Microbial community structure from southern High Plains beef cattle feedyard manure and relationship with nitrous oxide emissions

Modern molecular techniques enable characterization of the microbial biome in livestock manure, from which there is particular concern over emission of greenhouse gases. This study evaluated how sampling depth, time, temperature, and artificial rainfall affected microbial community structure in feedyard manure, and relationships between the manure biome and known parameters related to nitrous oxide (N2O) emissions. In three large incubation chambers, maintained at different temperatures that received two applications of artificial rainfall, we evaluated manure microbiome composition and abundance of N2O-producing enzymes (nirK and nirS) using quantitative polymerase chain reaction (qPCR). These data were used with previously published data from the same study on N2O emissions and assessment of manure physicochemical properties, denitrification enzyme activity (DEA), and nitrification activity (NA). Microbiome composition was Firmicutes (50%), followed by 32% Actinobacteria, 11% Proteobacteria, 5% Bacteroidetes, 1% Chloroflexi, and small populations (\u3c0.5%) of Planctomycetes, Deinococcus-Thermus, Gemmatimonadetes, Verrucomicrobia, Tenericutes, and other organisms. Average bacterial populations varied largely as a function of sampling depth and time. Firmicutes increases tended to coincide with high N2O emissions. Overall, the largest change observed was increased Proteobacteria at 5–10 cm, where relative abundance increased from 10% (17.2 °C) to 24% (46.2 °C) over time and with increased temperature. Firmicutes and Actinobacteria predominated the microbial community of manure, but favorable conditions may lead to increases in Bacteroidetes, Proteobacteria, and Chloroflexi, which could influence N cycling and N2O emissions from feedyards. Copy numbers of nirS at the beginning of the experiment were higher than nirK. Differences in concentrations of nirK and nirS indicated that denitrifying enzymes in feedyard manure, particularly nirS, were sensitive to environmental changes

Identifying challenges and opportunities for improved nutrient management through U.S.D.A's Dairy Agroecosystem Working Group

Nutrient management is a priority of U.S. dairy farms, although specific concerns vary across regions and management systems. To elucidate challenges and opportunities to improving nutrient use efficiencies, the USDA’s Dairy Agroecosystems Working Group investigated 10 case studies of confinement (including open lots and free stall housing) and grazing operations in the seven major U.S. dairy producing states. Simulation modeling was carried out using the Integrated Farm Systems Model over 25 years of historic weather data. Dairies with a preference for importing feed and exporting manure, common for simulated dry lot dairies of the arid west, had lower nutrient use efficiencies at the farm gate than freestall and tie-stall dairies in humid climates. Phosphorus (P) use efficiencies ranged from 33 to 82% of imported P, while N use efficiencies were 25 to 50% of imported N. When viewed from a P budgeting perspective, environmental losses of P were generally negligible, especially from dry lot dairies. Opportunities for greater P use efficiency reside primarily in increasing on-farm feed production and reducing excess P in diets. In contrast with P, environmental losses of nitrogen (N) were 50 to 75% of annual farm N inputs. For dry lot dairies, the greatest potential for N conservation is associated with ammonia (NH3) control from housing, whereas for freestall and tie-stall operations, N conservation opportunities vary with soil and manure management system. Given that fertilizer expenses are equivalent to 2 to 6% of annual farm profits, cost incentives do exist to improve nutrient use efficiencies. However, augmenting on-farm feed production represents an even greater opportunity, especially on large operations with high animal unit densities

Forms, Distribution, and Potential Availability of Phosphorous from Poultry Manure

Phosphorus (P) solubility following soil application of poultry manure (PM) is influenced by manure composition, soil physicochemical properties, and rhizosphere processes. As more than 42% of P in PM is insoluble, high application rates may result in accumulation of P that is not plant available. Objectives were to (1) use sequential fractionation to characterize inorganic (Pi) and organic (Po) phosphorus from PM, and determine the effects of laboratory drying, (2) examine short-term changes in soil P fractions following PM application, and (3) identify changes in soil P fractions and soil phosphatase activities induced by both rhizosphere processes and PM in soil planted with ryegrass. Sequential fractionation of PM revealed that most Pi was in the labile (H2O + NaHCO3) and relatively stable HCl fractions, representing 46 and 48%, respectively. HCl-Po represented 53% of total manure organic P. Drying induced transformation of NaHCO3-Pi to H2O-Pi, increasing P solubility 42-97% in the order of 65°C\u3e22°C\u3efreeze-dry\u3eundried; therefore, freeze-drying is recommended as the method least likely to overestimate H2O-Pi. Incubation of soil with PM at 100 and 200 mg P kg-1 of soil revealed that H2O- and HCl-Pi from PM increased Pi in corresponding fractions from soil. However, whereas H2O-Pi from PM was rapidly transformed to other fractions, HCl-Pi remained elevated when soil received the high rate of PM (200 mg kg-1). This was not observed in soil that received PM at 100 mg kg-1, suggesting that moderate application may not increase relatively stable P in soil, or may increase this fraction more slowly than high application rates. In a greenhouse study, when ryegrass was grown with PM at 112 mg P kg-1 of soil, labile-Pi in the rhizosphere was 30% higher than unamended soil, increasing root P concentrations by 37% and total P uptake by 59%. While PM initially increased NaOH- and HCl-Pi, these fractions did not differ between treatments after 16 weeks. In conclusion, chemical and biotic processes may readily transform H2O-extractable Pi from PM into more stable forms, and transform slowly available Pi and Po into labile-P; therefore, a single PM application does not necessarily lead to accumulation of recalcitrant P

Antibiotic resistance gene profile changes in cropland soil after manure application and rainfall

Land application of manure introduces gastrointestinal microbes into the environment, including bacteria carrying antibiotic resistance genes (ARGs). Measuring soil ARGs is important for active stewardship efforts to minimize gene flow from agricultural production systems; however, the variety of sampling protocols and target genes makes it difficult to compare ARG results between studies. We used polymerase chain reaction (PCR) methods to characterize and/or quantify 27 ARG targets in soils from 20 replicate, long-term no-till plots, before and after swine manure application and simulated rainfall and runoff. All samples were negative for the 10 b-lactamase genes assayed. For tetracycline resistance, only source manure and post-application soil samples were positive. The mean number of macrolide, sulfonamide, and integrase genes increased in post-application soils when compared with source manure, but at plot level only, 1/20, 5/20, and 11/20 plots post-application showed an increase in erm(B), sulI, and intI1, respectively. Results confirmed the potential for temporary blooms of ARGs after manure application, likely linked to soil moisture levels. Results highlight uneven distribution of ARG targets, even within the same soil type and at the farm plot level. This heterogeneity presents a challenge for separating effects of manure application from background ARG noise under field conditions and needs to be considered when designing studies to evaluate the impact of best management practices to reduce ARG or for surveillance. We propose expressing normalized quantitative PCR (qPCR) ARG values as the number of ARG targets per 100,000 16S ribosomal RNA genes for ease of interpretation and to align with incidence rate data

Enzymatic Quantification of Phytate in Animal Manure

Phytate (inositol hexaphosphate) has been identified as a major organic phosphorus (P) form in soil, animal manure, and other environmental samples. Although a number of methods are available for quantitative isolation and determination of phytate, they are time-consuming and not amenable to routine analysis. We developed a simple, rapid method for enzymatic determination of phytate in animal manure. Animal manure was extracted by H2O, 1M hydrochloric acid (HCl), 0.1M sodium acetate (NaOAc, pH5.0) with or without 0.05M ethylenediaminetetraacetate (EDTA), and 0.25M or 0.5M sodium hydroxide (NaOH)–0.05M EDTA. Extracts were diluted (1/10–1/150) and adjusted to pH5.0 in sodium acetate buffer. The diluted extracts were then incubated at 37 °C for 1 h in the absence and presence of fungal 3-phytase (PHY) and potato acid phosphatase (PAP). Enzymatic hydrolyzable organic P was calculated as the difference in inorganic P (Pi) between the mixtures with and without enzymes. Our data indicated that enzymatic incubation of properly diluted and pH-adjusted HCl or NaOH/EDTA extracts released phytate P. The complementary substrate specificity of the two enzymes is considered to enhance the effectiveness of enzymatic hydrolysis. Consequently, we recommend this method of combining PAP and PHY for quantifying phytate P. Additional research is being conducted to verify the effectiveness of this method for general use across a wider range of soils and manures

Antibiotic resistance gene profile changes in cropland soil after manure application and rainfall

Land application of manure introduces gastrointestinal microbes into the environ- ment, including bacteria carrying antibiotic resistance genes (ARGs). Measuring soil ARGs is important for active stewardship efforts to minimize gene flow from agri- cultural production systems; however, the variety of sampling protocols and target genes makes it difficult to compare ARG results between studies. We used polymerase chain reaction (PCR) methods to characterize and/or quantify 27 ARG targets in soils from 20 replicate, long-term no-till plots, before and after swine manure application and simulated rainfall and runoff. All samples were negative for the 10 b-lactamase genes assayed. For tetracycline resistance, only source manure and post-application soil samples were positive. The mean number of macrolide, sulfonamide, and inte- grase genes increased in post-application soils when compared with source manure, but at plot level only, 1/20, 5/20, and 11/20 plots post-application showed an increase in erm(B), sulI, and intI1, respectively. Results confirmed the potential for tempo- rary blooms of ARGs after manure application, likely linked to soil moisture lev- els. Results highlight uneven distribution of ARG targets, even within the same soil type and at the farm plot level. This heterogeneity presents a challenge for separating effects of manure application from background ARG noise under field conditions and needs to be considered when designing studies to evaluate the impact of best manage- ment practices to reduce ARG or for surveillance. We propose expressing normalized quantitative PCR (qPCR) ARG values as the number of ARG targets per 100,000 16S ribosomal RNA genes for ease of interpretation and to align with incidence rate dat

Tetracycline and Sulfonamide Antibiotic Resistance Genes in Soils From Nebraska Organic Farming Operations

There is widespread agreement that agricultural antibiotic resistance should be reduced, however, it is unclear from the available literature what an appropriate target for reduction would be. Organic farms provide a unique opportunity to disentangle questions of agricultural antibiotic drug use from questions of antibiotic resistance in the soil. In this study, soil was collected from 12 certified organic farms in Nebraska, evaluated for the presence of tetracycline and sulfonamide resistance genes (n = 15 targets), and correlated to soil physical, chemical, and biological parameters. Tetracycline and sulfonamide antibiotic resistance genes (ARGs) were found in soils from all 12 farms, and 182 of the 196 soil samples (93%). The most frequently detected gene was tetG (55% of samples), followed by tet(Q) (49%), tet(S) (46%), tet(X) (30%), and tetA(P) (29%). Soil was collected from two depths. No differences in ARGs were observed based on soil depth. Positive correlations were noted between ARG presence and soil electrical conductivity, and concentrations of Ca, Na, and Mehlich-3 phosphorus. Data from this study point to possible relationships between selected soil properties and individual tetracycline resistance genes, including tet(O) which is a common target for environmental samples. We compared organic farm results to previously published data from prairie soils and found significant differences in detection frequency for 12 genes, eight of which were more commonly detected in prairie soils. Of interest, when tetracycline ARG results were sorted by gene mechanism, the efflux genes were generally present in higher frequency in the prairie soils, while the ribosomal protection and enzymatic genes were more frequently detected in organic farm soils, suggesting a possible ecological role for specific tetracycline resistance mechanisms. By comparing soil from organic farms with prairie soils, we can start to determine baseline effects of low-chemical input agricultural production practices on multiple measures of resistance

Antibiotic resistance gene profile changes in cropland soil after manure application and rainfall

Land application of manure introduces gastrointestinal microbes into the environment, including bacteria carrying antibiotic resistance genes (ARGs). Measuring soil ARGs is important for active stewardship efforts to minimize gene flow from agricultural production systems; however, the variety of sampling protocols and target genes makes it difficult to compare ARG results between studies. We used polymerase chain reaction (PCR) methods to characterize and/or quantify 27 ARG targets in soils from 20 replicate, long-term no-till plots, before and after swine manure application and simulated rainfall and runoff. All samples were negative for the 10 b-lactamase genes assayed. For tetracycline resistance, only source manure and post-application soil samples were positive. The mean number of macrolide, sulfonamide, and integrase genes increased in post-application soils when compared with source manure, but at plot level only, 1/20, 5/20, and 11/20 plots post-application showed an increase in erm(B), sulI, and intI1, respectively. Results confirmed the potential for temporary blooms of ARGs after manure application, likely linked to soil moisture levels. Results highlight uneven distribution of ARG targets, even within the same soil type and at the farm plot level. This heterogeneity presents a challenge for separating effects of manure application from background ARG noise under field conditions and needs to be considered when designing studies to evaluate the impact of best management practices to reduce ARG or for surveillance. We propose expressing normalized quantitative PCR (qPCR) ARG values as the number of ARG targets per 100,000 16S ribosomal RNA genes for ease of interpretation and to align with incidence rate data

Condensed and Hydrolyzable Tannins for Reducing Methane and Nitrous Oxide Emissions in Dairy Manure—A Laboratory Incubation Study

The objectives of this study were to (1) examine the effects of plant condensed (CT) and hydrolyzable tannin (HT) extracts on CH4 and N2O emissions; (2) identify the reactions responsible for manure-derived GHG emissions, and (3) examine accompanying microbial community changes in fresh dairy manure. Five treatments were applied in triplicate to the freshly collected dairy manure, including 4% CT, 8% CT, 4% HT, 8% HT (V/V), and control (no tannin addition). Fresh dairy manure was placed into 710 mL glass incubation chambers. In vitro composted dairy manure samples were collected at 0, 24, 48, and 336 h after the start of incubation. Fluxes of N2O and CH4 were measured for 5-min/h for 14 d at a constant ambient incubation temperature of 39 °C. The addition of quebracho CT significantly decreased the CH4 flux rates compared to the tannin-free controls (215.9 mg/m2/h), with peaks of 75.6 and 89.6 mg/m2/h for 4 and 8% CT inclusion rates, respectively. Furthermore, CT significantly reduced cumulative CH4 emission by 68.2 and 57.3% at 4 and 8% CT addition, respectively. The HT treatments failed to affect CH4 reduction. However, both CT and HT reduced (p 2O emissions. The decrease in CH4 flux with CT was associated with a reduction in the abundance of Bacteroidetes and Proteobacteria