Shane Rogers

Decay of bacterial pathogens, fecal indicators, and real-time quantitative PCR genetic markers in manure amended soils

ABSTRACT This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria (FIB), and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manure-amended agricultural soils. Known concentrations of transformed green fluorescent protein-expressing Escherichia coli O157:H7/pZs and red fluorescent protein-expressing Salmonella enterica serovar Typhimurium/pDs were added to laboratory-scale manure-amended soil microcosms with moisture contents of 60% or 80% field capacity and incubated at temperatures of −20°C, 10°C, or 25°C for 120 days. A two-stage first-order decay model was used to determine stage 1 and stage 2 first-order decay rate coefficients and transition times for each organism and qPCR genetic marker in each treatment. Genetic markers for FIB (Enterococcus spp., E. coli, and Bacteroidales) exhibited decay rate coefficients similar to that of E. coli O157:H7/pZs but not of S. enterica serovar Typhimurium/pDs and persisted at detectable levels longer than both pathogens. Concentrations of these two bacterial pathogens, their counterpart qPCR genetic markers (stx1 and ttrRSBCA, respectively), and FIB genetic markers were also correlated (r = 0.528 to 0.745). This suggests that these qPCR genetic markers may be reliable conservative surrogates for monitoring fecal pollution from manure-amended land. Host-associated qPCR genetic markers for microbial source tracking decayed rapidly to nondetectable concentrations, long before FIB, Salmonella enterica serovar Typhimurium/pDs, and E. coli O157:H7/pZs. Although good indicators of point source or recent nonpoint source fecal contamination events, these host-associated qPCR genetic markers may not be reliable indicators of nonpoint source fecal contamination events that occur weeks following manure application on land.

Anaerobic digestion of food waste through the operation of a mesophilic two-phase pilot scale digester – Assessment of variable loadings on system performance

Single and two-phase operations were compared at mesophilic operating conditions using a digester system consisting of three 5-m(3) reactors treating food waste generated daily within the university campus kitchens. When normalizing the methane production to the daily feedstock characteristics, significantly greater methane was produced during two-phase mesophilic digestion compared to the single-stage operation (methane yield of 380 vs 446-L CH4 kg VS(-1); 359 vs 481-L CH4 kg COD(-1) removed for single vs two stage operation). The fermentation reactor could be maintained reliably even under very low loading rates (0.79±0.16 kg COD m(-3) d(-1)) maintaining a steady state pH of 5.2.

Emission and Dispersion of Bioaerosols from Dairy Manure Application Sites: Human Health Risk Assessment

In this study, we report the human health risk of gastrointestinal infection associated with inhalation exposure to airborne zoonotic pathogens emitted following application of dairy cattle manure to land. Inverse dispersion modeling with the USEPAs AERMOD dispersion model was used to determine bioaerosol emission rates based on edge-of-field bioaerosol and source material samples analyzed by real-time quantitative polymerase chain reaction (qPCR). Bioaerosol emissions and transport simulated with AERMOD, previously reported viable manure pathogen contents, relevant exposure pathways, and pathogen-specific dose-response relationships were then used to estimate potential downwind risks with a quantitative microbial risk assessment (QMRA) approach. Median 8-h infection risks decreased exponentially with distance from a median of 1:2700 at edge-of-field to 1:13 000 at 100 m and 1:200 000 at 1000 m; peak risks were considerably greater (1:33, 1:170, and 1:2500, respectively). These results indicate that bioaerosols emitted from manure application sites following manure application may present significant public health risks to downwind receptors. Manure management practices should consider improved controls for bioaerosols in order to reduce the risk of disease transmission.

Real-time investigation of antibiotics-induced oxidative stress and superoxide release in bacteria using an electrochemical biosensor

The involvement of oxidative stress in the mechanism of antibiotics-meditated cell death is unclear and subject to debate. The kinetic profile and a quantitative relationship between the release of reactive oxygen species (ROS), bacteria and antibiotic type remain elusive. Here we report direct measurements and analytical quantification of the release of superoxide radicals (O2(·-)), a major contributor to ROS, in antibiotics-treated bacterial cultures using a cytochrome c electrochemical biosensor. The specificity of electrochemical measurements was established by the addition of superoxide dismutase (SOD) which decreased the O2(·-) signal. Measurements using a general ROS-specific fluorescence dye and colony forming units (CFU) assays were performed side-by-side to determine the total ROS and establish the relationship between ROS and the degree of lethality. Exposure of Escherichia coli and Listeria monocytogenes cultures to antibiotics increased the release of O2(·-) radicals in a dose-dependent manner, suggesting that the transmembrane generation of ROS may occur as part of the antibiotic action. The study provides a quantitative methodology and fundamental knowledge to further explore the role of oxidative stress in antibiotics-meditated bacterial death and to assess physiological changes associated with the complex metabolic events related to oxidative stress and bacterial resistance.

Comparative study of methanogens in one- and two-stage anaerobic digester treating food waste

Changes in methanogenic archaea were investigated in pilot-scale experiments during one- and two-stage mesophilic anaerobic digestion (AD) of food waste. Methane yields were 379.7±75.3 liters of methane per kg of volatile solids [L-CH 4 (kg-VS) −1 ] added to the system, during one-stage operation, and 446±922 L-CH 4 (kg-VS) −1 added during two-stage operation. Populations of methanogenic archaea were monitored quantitatively by targeting the functional gene for methyl-coenzyme-M reductase ( mcrA ) using real-time quantitative polymerase chain reaction techniques. During one-stage operation, mean mcrA gene concentrations were 2.48×10 9 ±2.7×10 9 copies ml −1 . Two-stage operation yielded mean mcrA gene concentrations of 9.85×10 8 ±8.2×10 8 copies ml −1 in the fermentation and 1.76×10 10 ±8.5×10 9 copies ml −1 in the methanogenesis reactors, respectively. Diversity of archaea in the methanogenic reactors was investigated by denaturing gradient gel electrophoresis targeting the V3 region of 16S rRNA of archaea. The Shannon index ( H ) was 2.98 for one-stage operation and 7.29 for two-stage operation, suggesting greater archaeal diversity in the two-stage AD. The fivefold increase in methanogenic archaea populations during the two-stage operation, as indicated by mcrA gene concentration, corresponded to an increase in methane production rates. While the diversity may also be related to the stability of the microbial bioprocesses and improved methane production rates, the correlation between diversity and production rates should be studied further.

Bioaerosol Deposition to Food Crops near Manure Application: Quantitative Microbial Risk Assessment

Production of both livestock and food crops are central priorities of agriculture; however, food safety concerns arise where these practices intersect. In this study, we investigated the public health risks associated with potential bioaerosol deposition to crops grown in the vicinity of manure application sites. A field sampling campaign at dairy manure application sites supported the emission, transport, and deposition modeling of bioaerosols emitted from these lands following application activities. Results were coupled with a quantitative microbial risk assessment model to estimate the infection risk due to consumption of leafy green vegetable crops grown at various distances downwind from the application area. Inactivation of pathogens ( spp., spp., and O157:H7) on both the manure-amended field and on crops was considered to determine the maximum loading of pathogens to plants with time following application. Overall median one-time infection risks at the time of maximum loading decreased from 1:1300 at 0 m directly downwind from the field to 1:6700 at 100 m and 1:92,000 at 1000 m; peak risks (95th percentiles) were considerably greater (1:18, 1:89, and 1:1200, respectively). Median risk was below 1:10,000 at >160 m downwind. As such, it is recommended that a 160-m setback distance is provided between manure application and nearby leafy green crop production. Additional distance or delay before harvest will provide further protection of public health.

Pathogen Reduction and Factors Responsible for Pathogen Reduction in Dairy Farm Operations Treating Dairy Manure

Manure management at large dairy operations presents a challenge; there is an increasing interest among farm managers for reuse of their manure and other farm residuals as low-cost bedding materials. A significant barrier preventing more widespread adoption of this technology is ambiguity in regards to the presence of disease-causing agents that may sicken animals and reduce farm milk production. In this study, pathogen reduction was correlated to the operational parameters of different manure management techniques at three different dairy farms in northern New York State. Two log order reductions of Campylobacter, Enterococcus, Klebsiella, and fecal Bacteroidetes concentrations were achieved during composting or mesophilic anaerobic digestion of organic material. Effluent concentrations for the specific pathogens ranged between 1E3 to 1E2 g-1 dry solids and 1E6 g-1 dry solids for fecal Bacteroidetes for both treatment systems. Bacterial concentrations in freestall barn bedding was lowest for the farm using clean sand only, followed by the farm that recycles bedding sand after sand separation, and the farm that uses bedding with composted manure solids. Pathogen concentrations were strongly correlated to moisture content, volatile solids content, and sample type (organic versus inorganic samples).

Antibiotic-Resistant Genes and Pathogens Shed by Wild Deer Correlate with Land Application of Residuals

The purpose of this study was to investigate genetic biomarkers of zoonotic enteric pathogens and antibiotic-resistant genes (ARGs) in the feces of white-tailed deer (Odocoileus virginianus) as related to proximity of deer to land that receives livestock manure or human waste biosolid fertilizers. Deer feces were collected in the St. Lawrence River Valley and Adirondack State Park of New York. Campylobacter spp. 16S rDNA was detected in 12 of 232 fecal samples (8 of 33 sites). Salmonellae were cultivated from 2 of 182 fecal samples (2 of 29 sites). Genetic virulence markers for Shiga-like toxin I (stx1) and enterohemolysin (hylA) were each detected in one isolate of Escherichia coli; E. coli O157 was not detected in any of 295 fecal samples. ARGs detected in deer feces included ermB (erythromycin-resistant gene; 9 of 295 fecal samples, 5 of 38 sites), vanA (vancomycin-resistant gene; 93 of 284 samples, 33 of 38 sites), tetQ (tetracycline-resistant gene; 93 of 295 samples, 25 of 38 sites), and sul(I) (sulfonamide-resistant gene; 113 of 292 samples, 28 of 38 sites). Genetic markers of pathogens and ARGs in deer feces were spatially associated with collection near concentrated animal feeding operations (CAFOs; Campylobacter spp., tetQ, and ermB) and land-applied biosolids (tetQ). These results indicate that contact with human waste biosolids or animal manure may be an important method of pathogen and ARG transmission and that deer in proximity to land-applied manure and human waste biosolids pose increased risk to nearby produce and water quality.

Effect of land-applied biosolids on surface-water nutrient yields and groundwater quality in Orange County, North Carolina

Land application of municipal wastewater biosolids is the most common method of biosolids management used in North Carolina and the United States. Biosolids have characteristics that may be beneficial to soil and plants. Land application can take advantage of these beneficial qualities, whereas disposal in landfills or incineration poses no beneficial use of the waste. Some independent studies and laboratory analysis, however, have shown that land-applied biosolids can pose a threat to human health and surface-water and groundwater quality. The effect of municipal biosolids applied to agriculture fields is largely unknown in relation to the delivery of nutrients, bacteria, metals, and contaminants of emerging concern to surface-water and groundwater resources. Therefore, the North Carolina Department of Environment and Natural Resources (NCDENR) collaborated with the U.S. Geological Survey (USGS) through the 319 Nonpoint Source Program to better understand the transport of nutrients and bacteria from biosolids application fields to groundwater and surface water and to provide a scientific basis for evaluating the effectiveness of the current regulations. The USGS conducted a paired agricultural watershed study in the Collins Creek and Cane Creek Reservoir watersheds in Orange County, North Carolina. Field activities were conducted from March 2011 through May 2013 at two field study sites, including biosolids field application sites owned by Orange County Water and Sewer Authority (OWASA) in the Collins Creek watershed and a background study site in the Cane Creek watershed that has no fields receiving biosolids applications. Samples of biosolids source material and soil were collected from the land-application fields for laboratory analyses. Soil samples were also collected from a background agricultural field in the Cane Creek watershed that has never received land-applied municipal biosolids. Shallow groundwater samples were collected quarterly from new monitoring wells installed by NCDENR along the edge of the biosolids land-application fields and a background agricultural field for laboratory analyses. Two surface-water monitoring sites were established on Collins Creek to compute continuous streamflow and collect discrete baseflow and stormwater runoff water-quality data upstream and downstream from the biosolids land-application fields. Surface waterquality samples were also collected for baseflow and stormwater runoff conditions at an existing USGS streamgage on Cane Creek to monitor water-quality conditions in the background study watershed. The study primarily focused on nutrients and bacteria; however, data for field properties and water-quality constituents, including metals, major ions, and contaminants of emerging concern (household-, industrial-, and agricultural-use compounds, pharmaceutical compounds, hormones, and antibiotics) also were collected and used in the analyses. There were no exceedances of the 10 elements with designated U.S. Environmental Protection Agency (EPA) ceiling concentrations for land-applied biosolids in any of the biosolids samples. Treatment processes and storage techniques used by OWASA are effective in eliminating Escherichia coli and fecal coliform bacteria from biosolids. Copper, molybdenum, total Kjeldahl nitrogen, and total phosphorus were elevated in the soil from biosolids land-application fields relative to the background field. The relative richness of these constituents in the biosolids land-application fields is consistent with biosolids being the source of the elevated concentrations given the relatively high concentrations of these constituents in the biosolids samples that were collected. Shallow groundwater in the transitional zone wells, which were located adjacent to and topographically downgradient from all the biosolids land-application fields, were found to be statistically different and had higher nitrate concentrations (medians greater than 12 milligrams per liter) than all the other wells sampled as part of the study. Surface-water nutrient concentrations and yields, primarily nitrate, were higher at the 1U.S. Geological Survey. 2Clarkson University. 2 Effect of Land-Applied Biosolids on Surface-Water Nutrient Yields and Groundwater Quality in Orange County, North Carolina monitoring site on Collins Creek downstream from the biosolids land-application fields than the other study sites that drained watersheds without biosolids land application. The largest differences in concentrations between sites were measured at baseflow conditions, which indicate that the main cause of these differences, particularly between Cane Creek and the Collins Creek site downstream from the OWASA application fields, is related to nitrate contribution from the shallow groundwater. Contaminants of emerging concern were detected in approximately 40 percent of the laboratory analyses of the biosolids samples and more frequently in soil samples from the biosolids land-application fields (approximately 40 percent of laboratory analyses) relative to the soil samples from the background field (approximately 12 percent of laboratory analyses). However, contaminants of emerging concern detected in the laboratory analysis for this study do not appear to be good indicators of human-waste contaminants derived from landapplied biosolids in groundwater or surface-water because the number of detections and concentrations at the background wells and surface-water monitoring sites are similar to or higher than those at wells and monitoring sites adjacent to or downstream from the biosolids land-application fields. The data, analysis, and conclusions associated with this study can be used by regulatory agencies, resource managers, and wastewater-treatment operators to (1) better understand the quantity and characteristics of nutrients, bacteria, metals, and contaminants of emerging concern that are transported away from biosolids land-application fields to surface water and groundwater under current regulations for the purposes of establishing effective total maximum daily loads (TMDLs) and restoring impaired water resources, (2) assess how well existing regulations protect waters of the State and potentially recommend effective changes to regulations or land-application procedures, and (3) establish a framework for developing guidance on effective techniques for monitoring and regulatory enforcement of permitted biosolids land-application fields. Introduction Biosolids are defined as any solid, semi-solid, or liquid waste, other than raw effluent or residues from agricultural products and processing, generated from a wastewater-treatment facility, water-supply treatment facility, or air pollution control facility permitted under the authority of the North Carolina Environmental Management Commission (EMC). Depending on the level of treatment that the biosolids receive, the biosolids may be applied to land for fertilizer or disposed of in a surface disposal unit (such as a land fill or incineration). As of December 2009, a total of 260 facilities in North Carolina were permitted to apply biosolids to land and only 15 facilities were permitted for surface disposal. Land application of municipal wastewater biosolids is the most common method of biosolids management used in North Carolina and the United States (National Research Council of the National Academy of Sciences, 2002). Biosolids have characteristics that may be beneficial to soil and plants. Land application can take advantage of these beneficial qualities, whereas disposal in landfills or incineration poses no beneficial use of the waste (National Research Council of the National Academy of Sciences, 2002; Lu and others, 2012). For these reasons, the State of North Carolina and the U.S. Environmental Protection Agency (EPA) consider controlled land application a “beneficial use” of biosolids. However, some independent studies and laboratory analyses (National Research Council of the National Academy of Sciences, 2002; Rudo, 2005; U.S. Environmental Protection Agency, 2009) have shown that biosolids can pose a threat to human health as well as surface-water and groundwater quality. Hence, biosolids are defined as waste under North Carolina General Statute (NCGS) 142–213, and any system that collects, treats, or disposes of waste cannot be constructed or operated without a permit (NCGS 143–215.1(a)). The statute (NCGS 143–215.1(a)) authorizes the EMC and the North Carolina Department of Environment and Natural Resources (NCDENR) to develop and implement State regulations and to issue permits for the generation and disposal of residual biosolids; however, only NCDENR’s Division of Water Resources (DWR) carries out these functions. The North Carolina rules (15A NCAC 02T.1109) for biosolids management meet or exceed the Title 40 Code of Federal Regulations part 503 (40 CFR 503) standards for the use or disposal of sewage sludge. The permitting request and approval process implemented by DWR is designed to provide relevant information to concerned parties before, during, and after the application of biosolids in an attempt to assure that land applications of biosolids are managed safely and effectively. The DWR conducts reviews of all permit applications, performs site visits to proposed application fields to verify site conditions, and works with county managers and health departments to address local concerns with proposed biosolids land-application activities. Permit holders are required to provide DWR with annual reports that summarize the past year’s activities and document that biosolids quality and nutrient management requirements were met. Permit holders also are responsible for meeting the requirements of their permit and for reporting any permit violations. Monitoring and enforcement of permit requirements is challenging and relies primarily on self-reporting of permittees (supplier of the biosolids) and citizen complaints

Case Study: Occasional excessive ammonia emissions following dairy manure application to land: causes, impacts, and management recommendations

Abstract. Ammonia is being monitored and modeled following land application of dairy manure to determine the impacts of application method on its emission, transport, and deposition. Ammonia emission flux from manure applied fields and downwind concentrations are measured following conventional splash plate spreading, direct injection, and drag-hose application of liquid dairy manure. Conventional broadcast spreading has produced the greatest nitrogen emissions from both the field and lab soil experiments; exposure concentrations in excess of OSHA’s Permissible Exposure Limit (PEL) of 35 mg/m 3 have been observed. On two instances, ammonia emissions flux exceeded 111 mg/m 2 /hr, the maximum emissions rate that our instrument could monitor. This equated to 3.6 times the average ammonia emissions flux of the remaining sampling events. Atmospheric ammonia concentrations on those dates reached 56 mg/m 3 , far exceeding OSHA’s PEL. Here, we report the causes of these excessive ammonia-N fluxes, potential impacts, and management recommendations to avoid high ammonia emissions events and preserve valuable nitrogen fertilizer.