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Item Treatment performance and microbial community structure in an aerobic granular sludge sequencing batch reactor amended with diclofenac, erythromycin, and gemfibrozil(Frontiers Media SA, 2023-09) Bodle, Kylie B.; Mueller, Rebecca C.; Pernat, Madeline R.; Kirkland, Catherine M.This study characterizes the effects of three commonly detected pharmaceuticals—diclofenac, erythromycin, and gemfibrozil—on aerobic granular sludge. Approximately 150 µg/L of each pharmaceutical was fed in the influent to a sequencing batch reactor for 80 days, and the performance of the test reactor was compared with that of a control reactor. Wastewater treatment efficacy in the test reactor dropped by approximately 30-40%, and ammonia oxidation was particularly inhibited. The relative abundance of active Rhodocyclaceae, Nitrosomonadaceae, and Nitrospiraceae families declined throughout exposure, likely explaining reductions in wastewater treatment performance. Pharmaceuticals were temporarily removed in the first 12 days of the test via both sorption and degradation; both removal processes declined sharply thereafter. This study demonstrates that aerobic granular sludge may successfully remove pharmaceuticals in the short term, but long-term tests are necessary to confirm if pharmaceutical removal is sustainable.Item Rumen microbiome response to sustained release mineral bolus supplement with low- and high-quality forages(Frontiers Media SA, 2023-06) Eberly, Jed O.; Wyffels, Samuel A.; Carlisle, Tanner J.; DelCurto, TimothyIntroduction: Limited forage quantity and quality are challenges faced in livestock production systems in semi-arid rangelands of the western United States, particularly when livestock face stressors such as cold weather or have increased nutritional requirements such as during pregnancy and lactation. To meet livestock nutrient requirements, producers frequently provide supplemental nutrition, however there is limited knowledge regarding the effects of these practices on the rumen microbiome in these environments. Methods: A study was conducted to evaluate changes in the rumen microbiome in response to high- and low- quality forage with sustained release mineral boluses. The study consisted of 16 ruminally-cannulated 2–3-year-old black angus cows fed high quality grass alfalfa hay or low-quality grass hay with a 90 or 180 day sustained release mineral bolus. Rumen samples were collected pre-feeding and 8 hours post feeding and bacterial 16S rRNA gene amplicons were sequenced from the rumen fluid. Results: Alpha diversity as measured by Shannon’s diversity index decreased significantly over time (p<0.01) and averaged 5.6 pre-feeding and 5.4 post- feeding and was not significantly different between high- and low-quality forages or between mineral bolus types (p>0.05). Principal coordinates analysis (PCoA) of the Bray-Curtis dissimilarity matrix showed distinct grouping by feed quality and time but not by mineral bolus type. Bacteroidetes and Firmicutes were the dominant phyla in all treatments and significant increases (p<0.05) in the relative abundance of the family Lachnospiraceae and the genus Prevotella were observed in high quality forage diets. Rumen VFA and NH3-N concentrations were also strongly associated with the high-quality forage diet. Predictive functional profiling indicated that functions associated with methanogenesis were negatively correlated with feed quality. Discussion: The results of this study suggest that mineral bolus type is unlikely to affect rumen bacterial community structure or function while forage quality can significantly alter community structure and predicted functions associated with methanogenesis and VFA production.Item Rumen microbiome response to sustained release mineral bolus supplement with low- and high-quality forages(Frontiers Media SA, 2023-06) Eberly, Jed O.; Wyffels, Samuel A.; Carlisle, Tanner J.; DelCurto, TimothyIntroduction: Limited forage quantity and quality are challenges faced in livestock production systems in semi-arid rangelands of the western United States, particularly when livestock face stressors such as cold weather or have increased nutritional requirements such as during pregnancy and lactation. To meet livestock nutrient requirements, producers frequently provide supplemental nutrition, however there is limited knowledge regarding the effects of these practices on the rumen microbiome in these environments. Methods: A study was conducted to evaluate changes in the rumen microbiome in response to high- and low- quality forage with sustained release mineral boluses. The study consisted of 16 ruminally-cannulated 2–3-year-old black angus cows fed high quality grass alfalfa hay or low-quality grass hay with a 90 or 180 day sustained release mineral bolus. Rumen samples were collected pre-feeding and 8 hours post feeding and bacterial 16S rRNA gene amplicons were sequenced from the rumen fluid. Results: Alpha diversity as measured by Shannon’s diversity index decreased significantly over time (p<0.01) and averaged 5.6 pre-feeding and 5.4 post- feeding and was not significantly different between high- and low-quality forages or between mineral bolus types (p>0.05). Principal coordinates analysis (PCoA) of the Bray-Curtis dissimilarity matrix showed distinct grouping by feed quality and time but not by mineral bolus type. Bacteroidetes and Firmicutes were the dominant phyla in all treatments and significant increases (p<0.05) in the relative abundance of the family Lachnospiraceae and the genus Prevotella were observed in high quality forage diets. Rumen VFA and NH3-N concentrations were also strongly associated with the high-quality forage diet. Predictive functional profiling indicated that functions associated with methanogenesis were negatively correlated with feed quality. Discussion: The results of this study suggest that mineral bolus type is unlikely to affect rumen bacterial community structure or function while forage quality can significantly alter community structure and predicted functions associated with methanogenesis and VFA production.Item Associations among the genome, rumen metabolome, ruminal bacteria, and milk production in early-lactation Holsteins(Elsevier, 2023-03) Golder, H.M.; Thomson, J.; Rehberger, J.; Smith, A.H.; Block, E.; Lean, I.J.A multicenter observational study to evaluate genome-wide association was conducted in early-lactation Holstein cows (n = 293) from 36 herds in Canada, the USA, and Australia. Phenotypic observations included rumen metabolome, acidosis risk, ruminal bacterial taxa, and milk composition and yield measures. Diets ranged from pasture supplemented with concentrates to total mixed rations (nonfiber carbohydrates = 17 to 47, and neutral detergent fiber = 27 to 58% of dry matter). Rumen samples were collected <3 h after feeding and analyzed for pH, ammonia, d- and l-lactate, volatile fatty acid (VFA) concentrations, and abundance of bacterial phyla and families. Eigenvectors were produced using cluster and discriminant analyses from a combination of pH and ammonia, d-lactate, and VFA concentrations, and were used to estimate the probability of the risk of ruminal acidosis based on proximity to the centroid of 3 clusters, termed high (24.0% of cows), medium (24.2%), and low risk (51.8%) for acidosis. DNA of sufficient quality was successfully extracted from whole blood (218 cows) or hair (65 cows) collected simultaneously with the rumen samples and sequenced using the Geneseek Genomic Profiler Bovine 150K Illumina SNPchip. Genome-wide association used an additive model and linear regression with principal component analysis (PCA) population stratification and a Bonferroni correction for multiple comparisons. Population structure was visualized using PCA plots. Single genomic markers were associated with milk protein percent and the center logged ratio abundance of the phyla Chloroflexi, SR1, and Spirochaetes, and tended to be associated with milk fat yield, rumen acetate, butyrate, and isovalerate concentrations and with the probability of being in the low-risk acidosis group. More than one genomic marker was associated or tended to be associated with rumen isobutyrate and caproate concentrations, and the center log ratio of the phyla Bacteroidetes and Firmicutes and center log ratio of the families Prevotellaceae, BS11, S24-7, Acidaminococcaceae, Carnobacteriaceae, Lactobacillaceae, Leuconostocaceae, and Streptococcaceae. The provisional NTN4 gene, involved in several functions, had pleiotropy with 10 bacterial families, the phyla Bacteroidetes and Firmicutes, and butyrate. The ATP2CA1 gene, involved in the ATPase secretory pathway for Ca2+ transport, overlapped for the families Prevotellaceae, S24-7, and Streptococcaceae, the phylum Bacteroidetes, and isobutyrate. No genomic markers were associated with milk yield, fat percentage, protein yield, total solids, energy-corrected milk, somatic cell count, rumen pH, ammonia, propionate, valerate, total VFA, and d-, l-, or total lactate concentrations, or probability of being in the high- or medium-risk acidosis groups. Genome-wide associations with the rumen metabolome, microbial taxa, and milk composition were present across a wide geographical and management range of herds, suggesting the existence of markers for the rumen environment but not for acidosis susceptibility. The variation in pathogenesis of ruminal acidosis in the small population of cattle in the high risk for acidosis group and the dynamic nature of the rumen as cows cycle through a bout of acidosis may have precluded the identification of markers for acidosis susceptibility. Despite a limited sample size, this study provides evidence of interactions between the mammalian genome, the rumen metabolome, ruminal bacteria, and milk protein percentage.Item Characterizing ruminal acidosis risk: A multiherd, multicountry study(Elsevier, 2023-03) Golder, H. M.; LeBlanc, S.J.; Duffield, T.; Rossow, H.A.; Bogdanich, R.; Hernandez, L.; Block, E.; Rehberger, J.; Smith, A.H.; Thomson, J.A multicenter observational study was conducted on early lactation Holstein cows (n = 261) from 32 herds from 3 regions (Australia, AU; California, CA; and Canada, CAN) to characterize their risk of acidosis into 3 groups (high, medium, or low) using a discriminant analysis model previously developed. Diets ranged from pasture supplemented with concentrates to total mixed ration (nonfiber carbohydrates = 17 to 47 and neutral detergent fiber = 27 to 58% of dry matter). Rumen fluid samples were collected <3 h after feeding and analyzed for pH, and ammonia, d- and l-lactate, and volatile fatty acid (VFA) concentrations. Eigenvectors were produced using cluster and discriminant analysis from a combination of rumen pH, and ammonia, d-lactate, and individual VFA concentrations and were used to calculate the probability of the risk of ruminal acidosis based on proximity to the centroid of 3 clusters. Bacterial 16S ribosomal DNA sequence data were analyzed to characterize bacteria. Individual cow milk volume, fat, protein, and somatic cell count values were obtained from the closest herd test to the rumen sampling date (median = 1 d before rumen sampling). Mixed model analyses were performed on the markers of rumen fermentation, production characteristics, and the probability of acidosis. A total of 26.1% of the cows were classified as high risk for acidosis, 26.8% as medium risk, and 47.1% as low risk. Acidosis risk differed among regions with AU (37.2%) and CA (39.2%) having similar prevalence of high-risk cows and CAN only 5.2%. The high-risk group had rumen phyla, fermentation, and production characteristics consistent with a model of acidosis that reflected a rapid rate of carbohydrate fermentation. Namely, an acetate to propionate (1.98 ± 0.11), concentrations of valerate (2.93 ± 0.14 mM), milk fat to protein ratio (1.11 ± 0.047), and a positive association with abundance of phylum Firmicutes. The medium-risk group contains cows that may be inappetant or that had not eaten recently or were in recovery from acidosis. The low-risk group may represent cattle that are well fed with a stable rumen and a slower rumen fermentation of carbohydrates. The high risk for acidosis group had lower diversity of bacteria than the other groups, whereas CAN had a greater diversity than AU and CA. Rumen fermentation profile, abundance of ruminal bacterial phyla, and production characteristics of early lactation dairy cattle from 3 regions were successfully categorized in 3 different acidosis risk states, with characteristics differing between acidosis risk groups. The prevalence of acidosis risk also differed between regions.Item Soil bacterial community response to cover crop introduction in a wheat-based dryland cropping system(Frontiers Media SA, 2022-11) Eberly, Jed O.; Bourgault, Maryse; Dafo, Julia M.; Yeoman, Carl J.; Wyffels, Samuel A.; Lamb, Peggy F.; Boss, Darrin L.The incorporation of cover crops into cropping systems is important for enhancing soil health in agricultural systems. Soil microbes contribute to soil health by supplying key nutrients and providing protection against plant pests, diseases, and abiotic stress. While research has demonstrated the connection between cover crops and the soil microbiology, less is known regarding the impact of cover crops on the soil microbial community in semi-arid regions of the Northern Great Plains. Our objectives were to evaluate changes in the soil bacterial community composition and community networks in wheat grown after multi-species cover crops. Cover crops were compared to continuous cropping and crop/fallow systems and the effects of cover crop termination methods were also evaluated. Cover crops consisted of a cool season multispecies mix, mid-season multispecies mix, and a warm season multispecies mix, which were grown in rotation with winter wheat. A continuous cropping (wheat/barley) and wheat/fallow system were also included along with cover crop termination by grazing, herbicide application, and haying. Cover crop treatments and termination methods had no significant impact on microbial community alpha diversity. Cover crop termination methods also had no significant impact on microbial community beta diversity. Families belonging to the phyla Actinobacteria, Bacterioidota, and Proteobacteria were more abundant in the cool season cover crop treatment compared to the warm season cover crop treatment. Co-occurrence network analysis indicated that incorporation of cool season cover crops or mid-season mixes in a wheat-based cropping system led to greater complexity and connectivity within these microbial networks compared to the other treatments which suggests these communities may be more resilient to environmental disturbances.Item Climate mitigation potential and soil microbial response of cyanobacteria‐fertilized bioenergy crops in a cool semi‐arid cropland(Wiley, 2022-10) Gay, Justin D.; Goemann, Hannah M.; Currey, Bryce; Stoy, Paul C.; Christiansen, Jesper Riis; Miller, Perry R.; Poulter, Benjamin; Peyton, Brent M.; Brookshire, E. N. JackBioenergy carbon capture and storage (BECCS) systems can serve as decarbonization pathways for climate mitigation. Perennial grasses are a promising second-generation lignocellulosic bioenergy feedstock for BECCS expansion, but optimizing their sustainability, productivity, and climate mitigation potential requires an evaluation of how nitrogen (N) fertilizer strategies interact with greenhouse gas (GHG) and soil organic carbon (SOC) dynamics. Furthermore, crop and fertilizer choice can affect the soil microbiome which is critical to soil organic matter turnover, nutrient cycling, and sustaining crop productivity but these feedbacks are poorly understood due to the paucity of data from certain agroecosystems. Here, we examine the climate mitigation potential and soil microbiome response to establishing two functionally different perennial grasses, switchgrass (Panicum virgatum, C4) and tall wheatgrass (Thinopyrum ponticum, C3), in a cool semi-arid agroecosystem under two fertilizer applications, a novel cyanobacterial biofertilizer (CBF) and urea. We find that in contrast to the C4 grass, the C3 grass achieved 98% greater productivity and had a higher N use efficiency when fertilized. For both crops, the CBF produced the same biomass enhancement as urea. Non-CO2 GHG fluxes across all treatments were low and we observed a 3-year net loss of SOC under the C4 crop and a net gain under the C3 crop at a 0–30 cm soil depth regardless of fertilization. Finally, we detected crop-specific changes in the soil microbiome, including an increased relative abundance of arbuscular mycorrhizal fungi under the C3, and potentially pathogenic fungi in the C4 grass. Taken together, these findings highlight the potential of CBF-fertilized C3 crops as a second-generation bioenergy feedstock in semi-arid regions as a part of a climate mitigation strategy.Item Aboveground and belowground responses to cyanobacterial biofertilizer supplement in a semi-arid, perennial bioenergy cropping system(Wiley, 2021-08) Goemann, Hannah M.; Gay, Justin D.; Mueller, Rebecca C.; Brookshire, E. N. Jack; Miller, Perry; Poulter, Benjamin; Peyton, Brent M.The need for sustainable agricultural practices to meet the food, feed, and fuel demands of a growing global population while reducing detrimental environmental impacts has driven research in multi‐faceted approaches to agricultural sustainability. Perennial cropping systems and microbial biofertilizer supplements are two emerging strategies to increase agricultural sustainability that are studied in tandem for the first time in this study. During the establishment phase of a perennial switchgrass stand in SW Montana, USA, we supplemented synthetic fertilization with a nitrogen‐fixing cyanobacterial biofertilizer (CBF) and were able to maintain aboveground crop productivity in comparison to a synthetic only (urea) fertilizer treatment. Soil chemical analysis conducted at the end of the growing season revealed that late‐season nitrogen availability in CBF‐supplemented field plots increased relative to urea‐only plots. High‐throughput sequencing of bacterial/archaeal and fungal communities suggested fine‐scale responses of the microbial community and sensitivity to fertilization among arbuscular mycorrhizal fungi, Planctomycetes, Proteobacteria, and Actinobacteria. Given their critical role in plant productivity and soil nutrient cycling, soil microbiome monitoring is vital to understand the impacts of implementation of alternative agricultural practices on soil health.Item Comparison of Preservation and Extraction Methods on Five Taxonomically Disparate Coral Microbiomes(Frontiers Media SA, 2021-07) Pratte, Zoe A.; Kellogg, Christina A.All animals are host to a multitude of microorganisms that are essential to the animal’s health. Host-associated microbes have been shown to defend against potential pathogens, provide essential nutrients, interact with the host’s immune system, and even regulate mood. However, it can be difficult to preserve and obtain nucleic acids from some host-associated microbiomes, making studying their microbial communities challenging. Corals are an example of this, in part due to their potentially remote, underwater locations, their thick surface mucopolysaccharide layer, and various inherent molecular inhibitors. This study examined three different preservatives (RNAlater, DNA/RNA Shield, and liquid nitrogen) and two extraction methods (the Qiagen PowerBiofilm kit and the Promega Maxwell RBC kit with modifications) to determine if there was an optimum combination for examining the coral microbiome. These methods were employed across taxonomically diverse coral species, including deep-sea/shallow, stony/soft, and zooxanthellate/azooxanthellate: Lophelia pertusa, Paragorgia johnsoni, Montastraea cavernosa, Porites astreoides, and Stephanocoenia intersepta. Although significant differences were found between preservative types and extraction methods, these differences were subtle, and varied in nature from coral species to coral species. Significant differences between coral species were far more profound than those detected between preservative or extraction method. We suggest that the preservative types presented here and extraction methods using a bead-beating step provide enough consistency to compare coral microbiomes across various studies, as long as subtle differences in microbial communities are attributed to dissimilar methodologies. Additionally, the inclusion of internal controls such as a mock community and extraction blanks can help provide context regarding data quality, improving downstream analyses.