Application of animal manure to soils results in the introduction of manure-derived bacteria and their antimicrobial resistance genes (ARGs) into soils. ResCap is a novel targeted-metagenomic approach that allows the detection of minority components of the resistome gene pool without the cost-prohibitive coverage depths and can provide a valuable tool to study the spread of antimicrobial resistance (AMR) in the environment. We used high-throughput sequencing and qPCR for 16S rRNA gene fragments as well as ResCap to explore the dynamics of bacteria, and ARGs introduced to soils and adjacent water ditches, both at community and individual scale, over a period of three weeks. The soil bacteriome and resistome showed strong resilience to the input of manure, as manuring did not impact the overall structure of the bacteriome, and its effects on the resistome were transient. Initially, manure application resulted in a substantial increase of ARGs in soils and adjacent waters, while not affecting the overall bacterial community composition. Still, specific families increased after manure application, either through the input of manure (e.g., Dysgonomonadaceae) or through enrichment after manuring (e.g., Pseudomonadaceae). Depending on the type of ARG, manure application resulted mostly in an increase (e.g., aph(6)-Id), but occasionally also in a decrease (e.g., dfrB3) of the absolute abundance of ARG clusters (FPKM/kg or L). This study shows that the structures of the bacteriome and resistome are shaped by different factors, where the bacterial community composition could not explain the changes in ARG diversity or abundances. Also, it highlights the potential of applying targeted metagenomic techniques, such as ResCap, to study the fate of AMR in the environment.
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This study evaluated the performance of anaerobic co-digestion of cow manure (CM) and sheep manure (SM) in both batch and continuous digesters at 37 °C. Synergistic effects of co-digesting CM and SM at varying volatile solids (VS) ratios (1:0, 0:1, 3:1, 1:1, 1:3) were observed in the batch experiment, with the most effective degradation of cellulose (56%) and hemicellulose (55%), and thus, the highest cumulative methane yield (210 mL/gVSadded) obtained at a CM:SM ratio of 1:3. Co-digesting CM and SM improved the hydrolysis, as evidenced by the cellulase brought by SM and the increases of cellulolytic bacteria Clostridium. Besides, co-digestion enhanced the acidogenesis and methanogenesis, reflected by the enrichment of syntrophic bacteria Candidatus Cloacimonas and hydrogenotrophic archaea Methanoculleus (Coenzyme-B sulfoethylthiotransferase). When testing continuous digestion, the methane yield increased from 146 mL/gVS/d (CM alone) to 179 mL/gVS/d (CM:SM at 1:1) at a constant organic loading rate (OLR) of 1g VS/L/d and a hydraulic retention time (HRT) of 25 days. Furthermore, the anaerobic digestion process was enhanced when the daily feed changed back to CM alone, reflected by the improved daily methane yield (159 mL/VS/d). These results provided insights into the improvement of methane production during the anaerobic digestion of animal manure.
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Manure application can spread antimicrobial resistance (AMR) from manure to soil and surface water. This study evaluated the role of the soil texture on the dynamics of antimicrobial resistance genes (ARGs) in soils and surrounding surface waters. Six dairy farms with distinct soil textures (clay, sand, and peat) were sampled at different time points after the application of manure, and three representative ARGs sul1, erm(B), and tet(W) were quantified with qPCR. Manuring initially increased levels of erm(B) by 1.5 ± 0.5 log copies/kg of soil and tet(W) by 0.8 ± 0.4 log copies/kg across soil textures, after which levels gradually declined. In surface waters from clay environments, regardless of the ARG, the gene levels initially increased by 2.6 ± 1.6 log copies/L, after which levels gradually declined. The gene decay in soils was strongly dependent on the type of ARG (erm(B) < tet(W) < sul1; half-lives of 7, 11, and 75 days, respectively), while in water, the decay was primarily dependent on the soil texture adjacent to the sampled surface water (clay < peat < sand; half-lives of 2, 6, and 10 days, respectively). Finally, recovery of ARG levels was predicted after 29–42 days. The results thus showed that there was not a complete restoration of ARGs in soils between rounds of manure application. In conclusion, this study demonstrates that rather than showing similar dynamics of decay, factors such as the type of ARG and soil texture drive the ARG persistence in the environment.
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Biogas plays an important role in many future renewable energy scenarios as a source of storable and easily extracted form of renewable energy. However, there remains uncertainty as to which sources of biomass can provide a net energy gain while being harvested in a sustainable, ecologically friendly manner. This study will focus on the utilization of common, naturally occurring grass species which are cut during landscape management and typically treated as a waste stream. This waste grass can be valorized through co-digestion with cow manure in a biogas production process. Through the construction of a biogas production model based on the methodology proposed by (Pierie, Moll, van Gemert, & Benders, 2012), a life cycle analysis (LCA) has been performed which determines the impacts and viability of using common grass in a digester to produce biogas. This model performs a material and energy flow analysis (MEFA) on the biogas production process and tracks several system indicators (or impact factors), including the process energy return on energy investment ((P)EROI), the ecological impact (measured in Eco Points), and the global warming potential (GWP, measured in terms of kg of CO2 equivalent). A case study was performed for the village of Hoogkerk in the north-east Netherlands, to determine the viability of producing a portion of the village’s energy requirements by biogas production using biomass waste streams (i.e. common grass and cow manure in a co-digestion process). This study concludes that biogas production from common grass can be an effective and sustainable source of energy, while reducing greenhouse gas emissions and negative environmental impacts when compared to alternate methods of energy production, such as biogas produced from maize and natural gas production.
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A bioaugmentation approach was used to enhance the performance of anaerobic digestion (AD) using cow manure (CM) as the substrate in a continuous system. To obtain the desirable microbial culture for bioaugmentation, a biochemical methane potential test (BMP) was used to evaluate three commonly used inocula namely (1) municipal solid waste (MSW), (2) wastewater treatment plant (WWTP), and (3) cow manure digester (CMMD) for their hydrolytic capacity. The highest lignocellulose removal (56% for cellulose and 50% for hemicellulose) and the most profusion of cellulolytic bacteria were obtained when CM was inoculated with CMMD. CMMD was thus used as the seed inoculum in a continuously operated reactor (Ra) with the fiber fraction of CM as the substrate to further enrich cellulolytic microbes. After 100 days (HRT: 30 days), the Bacteria fraction mainly contained Ruminofilibacter, norank_o_SBR1031, Treponema, Acetivibrio. Surprisingly, the Archaea fraction contained 97% ‘cellulolytic archaea’ norank_c_Bathyarchaeia (Phylum Bathyarchaeota). This enriched consortium was used in the bioaugmentation experiment. A positive effect of bioaugmentation was verified, with a substantial daily methane yield (DMY) enhancement (24.3%) obtained in the bioaugmented reactor (Rb) (179 mL CH4/gVS/d) than that of the control reactor (Rc) (144 mL CH4/gVS/d) (P < 0.05). Meanwhile, the effluent of Rb enjoyed an improved cellulose reduction (14.7%) than that of Rc, whereas the amount of hemicellulose remained similar in both reactors' effluent. When bioaugmentation stopped, its influence on the hydrolysis and methanogenesis sustained, reflected by an improved DMY (160 mL CH4/gVS/d) and lower cellulose content (53 mg/g TS) in Rb than those in Rc (DMY 144 mL/CH4/gVS/d and cellulose content 63 mg/g TS, respectively). The increased DMY of the continuous reactor seeded with a specifically enriched consortium able to degrade the fiber fraction in CM shows the feasibility of applying bioaugmentation in AD of CM.
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The main objectives of this review were to: (1) review different methods/techniques to assess gaseous N-losses from manure (2) review N-gaps, attributed to dinitrogen loss as the difference between directly measured N compounds summed as total N loss and indirectly measured N loss through a mass balance in livestock manure systems, and (3) provide approaches to close the N-gap. In literature, N-gaps run up to 80% of total N loss, this undermines N emission assessments and leaves a huge part of the emission unexplained. However, studies that measure N-gaps are scarcely available or are limited in their evaluation, hence more study is needed. Three approaches are introduced to research N-gaps: (1) measure N2 through a suggested Gas Flow Soil Core (GFSC) technique and compare the sum of all measured N losses with the indirect method, (2) assume N2 loss as being the N-gap and (3) include N2 as an estimate based on ratios from literature. In a hypothetical example for poultry manure, assumed values for measurement error of 50% and variance due to physical differences between the experimental units of 50% led to a total standard deviation of 131% in the N-gap. Variance of N-gap was reduced with 80% point when assuming 16 vessels compared to single vessel. Using literature-based-ratios to estimate losses of N compounds led to variation of N-gap from 0.06% initial N overestimation to 26% of initial N underestimation. Future research should address this variance and apply methods to measure N2 to close N-gaps.
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The quantification and identification of new plasmid-acquiring bacteria in representative mating conditions is critical to characterize the risk of horizontal gene transfer in the environment. This study aimed to quantify conjugation events resulting from manure application to soils and identify the transconjugants resulting from these events. Conjugation was quantified at multiple time points by plating and flow cytometry, and the transconjugants were recovered by fluorescence-activated cell sorting and identified by 16S rRNA sequencing. Overall, transconjugants were only observed within the first 4 days after manure application and at values close to the detection limits of this experimental system (1.00–2.49 log CFU/g of manured soil, ranging between 10–5 and 10–4 transconjugants-to-donor ratios). In the pool of recovered transconjugants, we found amplicon sequence variants (ASVs) of genera whose origin was traced to soils (Bacillus and Nocardioides) and manure (Comamonas and Rahnella). This work showed that gene transfer from fecal to soil bacteria occurred despite the less-than-optimal conditions faced by manure bacteria when transferred to soils, but these events were rare, mainly happened shortly after manure application, and the plasmid did not colonize the soil community. This study provides important information to determine the risks of AMR spread via manure application.
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The 'AgroCycle' project investigates whether a cooperation of farms can become self-sufficient in energy and fertilization by using manure and organic waste streams for the production of energy, green fuel and green fertilizers by means of anaerobic digestion (AD). In the project, the project partners aim to link the nutrient cycle (from manure to digestate to green fertilizer) to a self-sufficient energy system (biomass to biogas to green fuel for processing the land) through the combined production of biogas and green fertilizers. The financial feasibility of a bio-digester is highly dependent on the use and economic value of the digestate. This combined approach increases both feasibility and sustainability (environmental impacts and CO2 emissions). To explore the feasibility of the aforementioned concept, use is made of the existing 'BioGas simulator' model developed by Hanze UAS to simulate the technical process of decentralized production of biogas and the economic cost.
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Agricultural by-products, that is primary residue, industrial by-products and animal manure, are an important source of nutrients and carbon for maintaining soil quality and crop production but can also be valorised through treatment pathways such as fermentation, incineration or a combination of these called bio-refinery. Here, we provide an overview of opportunity to reduce environmental impact of valorising agricultural by-products. We estimate the available by-products in Northwestern Europe as a case study and the maximum and realistic greenhouse gas reduction potentials. Availability, collectability, the original use and environmental impact including land use changes, soil carbon sequestration and pollution swapping are discussed as critical factors when valorising agricultural by-products.
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The dairy sector in the Netherlands aims for a 30% increase in efficiency and 30% carbon dioxide emission reduction compared to the reference year of 1990, and a 20% share of renewable energy, all by the year 2020. Anaerobic Digestion (AD) can play a substantial role in achieving these aims. However, results from this study indicate that the AD system is not fully optimized in combination with farming practices regarding sustainability. Therefore, the Industrial Symbiosis concept, combined with energy and environmental system analysis, Life Cycle Analysis and modeling is used to optimize a farm-scale AD system on four indicators of sustainability (i.e., energy efficiency, carbon footprint, environmental impacts and costs). Implemented in a theoretical case, where a cooperation of farms share biomass feedstocks, a symbiotic AD system can significantly lower external energy consumption by 72 to 92%, carbon footprint by 71 to 91%, environmental impacts by 68 to 89%, and yearly expenditures by 56 to 66% compared to a reference cooperation. The largest reductions and economic gains can be achieved when a surplus of manure is available for upgrading into organic fertilizer to replace fossil fertilizers. Applying the aforementioned symbiotic concept to the Dutch farming sector can help to achieve the stated goals indicated by the Dutch agricultural sector for the year 2020.
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