To determine the evolution of microbial community and microbial shift under anaerobic processes, this study investigates the use of denaturing gradient gel electrophoresis (DGGE). In the DGGE, short- and medium-sized DNA fragments are separated based on their melting characteristics, and this technique is used in this study to understand the dominant bacterial community in mesophilic and thermophilic anaerobic digestion processes. Dairy manure is known for emitting greenhouse gases (GHGs) such as methane, and GHG emissions from manure is a biological process that is largely dependent on the manure conditions, microbial community presence in manure, and their functions. Additional efforts are needed to understand the GHG emissions from manure and develop control strategies to minimize the biological GHG emissions from manure. To study the microbial shift during anaerobic processes responsible for GHG emission, we conducted a series of manure anaerobic digestion experiments, and these experiments were conducted in lab-scale reactors operated under various temperature conditions (28 °C, 36 °C, 44 °C, and 52 °C). We examined the third variable region (V3) of the 16S rRNA gene fingerprints of bacterial presence in anaerobic environment by PCR amplification and DGGE separation. Results showed that bacterial community was affected by the temperature conditions and anaerobic incubation time of manure. The microbial community structure of the original manure changed over time during anaerobic processes, and the community composition changed substantially with the temperature of the anaerobic process. At Day 0, the sequence similarity confirmed that most of the bacteria were similar (>95%) to Acinetobacter sp. (strain: ATCC 31012), a Gram-negative bacteria, regardless of temperature conditions. At day 7, the sequence similarity of DNA fragments of reactors (28 °C) was similar to Acinetobacter sp.; however, the DNA fragments of effluent of reactors at 44 °C and 52 °C were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 97%) and Tepidimicrobium ferriphilum (strain: DSM 16624) (similarity: 100%), respectively. At day 60, the analysis showed that DNA fragments of effluent of 28 °C reactor were similar to Galbibacter mesophilus (strain: NBRC 10162) (similarity: 87%), and DNA fragments of effluent of 36 °C reactors were similar to Syntrophomonas curvata (strain: GB8-1) (similarity: 91%). In reactors with a relatively higher temperature, the DNA fragments of effluent of 44 °C reactor were similar to Dielma fastidiosa (strain: JC13) (similarity: 86%), and the DNA fragments of effluent of 52 °C reactor were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 99%). To authors’ knowledge, this is one of the few studies where DGGE-based approach is utilized to study and compare microbial shifts under mesophilic and thermophilic anaerobic digestions of manure simultaneously. While there were challenges in identifying the bands during gradient gel electrophoresis, the joint use of DGGE and sequencing tool can be potentially useful for illustrating and comparing the change in microbial community structure under complex anaerobic processes and functionality of microbes for understanding the consequential GHG emissions from manure.

AbstractThis paper explores growers’ supply response to the 2005 “Sideways effect” demand shock (Cuellar, Karnowsky, and Acosta, 2009) triggered by the 2004 release of the movie Sideways. We use a modified difference-in-difference approach to evaluate the supply response in California and regional supply response differences within California. We use U.S. Department of Agriculture data for the period 1999–2012 and find evidence of a supply response in the post-release period that is consistent with the “Sideways effect” on wine demand. The positive supply response for Pinot Noir is stronger than the negative response for Merlot and concentrated in lower value Central Valley vineyards. (JEL Classifications: D25, Q12)

Antibiotic resistance is an emerging risk for human and animal health, and mitigating the risk requires an improved understanding of various sources of risks and identifying the level of threats for each source. Many antibiotics are currently used against pathogens for treating infections in animals and humans, and it is considered that antibiotic resistance genes (ARGs) acquired by pathogens may have an environmental origin. Because of contamination in ambient waterways, it is likely that ARGs may affect both organic and non-organic farming. While health risk as a consequence of ARGs is difficult to determine because of lack of understanding of dose-response, the presence of ARGs in human waste and animal manure, and the subsequent application of these organic wastes as fertilizers has a potential of spreading ARGs in the environment. Additional research is needed to understand the presence, growth, and transport of ARGs through animal wastes such as dairy manure. In this review, we focus on synthesizing information regarding the occurrence of ARGs in dairy manure, potential transport pathways, and factors responsible for the spread of ARGs in the environment. Besides, we also explore potential treatment methods that may contribute to the ARG removal in dairy manure and help alleviate ARG contamination.

Cyanobacteria (blue-green algae) have been present on Earth for over 2 billion years, and can produce a variety of bioactive molecules, such as cyanotoxins. Microcystins (MCs), the most frequently detected cyanotoxins, pose a threat to the aquatic environment and to human health. The classic toxic mechanism of MCs is the inhibition of the protein phosphatases 1 and 2A (PP1 and PP2A). Immunity is known as one of the most important physiological functions in the neuroendocrine-immune network to prevent infections and maintain internal homoeostasis in fish. The present review aimed to summarize existing papers, elaborate on the MC-induced immunotoxicity in fish, and put forward some suggestions for future research. The immunomodulatory effects of MCs in fish depend on the exposure concentrations, doses, time, and routes of exposure. Previous field and laboratory studies provided strong evidence of the associations between MC-induced immunotoxicity and fish death. In our review, we summarized that the immunotoxicity of MCs is primarily characterized by the inhibition of PP1 and PP2A, oxidative stress, immune cell damage, and inflammation, as well as apoptosis. The advances in fish immunoreaction upon encountering MCs will benefit the monitoring and prediction of fish health, helping to achieve an ecotoxicological goal and to ensure the sustainability of species. Future studies concerning MC-induced immunotoxicity should focus on adaptive immunity, the hormesis phenomenon and the synergistic effects of aquatic microbial pathogens.

BACKGROUND:Antibiotic resistance genes (ARGs) are considered to be emerging environmental contaminants of concern potentially posing risks to human and animal health, and this research studied the prevalence of antimicrobial resistance in dairy manure.METHODS:This study is focused on investigating prevalence of ARGs in California dairy farm manure under current common different manure management. A total of 33 manure samples were collected from multiple manure treatment conditions: (1) flushed manure (FM), (2) fresh pile (FP), (3) compost pile (CP), (4) primary lagoon (PL), and (5) secondary lagoon (SL). After DNA extraction, all fecal samples were screened by PCR for the presence of eight ARGs: four sulfonamide ARGs (sulI, sulII, sulIII, sulA), two tetracycline ARGs (tetW, tetO), two macrolide-lincosamide-streptogramin B (MLSB) ARGs (ermB, ermF). Samples were also screened for two mobile genetic elements (MGEs) (intI1, tnpA), which are responsible for dissemination of ARGs. Quantitative PCR was then used to screen all samples for five ARGs (sulII, tetW, ermF, tnpA and intI1).RESULTS:Prevalence of genes varied among sample types, but all genes were detectable in different manure types. Results showed that liquid-solid separation, piling, and lagoon conditions had limited effects on reducing ARGs and MGEs, and the effect was only found significant on tetW (p = 0.01). Besides, network analysis indicated that sulII was associated with tnpA (p < 0.05), and Psychrobacter and Pseudomonas as opportunistic human pathogens, were potential ARG/MGE hosts (p < 0.05). This research indicated current different manure management practices in California dairy farms has limited effects on reducing ARGs and MGEs. Improvement of different manure management in dairy farms is thus important to mitigate dissemination of ARGs into the environment.

Antibiotic resistance genes (ARGs) are emerging contaminants causing serious global health concern. Interventions to address this concern include improving our understanding of methods for treating waste material of human and animal origin that are known to harbor ARGs. Anaerobic digestion is a commonly used process for treating dairy manure, and although effective in reducing ARGs, its mechanism of action is not clear. In this study, we used three ARGs to conducted a longitudinal bench scale anaerobic digestion experiment with various temperatures (28, 36, 44, and 52°C) in triplicate using fresh dairy manure for 30 days to evaluate the reduction of gene abundance. Three ARGs and two mobile genetic elements (MGEs) were studied: sulfonamide resistance gene ( sulII ), tetracycline resistance genes ( tetW ), macrolide-lincosamide-streptogramin B (MLSB) superfamily resistance genes ( erm F), class 1 integrase gene ( intI1 ), and transposase gene ( tnpA ). Genes were quantified by real-time quantitative PCR. Results show that the thermophilic anaerobic digestion (52°C) significantly reduced ( p < 0.05) the absolute abundance of sulII (95%), intI1 (95%), tnpA (77%) and 16S rRNA gene (76%) after 30 days of digestion. A modified Collins–Selleck model was used to fit the decay curve, and results suggest that the gene reduction during the startup phase of anaerobic digestion (first 5 days) was faster than the later stage, and reductions in the first five days were more than 50% for most genes.

Background.Antibiotic resistance genes (ARGs) are considered to be emerging environmental contaminants of concern potentially posing risks to human and animal health, and this research studied the prevalence of antimicrobial resistance in dairy manure.Methods.This study is focused on investigating prevalence of ARGs in California dairy farm manure under current common different manure management.A total of 33 manure samples were collected from multiple manure treatment conditions: 1) flushed manure (FM), 2) fresh pile (FP), 3) compost pile (CP), 4) primary lagoon (PL), and 5) secondary lagoon (SL).After DNA extraction, all fecal samples were screened by PCR for the presence of 8 ARGs: four sulfonamide ARGs (sulI, sulII, sulIII, sulA), two tetracycline ARGs (tetW, tetO), two macrolide-lincosamide-streptogramin B (MLS B ) ARGs (ermB, ermF).Samples were also screened for two mobile genetic elements (MGEs) (intI1, tnpA), which are responsible for dissemination of ARGs.Quantitative PCR was then used to screen all samples for five ARGs (sulII, tetW, ermF, tnpA and intI1).Results.Prevalence of genes varied among sample types, but all genes were detectable in different manure types.Results showed that liquid-solid separation, piling, and lagoon conditions had limited effects on reducing ARGs and MGEs, and the effect was only found significant on tetW (p = 0.01).Besides, network analysis indicated that sulII was associated with tnpA (p < 0.05), and Psychrobacter and Pseudomonas as opportunistic human pathogens, were potential ARG/MGE hosts (p < 0.05).This research indicated current different manure management practices in California dairy farms has limited effects on reducing ARGs and MGEs.Improvement of different manure management in dairy farms is thus important to mitigate dissemination of ARGs into the environment.

To characterize the drying of dairy manure during microwave (MW) heating, and to determine the impacts of microwave radiation on reductions of antibiotic resistance genes (ARGs), this study investigated on understanding the effects of microwave heating on solid streams of flushed dairy manure. A series of experiments were performed to determine the rate of drying, moisture removal percentages, change in solids, and the level of ARGs. Manure was exposed to microwave radiation for 30–300 s at a frequency of 2450 MHz. The results showed moisture removal rate (U) up to 0.63-g water per minute per gram of dairy manure. During MW treatment, volatile solid (VS) content was relatively stable. The MW treatment resulted in reduction of ARGs such as sulII, intI1, and tnpA. The ARG concentrations (sulII, intI1, and tnpA) were reduced by 2 orders of magnitude in less than 1 min of microwave heating. The preliminary results of this study showed that MW treatment can be a viable option for drying of dairy manure and reducing ARGs in manure.

Antibiotic resistance genes (ARGs) are emerging environmental contaminants of concern to both human and animal health. Dairy manure is considered reservoir of ARGs. This study is focused on investigating prevalence of ARGs in California dairy farm manure under current common manure management. A total of 33 manure samples were collected from multiple manure treatment conditions: 1) flushed manure (FM), 2) fresh pile (FP), 3) compost pile (CP), 4) primary lagoon (PL), and 5) secondary lagoon (SL). After DNA extraction, all fecal samples were screened by PCR for the presence of 8 ARGs: four sulfonamide ARGs (sulI, sulII, sulIII, sulA), two tetracycline ARGs (tetW, tetO), two macrolide-lincosamide-streptogramin B (MLSB) ARGs (ermB, ermF). Samples were also screened for two mobile genetic elements (MGEs) (intI1, tnpA), which are responsible for dissemination of ARGs. Quantitative PCR was then used to screen all samples for five ARGs (sulII, tetW, ermF, tnpA and intI1). Prevalence of genes varied among sample types, but all genes were detectable in different manure types. Results showed that liquid-solid separation, piling, and lagoon conditions had limited effects on reducing ARGs and MGEs, and the effect was only found significant on tetW (p = 0.01). Besides, network analysis indicated that sulII was associated with tnpA (p < 0.05), and Psychrobacter and Pseudomonas as opportunistic human pathogens, were potential ARG/MGE hosts (p < 0.05). This research indicated current manure management practices in California dairy farms has limited effects on reducing ARGs and MGEs. Improvement of manure management in dairy farms is thus important to mitigate dissemination of ARGs into the environment.

Purpose: In recreational water bodies, herbicides are widely used for controlling unwanted weeds, and impacts of herbicide residues on health risks to aquatic ecosystem is a serious concern. This study was aimed to improve the existing understanding of the deposition of herbicides from water column to bed sediment and leachate of herbicides from bed sediment to water column. We investigated the attachment of two herbicides with sediment and release from sediment: (1) Glyphosate; and (2) Fluridone. The goal of this study was to determine the deposition and release of Glyphosate and Fluridone in bed sediment of the Sacramento-San Joaquin River Delta. Materials and Methods: Field sampling was performed to collect water and sediment samples from Sacramento-San Joaquin River Delta. Bottom dredge sampler was used for collecting sediment samples and horizontal water bottle sampler was used for collecting water samples. A series of experiments were conducted to determine the attachment and release of Fluridone and Glyphosate from sediment at a different level of initial concentrations. For analyzing Fluridone and Glyphosate in sediment leachate and water, samples were processed using enzyme-linked immunosorbent assay (ELISA) based method. Results and Discussion: Observations showed that proportions of Glyphosate concentrations in water were higher than Fluridone concentrations in water, when both herbicides were inoculated in water in same quantity. On the contrary, the concentrations of Fluridone in sediment-bound leachate were higher than Glyphosate concentrations in sediment-bound leachate, regardless of the initial concentrations. Fluridone and Glyphosate concentrations in water column samples differed significantly (p < 0.05) over the time even initial concentrations of these herbicides were kept similar, which indicates that Fluridone interaction with water column was considerably different than the interaction of Glyphosate with the water column. Conclusions: Bed sediment can be an important sink and source for release of Fluridone and Glyphosate from bed sediment to the water column of an ambient water body. Significant concentrations of herbicides were deposited in bed sediment of Sacramento-San Joaquin Delta, and eventually the high concentrations of herbicides were observed in sediment leachate. Improved understanding of this important release pathway can provide much needed information to adequately address the impacts of particle attached herbicides on aquatic and ecological environment of a water body.

Gluconic acid, an oxidized cellulose degradation product, could be produced from cellulosic biomass. Glycerol is an inexpensive and renewable resource for fuels and chemicals production and is available as a byproduct of biodiesel production. Gluconate is a more oxidized substrate than glucose, whereas glycerol is a more reduced substrate than glucose. Although the production of homoethanol from glucose can be achieved, the conversion of gluconate to ethanol is accompanied by the production of oxidized byproduct such as acetate, and reduced byproducts such as 1,3-propanediol are produced, along with ethanol, when glycerol is used as the carbon source. When gluconate and glycerol are used as the sole carbon source by Klebsiella oxytoca BW21, the ethanol yield is about 62 to 64%. Coutilization of both gluconate and glycerol in batch fermentation increased the yield of ethanol to about 78.7% and decreased by-product accumulation (such as acetate and 1,3-propanediol) substantially. Decreasing by-product formation by deleting the pta, frd, ldh, pflA, and pduC genes in strain BW21 increased the ethanol yield to 89.3% in the batch fermentation of a glycerol-gluconate mixture. These deletions produced the strain K. oxytoca WT26. However, the utilization rate of glycerol was significantly slower than that of gluconate in batch fermentation. In addition, substantial amounts of glycerol remain unutilized after gluconate was depleted in batch fermentation. Continuous fed-batch fermentation was used to solve the utilization rate mismatch problem for gluconate and glycerol. An ethanol yield of 97.2% was achieved in continuous fed-batch fermentation of these two substrates, and glycerol was completely used at the end of the fermentation.IMPORTANCE Gluconate is a biomass-derived degradation product, and glycerol can be obtained as a biodiesel byproduct. Compared to glucose, using them as the sole substrate is accompanied by the production of by-products. Our study shows that through pathway engineering and adoption of a fed-batch culture system, high-yield homoethanol production that usually can be achieved by using glucose as the substrate is achievable using gluconate and glycerol as cosubstrates. The same strategy is expected to be able to achieve homofermentative production of other products, such as lactate and 2,3-butanediol, which can be typically achieved using glucose as the substrate and inexpensive biodiesel-derived glycerol and biomass-derived gluconate as the cosubstrates.

Pathogen contamination in stream water is a serious concern, which can pose risks to public and animal health, and reducing the risks of pathogens requires enhanced knowledge of pathogen sources and pathogen survival under various environmental and controlled conditions. One of the major factors, which affect pathogen survival, is temperature. To improve existing understanding of pathogen inactivation in stream water and sediment at various temperature conditions, we executed a series of microcosm studies in batch mode for assessing pathogen survival. Water and sediment samples from stream water column and bed sediment of Merced River Watershed, California were collected, and stored at 4°C prior to experiments. Subsequently, heat shock experiments were conducted under temperature controlled conditions. Initial pathogen levels in water and sediment samples were controlled by mixing the inoculum of E. coli, Salmonella, and Listeria, which are water and foodborne pathogens, known for causing illnesses in humans. We studied pathogen survival at 30°C, and 50°C. Results showed that the temperature of heat shock and incubation time under heat shock played crucial role in reducing pathogen levels. Low temperature (30°C) heat shocks and limited time heat shocks with relatively higher temperature (50°C) may not have considerable impacts in reducing pathogen levels.

Improved understanding of pathogen survival in the stream environment is needed to enhance existing predictive models of stream pathogen populations. Further, the increasing use of thermal springs for bathing necessitates additional studies focused on not only typical streams but also thermal spring conditions, where water temperature is relatively higher than typical streams. This study was conducted to assess the survival of E. coli O157:H7 and Salmonella Typhimurium in stream water under free floating and particle-attached conditions at a range of temperature. A series of microcosm studies were conducted to determine pathogen decay rates. In bench-scale experiments, water circulation and sediment resuspension mimicked natural stream and thermal spring conditions, with continuous air flow providing aeration, constant mixing and turbulent conditions, and improved water circulation. Data on E. coli O157:H7 and Salmonella survival were subsequently used to determine first-order decay equations for calculating the rate constant and decimal reduction time for the modeled experimental conditions. Results showed that at 40 °C, the survival of particle attached E. coli O157:H7 was longer than that of particle attached Salmonella. Under free floating condition, Salmonella survived longer than E. coli O157:H7. At 50 °C, survival of particle attached E. coli O157:H7 and Salmonella was longer than that of free floating E. coli and Salmonella. At 60 °C, survival of particle attached Salmonella was longer than that of free floating Salmonella. Similarly at 60 °C, the survival of E. coli O157:H7  under particle attached condition was longer than that of the free floating condition. The findings of this study suggest that the survival of E. coli O157:H7 differs than the survival of Salmonella in stream water and thermal spring conditions, and the assumption used in previous studies to estimate survival of bacteria in stream environment could result in over/underestimation if the impact of particle attachment on pathogen survival is not accounted for.

Increased public health risk caused by pathogen contamination in streams is a serious issue, and mitigating the risk requires improvement in existing microbial monitoring of streams. To improve understanding of microbial contamination in streams, we monitored in stream water columns and streambed sediment. Two distinct streams and their subwatersheds were studied: (i) a mountain stream (Merced River, California), which represents pristine and wild conditions, and (ii) an agricultural stream (Squaw Creek, Iowa), which represents an agricultural setting (i.e., crop, manure application, cattle access). Stream water column and sediment samples were collected in multiple locations in the Merced River and Squaw Creek watersheds. Compared with the mountain stream, water column concentrations in the agricultural stream were considerably higher. In both mountain and agricultural streams, concentrations in bed sediment were higher than the water column, and principal component analysis indicates that land use affected water column levels significantly ( < 0.05). The cluster analysis showed grouping of subwatersheds for each basin, indicating unique land use features of each watershed. In general, water column levels in the mountain stream were lower than the USEPA's existing water quality criteria for bacteria. However, the levels in the agricultural stream exceeded the USEPA's microbial water quality criteria by several fold, which substantiated that increased agricultural activities, use of animal waste as fertilizers, and combined effect of rainfall and temperature may act as potential determining factors behind the elevated levels in agriculture streams.

In the U.S., more than 21012 kg of dairy waste is generated annually, which can be a potential feedstock for producing renewable energy source such as biogas. The use of granules as an inoculum is reported to accelerate these processes, and biogas production. The goal of this study is to understand the impact of anaerobic digestion process on flushed dairy manure-borne Escherichia coli reductions and biogas production. A series of anaerobic experiments were performed using the flushed manure inoculated with granule inoculum. The anaerobic digestion batch experiments were performed at multiple temperatures including room temperature (~ 22 °C), 37 °C, and 55 °C. Results showed that under room temperature, no significant reduction of E.coli concentration was found at the end of 33 days compared with the initial condition. In 37 °C, E.coli concentration was reduced substantially on day 7 and subsequently low levels of E. coli was observed. At 55 °C, E. coli reduction was considerable higher than that of at room temperature and 37 °C within 24 hours of digestion. In addition to E. coli levels, biogas composition was measured using a gas chromatography (GC) with Flame Ionization Detector (FID) and Electron Capture Detector (ECD). CO2 concentration was relatively consistent over the time of digestion. Granule inoculum significantly enhanced methane concentration. Methane concentration was higher when the temperature is increased. We anticipate that this preliminary study provides information with regards to pathogen indicator survival during anaerobic digestion of flushed manure, and changes in biogas content.

To improve understanding of pathogen inactivation in flushed dairy manure during anaerobic digestion, lagoon systems, and mesophilic composting, we assessed pathogen inactivation in batch-scale anaerobic digesters, aerobic lagoons, and manure piles. Further, we assessed the impact of air injection in lagoon systems on pathogen inactivation. The inactivation of Salmonella and E. coli was studied for more than 70days. The change in pH, total solids, total carbon, and total nitrogen during manure treatment was determined. The results showed that the lagoon system with air injection to be more effective in pathogen removal compared to the lagoon system with no air injection, anaerobic digestion, and mesophilic composting. Both types of lagoon systems (with and without air injection) were found to be more effective in eliminating pathogens than anaerobic digester systems. Mesophilic manure pile extended the survival of both Salmonella and E. coli. In the anaerobic system, E. coli was detected beyond 60days of incubation, while in the aerobic system; it was eliminated in 30–35days. In anaerobic system, Salmonella reached to non-detectable level in 30–35days, while in aerobic system it took 4–12days. In the mesophilic manure pile, however, E. coli survival was extended beyond 78days. Less than one log reduction was obtained in 78days of mesophilic composting. Salmonella survival was also extended in mesophilic composting pile.

Viral systemic infection of a plant host involves two processes, cell-to-cell movement and long-distance transport. Molecular determinants associated with these two processes were probed by investigating the effects that alanine scanning mutations in the movement protein (MP) of red clover necrotic mosaic virus (RCNMV) had on viral infection in the plant hosts Nicotiana edwardsonii, Vigna unguiculata (cowpea), and the experimental plant Nicotiana benthamiana. Plants were inoculated with RCNMV expressing wild-type and mutant forms of the MP. Immunocytochemical studies at the light and electron microscope levels were performed on these plants, using a polyclonal antibody raised against the RCNMV capsid protein to identify the cells/tissues that RCNMV could infect. These experiments demonstrated that one cellular boundary at which the RCNMV MP functions to facilitate entry into the phloem long-distance transport system is located at the interfaces between the bundle sheath and phloem parenchyma cells and the companion cell-sieve element complex. Interestingly, in Nicotiana tabacum, a host that only allows a local infection, RCNMV cell-to-cell movement was found to be blocked at this same intercellular boundary. Four mutants that were able to systemically infect N. benthamiana were partially or completely defective for systemic infection of N. edwardsonii and cowpea, which indicated that these MP mutants exhibited host-specific defects. Thus, the roles of the RCNMV MP in cell-to-cell movement and in long-distance transport appear to be genetically distinct. These results are discussed in terms of the mechanism by which RCN MV enters the phloem to establish a systemic infection.