Mesophilic Anaerobic Digestion Research Articles

Mesophilic Anaerobic Digestion of Fruit and Vegetable Waste: Single-Versus Two-Stage Reactor, and Modeling of the Liquid and Solid Fractions of the Residue

Mesophilic Anaerobic Digestion of Fruit and Vegetable Waste: Single-<i>Versus</i> Two-Stage Reactor, and Modeling of the Liquid and Solid Fractions of the Residue

Unveiling antibiotic resistance dynamics in single and two-stage anaerobic digestion of dairy cow manure: Implications for environmental health

This study investigated antibiotic-resistant bacteria (ARB) survival during mesophilic anaerobic digestion (AD) of dairy cow manure with cefazolin (CEZ) antibiotic. Comparing single- and two-stage AD systems, we assessed ARB removal rates, biogas yield, volatile fatty acid concentration, and microbial populations. The results revealed that in the two-stage AD system, the methanogenic phase (MP) exhibited the highest removal rates of CEZ-resistant (CEZ-r) and oxytetracycline-resistant (OTC-r) bacteria at 9–16% and 69–72%, respectively. However, the MP had a higher proportion of ARB among the total culturable bacteria (24% to 30%) compared to the acidogenic phase (AP) and single-stage AD. CEZ negatively impacted ARB removal rates and biogas production in both systems, with a more pronounced effect in single-stage AD. Biogas yield in the two-stage AD ranged from 337.7–385.6mL/gVS, which was 10–20% higher than that of the single-stage AD, regardless of CEZ addition. The reduction in biogas production due to CEZ was primarily attributed to the suppression of VFA production. As for microbial populations, the changes in the percentages of Firmicutes, Bacteroidetes, and Proteobacteria were likely related to the variations in ARB numbers, and the abundance of Methanosaetaceae significantly influenced biogas production.

Enhancing anaerobic digestion of lignocellulosic biomass by mechanical cotreatment

BackgroundThe aim of this study was to increase the accessibility and accelerate the breakdown of lignocellulosic biomass to methane in an anaerobic fermentation system by mechanical cotreatment: milling during fermentation, as an alternative to conventional pretreatment prior to biological deconstruction. Effluent from a mesophilic anaerobic digester running with unpretreated senescent switchgrass as the predominant carbon source was collected and subjected to ball milling for 0.5, 2, 5 and 10 min. Following this, a batch fermentation test was conducted with this material in triplicate for an additional 18 days with unmilled effluent as the ‘status quo’ control.ResultsThe results indicate 0.5 – 10 min of cotreatment increased sugar solubilization by 5– 13% when compared to the unmilled control, with greater solubilization correlated with increased milling duration. Biogas concentrations ranged from 44% to 55.5% methane with the balance carbon dioxide. The total biogas production was statistically higher than the unmilled control for all treatments with 2 or more minutes of milling (α = 0.1). Cotreatment also decreased mean particle size. Energy consumption measurements of a lab-scale mill indicate that longer durations of milling offer diminishing benefits with respect to additional methane production.ConclusionsCotreatment in anaerobic digestion systems, as demonstrated in this study, provides an alternative approach to conventional pretreatments to increase biogas production from lignocellulosic grassy material.

Use of lyophilized and acclimated digestate dominated by Methanobrevibacter as a start-up inoculum in anaerobic digester led to higher methane production in biochemical methane potential assays

Transporting wet inoculum for full-scale anaerobic digester (AD) start-up is usually infeasible and costly, especially, for remote locations. To overcome these burdens lyophilized AD inoculum is thought to be used after on-site acclimation. For this reason, in this study, the impact of three different acclimated lyophilized AD inoculums collected from full-scale mesophilic AD installations treating different feedstocks was tested for 20 days to monitor AD start-up and methane production by using biochemical methane potential (BMP) assays. The lyophilized inoculums after acclimation were fed to corresponding triplicate digesters treating similar feedstocks as digestate (DG), waste activated sludge (WAS) plus landfill leachate (LL) and WAS, LL plus food waste from municipal solid waste (FWMSW). As a control, no inoculum added digesters with three different feedstocks collected freshly from full-scale mesophilic AD installations treating DG, WAS + LL and WAS + LL + FWMSW were also run in triplicates. All the digesters displayed enhanced methane production in two days of the incubation, the digesters fed with DG as an inoculum displayed shortened start-up and the highest methane production with 42.77 % comparing to control. BMP assays of the other two inoculums tested also displayed 4.73 % enhanced methane production for WAS plus LL and 4.51 % enhanced methane production for WAS, LL plus FWMSW comparing to their corresponding controls. Metagenome analyses of the inoculums used revealed that the dominant methanogens were Methanobacteriaceae (100 % Methanobrevibacter) for DG, %33 Methanosaetaceae (%100 Methanothrix) and %27 Methanobacteriaceae (%71 Methanobrevibacter and %29 Methanosphaera) for WAS + LL, %35 Methanosaetaceae (%100 Methanothrix) and %30 Methanobacteriaceae (%91 Methanobrevibacter and %9 Methanosphaera) for WAS + LL + FWMSW. The lyophilized DG dominated by hydrogenotrophic genus Methanobrevibacter seems to be promising inoculum after acclimation, however, its efficiency needs to be further analysed for the ADs treating various feedstocks.

Denaturing Gradient Gel Electrophoresis Approach for Microbial Shift Analysis in Thermophilic and Mesophilic Anaerobic Digestions.

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.

Biogas Production Potential of Mixed Banana and Pineapple Waste as Assessed by Long-Term Laboratory-Scale Anaerobic Digestion

Biogas is a renewable energy source generated through the anaerobic digestion (AD) of organic feedstocks. This study aims to quantify the biogas production potential (BPP) of fruit wastes via semi-continuous lab-scale mesophilic AD over a total of 100 days. The feed was composed of 80% banana peelings and 20% pineapple residues, mimicking the waste composition of a Costa Rican fruit processing facility used as a test case. The average loading rate of volatile suspended solids (VSS) corresponded to 3.6 kg VSS·m−3·d−1. Biogas yield and composition were monitored, along with the concentration of ammonium, volatile fatty acids, and pH. Discounting the start-up phase, the BPP averaged to 526 LN (kg VSS)−1 with a methane concentration of around 54%, suggesting suitability of the substrate for AD. We calculated that if upscaled to the Costa Rican test case facility, these values translate into a gross average heat and electricity production via AD of around 5100 MWhel·a−1 and 5100 MWhth·a−1, respectively. Deducting self-consumption of the AD treatment, this is equivalent to 73% of the facility’s electricity demand, and could save about 450,000 L of heavy oil per year for heat generation. To circumvent nitrogen shortage, the addition of a co-substrate such as dry manure seems advisable.

Alkali, thermal, or thermo-alkali pre-treatment to improve the anaerobic digestion of poly(lactic acid)?

Replacing petroleum-based plastics with biodegradable polymers is a major challenge for modern society especially for food packaging applications. To date, poly(lactic acid) represents 25 % of the total biodegradable plastics and it is estimated that, in the future, it could become the main contributor to the biodegradable plastics industry. Anaerobic digestion is an interesting way for the poly(lactic acid) end of life, even if its biodegradability is limited in mesophilic conditions. The aims of this study were to identify the best pre-treatment for maximizing the methane yield, minimizing the anaerobic digestion duration and limiting residual plastic fragments in the digestate. A systematic comparison was carried out between thermal, chemical, and thermo-chemical pre-treatment. Pre-treatment with 4 M KOH for 48 h at 35°C was effective in improving the mesophilic anaerobic digestion of the poly(lactic acid). Such pre-treatment allows obtaining 90 % of the theoretical methane potential, in 24 - 30 days. Importantly, such pre-treatment completely solubilized the poly(lactic acid), leaving no solid residues in the digestate. In addition, using KOH permits to avoid the sodication of the soil due to the digestate application as fertilizer.

Ammonia stripping by in situ biogas self-circulation to upgrade continuous thermophilic and mesophilic digestion of hydrothermal high-solid sludge

High-solid anaerobic digestion of hydrothermal sewage sludge has been developed. In order to upgrade the process by focusing on ammonia inhibition, a simply-equipped stripping system without additional alkali or heat supply was introduced by in situ biogas self-circulation. As the determined limit of total ammonia nitrogen at 1500 mg/L and 1000 mg/L for the mesophilic (MAD) and thermophilic anaerobic digestion (TAD) respectively and stripping rate at 5 L/min, continuous MAD and TAD was conducted in parallel. The stripping system successfully polished up the ammonia inhibition, and methanogenic capability of the TAD was promoted to approximately 90.0 % of the potential. Intermittent stripping mode proved usable. More frequent stripping was inevitable for the TAD as compared to the MAD. Hydraulic retention time below 20 d resulted in failure of the stripping mode due to rapid ammonia generation. Overall, this technology was practical in upgrading high-solid sludge digestion by effective ammonia control.

Archaeal community composition as key driver of H2 consumption rates at the start-up of the biomethanation process

The performance of hydrogen consumption by various inocula derived from mesophilic anaerobic digestion plants was evaluated under ex situ biomethanation. A panel of 11 mesophilic inocula was operated at a concentration of 15 gVS.L−1 at a temperature of 35 °C in batch system with two successive injections of H2:CO2 (4:1 mol:mol). Hydrogen consumption and methane production rates were monitored from 44 h to 72 h. Hydrogen consumption kinetics varies significantly based on the inoculum origin, with no accumulation of volatile fatty acids. Microbial community analyses revealed that microbial indicators such as the increase in Methanosarcina sp. abundance and the increase of the Archaea/Bacteria ratio were associated to high initial hydrogen consumption rates. The improvement in the hydrogen consumption rate between the two injections was correlated with the enrichment in hydrogenotrophic methanogens. This work provides new insights into the early response of microbial communities to hydrogen injection and on the microbial structures that may favor their adaptation to the biomethanation process.

Different effects of mesophilic and thermophilic anaerobic digestion on subsequent pyrolysis: Characteristics, kinetics and thermodynamic analysis

Anaerobic digestion (AD) combined with pyrolysis has been used as an innovative sludge-to-energy process that enhances energy recovery. This study investigated the different impacts of mesophilic and thermophilic AD on subsequent pyrolysis. The pyrolysis characteristics, kinetics, and thermodynamics of raw sludge (RS) and digested sludge (DS) after both mesophilic and thermophilic AD were investigated using thermogravimetric analysis (TGA) and three kinetic models (Starink, Flynn–Wall–Ozawa, Kissinger–Akahira–Sunose). TGA results indicated that thermophilic AD reduced the weight loss from 71.2-75.6 % for RS to 55.0–59.1 % for DS, more considerably than mesophilic AD (reducing from 70.6-75.2 % to 61.3–69.5 %), indicating higher organic matter removal. Kinetic analysis showed that the activation energy (E) of RS and DS pyrolysis ranged from 128.9 to 399.9 kJ/mol and 117.3 to 470.4 kJ/mol, respectively. Mesophilic AD generally increased the E, while thermophilic AD did not markedly alter E at low conversion rates (0.2–0.5) but increased it at high conversion rates (0.6–0.8), changing from 203.7–399.9 kJ/mol for RS to 248.9–470.4 kJ/mol for DS. Thermodynamic analysis showed that mesophilic AD generally enhanced pyrolysis favorability and reactivity but impeded activated complex formation. Thermophilic AD enhanced favorability and favored complex formation at low conversion rates but hindered it at high conversion rates. Reactivity varied with thermophilic AD extent, with excessive AD reducing reactivity. Pyrolysis–gas chromatography/mass spectrometry analysis confirmed that AD, on the one hand, decomposed biodegradable organics into low-molecular-weight organics, retained them in the DS, and facilitated subsequent pyrolysis. On the other hand, AD could accumulate large bio-refractory molecules, rendering DS less prone to pyrolytic decomposition. This effect of thermophilic AD was more significant than that of mesophilic AD. Given more favorable change was observed in kinetics and thermodynamics at lower conversion rates, low-temperature pyrolysis to produce biochar might be a promising strategy for DS treatment. The insights gained in this study may guide the optimization of AD conditions to maximize the efficiency of subsequent pyrolysis, providing more insight into the thermochemical conversion of DS and pyrolysis design.

Semi-continuous mesophilic anaerobic digestion of date palm (Phoenix dactelifyra L.) leaflets of the H'mira cultivar from the Adrar region of Southern Algeria using an alkaline pre-treatment

This research paper aim to investigate the enhancement potential of mesophilic Anaerobic Digestion (AD) of Algerian date palm (Phoenix dactylifera L.) leaflets from H'mira cultivars using an alkaline pretreatment approach. The experiment was conducted for the first time in semi-continuous digesters. NaOH was applied at various percentages (6 %, 12 % and 18 %) to chemically pretreat the substrate for 5 days at 37 °C. The chemical pretreatment results were interpreted for the first time with in-depth FTIR spectroscopic analyses. The Hydraulic Retention Time (HRT) was fixed at 15 days, and the Organic Loading Rate (OLR) at 2 g MO/L/day. The results showed that the methane (CH4) production values were stabilized with a clear distinction at averages of 211.85, 177.26, 143.36 and 123.07 ml of CH4/day, i.e. a CH4 yield of 105.92, 88.63, 71.68 and 61.53 ml of CH4/gVS/day for the reactors containing substrates pre-treated with 12 %, 6 %, 18 % NaOH and the substrate without pre-treatment respectively. Compared to the substrate without pre-treatment, the CH4 production rates were improved 1.72, 1.44 and 1.16 times for the reactors containing 12 %, 6 % and 18 % NaOH respectively. These findings were perfectly in accordance with FTIR spectroscopy results. These findings demonstrate that the semi-continuous AD process can be an attractive energy recovery solution from such waste compared to batch digesters, (on one day, it produce 77–105 % of the CH4 produced during 21–55 days in batch digesters), as well as the importance of adequate pre-treatment prior to the AD. FTIR Spectroscopic analysis technology can also be widely used to analyze pre-treatment data for lignocellulosic biomass, as it gives very encouraging results.

Anaerobic Co-Digestion of Agro-Industrial Waste Mixtures for Biogas Production: An Energetically Sustainable Solution

In a climate crisis, searching for renewable energy sources is urgent and mandatory to achieve a low-carbon society. The food industry is an attractive source for providing different organic waste with great potential for energy generation, avoiding the environmental impacts of its inadequate management at the disposal stage. This manuscript determines the feasibility of using three agro-industrial byproducts for biogas production with a mesophilic anaerobic digestion process. Three mixture samples such as tomato pulp with olive cake (TP-OC), apple pomace with olive cake (AP-OC), and tomato pulp with apple pomace (TP-AP) at a 1:1 w/w ratio were evaluated using bovine manure as inoculum. During 7 to 12 days of operation, results indicate that TP-OC achieved the highest biogas production yield with 1096 mL/L (with up to 70% methane), followed by AP-OC and TP-AP with 885 (62% methane) and 574 mL/L (69% methane), respectively. Experimentally, TP-OC consistently encompassed the highest biogas and methane production and fit the kinetic models, whereas the modified Gompertz model produced the best fit (R2 = 99.7%). This manuscript supports the preference for mixing byproducts from the agro-industrial sector rather than using them individually for biogas production.

Effect of the Inoculum-to-Substrate Ratio on Putative Pathogens and Microbial Kinetics during the Batch Anaerobic Digestion of Simulated Food Waste.

The effects of the inoculum (anaerobic digestion effluent) to substrate (simulated food waste) ratio (ISR) 4.00 to 0.25 on putative pathogens and microbial kinetics during batch mesophilic anaerobic digestion were investigated. Red fluorescent protein labelled (RFPAKN132) Escherichia coli JM105 was introduced as a marker species, and together with the indigenous Clostridium sp., Enterococcus sp., Escherichia coli, and total coliforms were used to monitor pathogen death kinetics. Quantitative polymerase chain reaction was also used to estimate the bacterial, fungal, and methanogenic gene copies. All the ISRs eliminated E. coli and other coliforms (4 log10 CFU/mL), but ISR 0.25 achieved this within the shortest time (≤2 days), while ISR 1.00 initially supported pathogen proliferation. Up to 1.5 log10 CFU/mL of Clostridium was reduced by acidogenic conditions (ISR 0.25 and 0.50), while Enterococcus species were resistant to the digestion conditions. Fungal DNA was reduced (≥5 log10 copies/mL) and was undetectable in ISRs 4.00, 2.00, and 0.50 at the end of the incubation period. This study has demonstrated that ISR influenced the pH of the digesters during batch mesophilic anaerobic digestion, and that acidic and alkaline conditions achieved by the lower (0.50 and 0.25) and higher (4.00 and 2.00) ISRs, respectively, were critical to the sanitisation of waste.

Temperature-phased anaerobic sludge digestion effectively removes antibiotic resistance genes in a full-scale wastewater treatment plant

Sludge is a major by-product and the final reservoir of antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs). Temperature-phased anaerobic digestion (TPAD), consisting of thermophilic anaerobic digestion (AD) (55 °C) and mesophilic AD processes (37 °C), has been implemented in WWTPs for sludge reduction while improving the biomethane production. However, the impact of TPAD on the ARGs' fate is still undiscovered in lab-scale experiments and full-scale WWTPs. This study, for the first time, investigated the fate of ARGs during the TPAD process across three seasons in a full-size WWTP. Ten typical ARGs and one integrase gene of class 1 integron (intI1) involving ARGs horizontal gene transfer were examined in sludge before and after each step of the TPAD process. TPAD reduced aac(6′)-Ib-cr, blaTEM, drfA1, sul1, sul2, ermb, mefA, tetA, tetB and tetX by 87.3–100.0 %. TPAD reduced the overall average absolute abundance of targeted ARGs and intI1 by 92.39 % and 92.50 %, respectively. The abundance of targeted ARGs in sludge was higher in winter than in summer and autumn before and after TPAD. During the TPAD processes, thermophilic AD played a major role in the removal of ARGs, contributing to >60 % removal of ARGs, while the subsequent mesophilic AD contributed to a further 31 % removal of ARGs. The microbial community analysis revealed that thermophilic AD reduced the absolute abundance of ARGs hosts, antibiotic resistant bacteria. In addition, thermophilic AD reduced the abundance of the intI1, while the intI1 did not reproduce during the mesophilic AD, also contributing to a decline in the absolute abundance of ARGs in TPAD. This study demonstrates that TPAD can effectively reduce the abundance of ARGs in sludge, which will suppress the transmission of ARGs from sludge into the natural environment and deliver environmental and health benefits to our society.

System stability associated with different lipid contents during mesophilic anaerobic digestion of lipid-rich food waste

Lipid content is an essential indicator which affects the operation performance and efficiency of an anaerobic digestion system. This study investigated the relationship between system stability and lipid degradation during mesophilic anaerobic digestion process of lipid-rich food waste. As the experiment designed, the hydraulic retention time was 30 days and influent total solid (TS) concentration of the substrates was kept at 10 %, in which lipid content gradually increased from 10 % to 60 % (on the TS basis). The anaerobic digestion process proceeded successfully, achieving a methane yield of 587 mL/g-VS (volatile solids) at lipid/TS ratio of 50 %. However, the system collapsed after the lipid/TS ratio exceeded 50 %. It was found that the accumulation of long-chain fatty acid was not the primary factor leading to system failure, as the total concentration remained below 500 mg/L even at lipid/TS ratio of 60 %. Instead, the C/N ratios resulting from different lipid contents significantly impacted system stability. As lipid/TS ratio increased to 60 %, the C/N ratio of substrate reached 45.47, causing system failure due to an efficiency imbalance between alkalinity production and VFAs accumulation. The mechanism was illustrated by carbon and nitrogen distributions. Additionally, the synergistic effect between lipids and food waste was also discussed, peaking at lipid/TS ratio of 40 %. These results highlighted that the C/N ratio had more prominent impacts on system stability than long-chain fatty acids accumulation during the designed lipid-rich food waste anaerobic digestion process.

Anaerobic Treatment of Food Waste with Biogas Recirculation under Psychrophilic Temperature

Food waste has emerged as a pressing concern, and thus advanced techniques to valorize food waste into nutrition rich materials as well as renewable energy are highly important. The exceptional biodegradability of food waste renders it a highly suitable substrate for anaerobic treatment. This leads to energy production and a reduction in the carbon footprint. Nevertheless, in frigid territories like Canada, the conventional mesophilic anaerobic digestion at 30–40 °C can require substantial amounts of energy. Consequently, this study introduces a new approach to treat food waste at psychrophilic temperatures (1–20 °C). Lower temperatures can negatively impact cellular processes during anaerobic treatment, rendering substrates less accessible to microscopic organisms. To address this challenge associated with lower temperatures, the study introduces an innovative biogas recirculation strategy. The primary objectives of this study are to assess the viability of anaerobic treatment for food waste at psychrophilic temperatures and to investigate the effectiveness of reintroduction of the produced biogas to the anaerobic system in enhancing biomethane generation and stability of the system. Batch experiments were conducted on food waste in various assessments, both with and without biogas recirculation. The outcomes revealed a methane concentration ranging from 68% to 93% when biogas recirculation was employed, whereas without this technique, methane concentration varied between 10% and 45%. Moreover, with biogas recirculation, the reduction in volatile solids reached a maximum of 92%, and there was an 82% decrease in chemical oxygen demand. In conclusion, the utilization of the recirculation of biogas at the psychrophilic temperature range enhanced biomethane production and reduction of volatile solids and chemical oxygen demand. This study underscores the potential of employing anaerobic treatment with reintroduction of produced biogas into the system in cold regions as an economically viable and sustainable choice for treating food waste with nominal energy consumption.

Metagenomic comparison of effects of mesophilic and thermophilic manure anaerobic digestion on antimicrobial resistance genes and mobile genetic elements

This study evaluated the long-term effects of mesophilic (MAD) and thermophilic (TAD) anaerobic digestion on the fate and evolution of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in cattle manure following a metagenomic approach. The results indicated that MAD and TAD lowered ARG levels in fresh cattle manure by over 50 % and MGEs by over 65 %. However, no statistically significant difference was observed between the MAD and TAD processes in terms of their ability to reduce ARG and MGE. Co-occurrence analysis suggested facultative anaerobic species belonging to the Bacillota and Actinomycetota phyla as the primary potential hosts of ARGs and MGEs. Analyses of co-assembled contigs revealed the presence of transposon and toxin-antitoxin systems in close proximity to ARGs typically found in anaerobic digesters. The identification of these systems near ARGs is particularly significant, as it highlights a potential mechanism for the persistence and spread of ARGs in such environments. Overall, the results indicated that digested cattle manure (whether under mesophilic or thermophilic conditions) would pose a much lower risk of dissemination of antimicrobial resistance than untreated manures. Since TAD did not outperform MAD at reducing ARGs and MGEs, thermophilic temperatures may not be necessary to improve ARG reduction rates in cattle manure AD. These valuable insights could help develop strategies to reduce the dissemination of antibiotic resistance due to manure treatment and disposal.

Adaptation of Anaerobic Digestion Microbial Communities to High Ammonium Levels: Insights from Strain-Resolved Metagenomics.

Ammonia release from proteinaceous feedstocks represents the main inhibitor of the anaerobic digestion (AD) process, which can result in a decreased biomethane yield or even complete failure of the process. The present study focused on the adaptation of mesophilic AD communities to a stepwise increase in the concentration of ammonium chloride in synthetic medium with casein used as the carbon source. An adaptation process occurring over more than 20 months allowed batch reactors to reach up to 20 g of NH4+ N/L without collapsing in acidification nor ceasing methane production. To decipher the microbial dynamics occurring during the adaptation and determine the genes mostly exposed to selective pressure, a combination of biochemical and metagenomics analyses was performed, reconstructing the strains of key species and tracking them over time. Subsequently, the adaptive metabolic mechanisms were delineated by following the single nucleotide variants (SNVs) characterizing the strains and prioritizing the associated genes according to their function. An in-depth exploration of the archaeon Methanoculleus bourgensis vb3066 and the putative syntrophic acetate-oxidizing bacteria Acetomicrobium sp. ma133 identified positively selected SNVs on genes involved in stress adaptation. The intraspecies diversity with multiple coexisting strains in a temporal succession pattern allows us to detect the presence of an additional level of diversity within the microbial community beyond the species level.

Biodegradability of PBAT/PLA coated paper and bioplastic bags under anaerobic digestion

Poly (lactic acid) (PLA) and Poly(butylene adipate-co-terephthalate) (PBAT) are two of biodegradable plastics with the highest production capacities in 2021. Bioplastic waste management can be easily integrated with organic waste management, especially when bioplastics are used as food packaging material, since they are potentially biodegradable. The aim of this study was to assess the biodegradability of biodegradable polymer-coated paper (BPCP) and bioplastic bags made from PBAT/PLA blend during mesophilic and thermophilic anaerobic digestion (AD) and to reveal the changes in the physicochemical properties of the bioplastics. BPCP obtained 155 NmL-CH4/g VS and 307.3 NmL-CH4/g VS under mesophilic and thermophilic conditions, respectively, but left bioplastic film residues. The bioplastic bags did not exhibit significant biodegradation during the AD processes. 1H NMR results indicated that the ratio of PLA to PBAT decreased significantly after AD of the BPCP film and that PLA monomers were formed from the bioplastic bags, leading to a decrease in the hydrophobicity on the surfaces of the materials. Methanoculleus was found to be enriched on the bioplastic surface after mesophilic AD. From the perspective of coupling bioplastic waste management with the food waste management, the incorporation of BPCP into the AD reactor not only enhances system stability and methane production to a greater extent than biodegradable plastic bags but also raises concerns regarding the residual biofilm when utilizing the digestate for direct land applications.

Influence of three different manure treatments on antimicrobial resistance genes and mobile genetic elements

There is a growing concern about the potential dissemination of antimicrobial resistance into agricultural fields due to the application of manure as crop fertilizer. While reducing the usage of antibiotics in livestock production stands as the first alternative to prevent this problem, there is evidence that this may not be enough to eliminate antimicrobial resistance elements already present in manure microbiomes. This study employed a metagenomic approach to investigate the impact of common manure treatments, including aerobic storage, mesophilic anaerobic digestion (MAD), and solid-liquid separation, on the presence and abundance of antimicrobial resistance genes (ARGs), bactericides, and heavy metal resistance genes (BacMet), and mobile genetic elements (MGEs) in manure from three different farms, including one operating in an antibiotic-free environment. The results indicated that MAD was the best method to reduce the numbers of ARGs, BacMet, and MGEs, achieving reduction rates greater than 40%, 89%, and 68%, respectively. Manure storage significantly reduced BacMet levels (over 30%) and MGEs (28%) but had no significant effect on total ARG levels. Solids recovered through solid-liquid separation exhibited elevated levels of ARGs, BacMet, and MGEs, while the liquid fraction displayed levels similar to untreated manures. Correlation and co-occurrence modeling analyses indicated that changes in microbial communities, particularly fluctuations in aerobic and facultative communities belonging to Bacillota, Actinomycetota, and Pseudomonadota phyla, played a significant role in driving changes in ARGs, BacMet, and MGEs. The results also showed the presence of toxin-antitoxin and transposon systems near different ARGs. Overall, the results confirmed that genes conferring resistance to various antimicrobials and MGE capable of mobilizing them are widely spread in dairy farms; that even under the absence of antibiotics, the use of heavy metals and disinfectants may promote the maintenance of ARGs and MGEs, and; that treatment such as anaerobic digestion could reduce the risk of the spread of antimicrobial resistance.