was to assess the removal of selected enteric viruses and a potential surrogate for virus removal at three full-scale MAR systems located in different regions of the United States (Arizona, Colorado, and California). Samples of source water (i.e., river water receiving treated wastewater and reclaimed water) before recharge and recovered groundwater at all three sites were tested for adenoviruses, enteroviruses, Aichi viruses and pepper mild mottle virus (PMMoV) by quantitative polymerase chain reaction (qPCR). Samples of groundwater positive for any virus were also tested for the presence of infectious virus by cell culture. PMMoV was the most commonly detected virus in the groundwater samples. Infectious enteric viruses (reovirus) were only detected in one groundwater sample with a subsurface residence time of 5 days. The results suggested that in groundwater with a residence time of greater than 14 days all of the viruses are removed below detection indicating a 1 to greater than 5 log removal depending upon the type of virus. Given its behavior, PMMoV may be suitable to serve as a conservative tracer of enteric virus removal in managed aquifer treatment systems.

Variant-specific SARS-CoV-2 shedding rates in wastewater.

Previous studies show that SARS-CoV-2 waste shedding rates vary by community and are influenced by multiple factors; however, differences in shedding rates across multiple variants have yet to be evaluated. The purpose of this work is to build on previous research that evaluated waste shedding rates for early SARS-CoV-2 and the Delta variant, and update population level waste shedding rates for the more-recent Omicron variant in six communities. Mean SARS-CoV-2 waste shedding rates were found to increase with the predominance of the Delta variant and subsequently decrease with Omicron infections. Interestingly, the Delta stage had the highest mean shedding rates and was associated with the most severe disease symptoms reported in other clinical studies, while Omicron, exhibiting reduced symptoms, had the lowest mean shedding rates. Additionally, shedding rates were most consistent across communities during the Omicron stage. This is the first paper to identify waste shedding rates specific to the Omicron variant and fills a knowledge gap critical to disease prevalence modeling.

Land application of manure and Class B biosolids: an occupational and public quantitative microbial risk assessment.

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. This 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. Recent contamination and high exposures doses increased most risks. 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 when using a 4-mo harvest delay for crop consumption. Occupational, more direct risks were greater than indirect public risks, which often occur after time and dilution have reduced pathogen loads to tolerable levels. 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 and maintained manure risk. Direct comparisons of shared pathogens resulted in greater manure risks. Overall, it appears that in the short term, risks were high for both types of residuals, but given treatment, attenuation, and dilution, risks can be reduced to near-insignificant levels. That being said, limited data sets, dose exposures, site-specific inactivation rates, pathogen spikes, environmental change, regrowth, and wildlife will increase risk and uncertainty and remain areas poorly understood.

Population level SARS-CoV-2 fecal shedding rates determined via wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) has been utilized as an early warning tool to anticipate disease outbreaks, especially during the COVID-19 pandemic. However, COVID-19 disease models built from wastewater-collected data have been limited by the complexities involved in estimating SARS-CoV-2 fecal shedding rates. In this study, wastewater from six municipalities in Arizona and Florida with distinct demographics were monitored for SARS-CoV-2 RNA between September 2020 and December 2021. Virus concentrations with corresponding clinical case counts were utilized to estimate community-wide fecal shedding rates that encompassed all infected individuals. Analyses suggest that average SARS-CoV-2 RNA fecal shedding rates typically occurred within a consistent range (7.53-9.29 log10 gc/g-feces); and yet, were unique to each community and influenced by population demographics. Age, ethnicity, and socio-economic factors may have influenced shedding rates. Interestingly, populations with median age between 30 and 39 had the greatest fecal shedding rates. Additionally, rates remained relatively constant throughout the pandemic provided conditions related to vaccination and variants were unchanged. Rates significantly increased in some communities when the Delta variant became predominant. Findings in this study suggest that community-specific shedding rates may be appropriate in model development relating wastewater virus concentrations to clinical case counts.

Evaluation of select sensors for real-time monitoring of Escherichia coli in water distribution systems.

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.

Survival of infectious prions in water.

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.

Survival of infectious prions in Class B biosolids.

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.

Incidence of Pfas in soil following long-term application of class B biosolids.

This field study investigated the impact of long-term land application of biosolids on PFAS presence in soils that received annual repetitive land application of Class B biosolids from 1984 to 2019. Soil samples were collected from three depths of 30.5, 91 and 183 cm below land surface. Biosolid and groundwater samples used for irrigation were also collected. Concentrations measured for 18 PFAS compounds were evaluated to assess incidence rates and potential impact on groundwater. No PFAS analytes were detected at the three sampling depths for soil samples collected from undisturbed sites with no history of agriculture, irrigation, or biosolids application (background control sites). Relatively low mean concentrations of PFAS ranging from non-detect to 1.9 µg/kg were measured in soil samples collected from sites that were used for agriculture and that received irrigation with groundwater, but never received biosolids. PFAS concentrations in soils amended with biosolids were similarly low, ranging from non-detect to a mean concentration of 4.1 µg/kg. PFOS was observed at the highest concentrations, followed by PFOA for all locations. PFAS detected in the irrigation water were also present in the soil. These results indicate that biosolids and irrigation water are both important sources of PFAS present in the soils for all of the study sites. Not all PFAS detected in the biosolids were detected in the soil. Very long chain PFAS present in the biosolids were not detected or were detected at very low levels for soil, suggesting potential preferential retention within the biosolids. The precursor NMeFOSAA was present at the second highest concentrations in the biosolids but not detected in soil, indicating possible occurrence of transformation reactions. The total PFAS soil concentrations exhibited significant attenuation with depth, with a mean attenuation of 73% at the 183 cm depth. Monotonically decreasing concentrations with depth were observed for the longer-chain PFAS.

Enumerating asymptomatic COVID-19 cases and estimating SARS-CoV-2 fecal shedding rates via wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) was utilized to monitor SARS-CoV-2 RNA in sewage collected from manholes specific to individual student dormitories (dorms) at the University of Arizona in the fall semester of 2020, which led to successful identification and reduction of SARS-CoV-2 transmission events. Positive wastewater samples triggered clinical testing of residents within that dorm; thus, SARS-CoV-2 infected individuals were identified regardless of symptom expression. This current study examined clinical testing data to determine the abundance of asymptomatic versus symptomatic cases in these defined communities. Nasal and nasopharyngeal swab samples processed via antigen and PCR tests indicated that 79.2% of SARS-CoV-2 infections were asymptomatic, and only 20.8% of positive cases reported COVID-19 symptoms at the time of testing. Clinical data was paired with corresponding wastewater virus concentrations, which enabled calculation of viral shedding rates in feces per infected person. Mean shedding rates averaged from positive wastewater samples across all dorms were 7.30 ± 0.67 log10 genome copies per gram of feces (gc/g-feces) based on the N1 gene. Quantification of SARS-CoV-2 fecal shedding rates from infected individuals has been the critical missing component necessary for WBE models to measure and predict SARS-CoV-2 infection prevalence in communities. The findings from this study can be utilized to create models that can be used to inform public health prevention and response actions.

COVID-19 containment on a college campus via wastewater-based epidemiology, targeted clinical testing and an intervention.

Wastewater-based epidemiology has potential as an early-warning tool for determining the presence of COVID-19 in a community. The University of Arizona (UArizona) utilized WBE paired with clinical testing as a surveillance tool to monitor the UArizona community for SARS-CoV-2 in near real-time, as students re-entered campus in the fall. Positive detection of virus RNA in wastewater lead to selected clinical testing, identification, and isolation of three infected individuals (one symptomatic and two asymptomatic) that averted potential disease transmission. This case study demonstrated the value of WBE as a tool to efficiently utilize resources for COVID-19 prevention and response. Thus, WBE coupled with targeted clinical testing was further conducted on 13 dorms during the course of the Fall semester (Table 3). In total, 91 wastewater samples resulted in positive detection of SARS-CoV-2 RNA that successfully provided an early-warning for at least a single new reported case of infection (positive clinical test) among the residents living in the dorm. Overall, WBE proved to be an accurate diagnostic for new cases of COVID-19 with an 82.0% positive predictive value and an 88.9% negative predictive value. Increases in positive wastewater samples and clinical tests were noted following holiday-related activities. However, shelter-in-place policies proved to be effective in reducing the number of daily reported positive wastewater and clinical tests. This case study provides evidence for WBE paired with clinical testing and public health interventions to effectively contain potential outbreaks of COVID-19 in defined communities.

Long-term effects of land application of class B biosolids on the soil microbial populations, pathogens, and activity.

This study evaluated the influence of 20 annual land applications of Class B biosolids on the soil microbial community. The potential benefits and hazards of land application were evaluated by analysis of surface soil samples collected following the 20th land application of biosolids. The study was initiated in 1986 at the University of Arizona Marana Agricultural Center, 21 miles north of Tucson, AZ. The final 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 final 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 significant 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 significantly higher microbial activity or potential for microbial transformations, including nitrification, sulfur oxidation, and dehydrogenase activity, than control plots and plots receiving inorganic fertilizers. Overall, the 20 annual land applications showed no long-term adverse effects, 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.