Mesophilic Anaerobic Digestion Research Articles

Thermophilic and mesophilic anaerobic digestion of soybean molasses: A performance vs. stability trade-off

One of the factors that has a direct impact on anaerobic digestion is the applied organic loading rate (OLRA). Increasing OLRA can boost methane production but can also cause process failure. As a result, establishing the appropriate OLRA for the procedure is critical. This study evaluated the effect of increasing the OLRA using soybean molasses in a thermophilic anaerobic reactor (R-Thermo), as well as the effect of feeding strategy and co-processing with okara. Furthermore, the performance versus stability trade-off between R-Thermo and mesophilic anaerobic digestion (R-Meso) was investigated. The increase of OLRA from 10 to 15 and 20 kg-COD/m³/d led to a decrease in COD removal efficiency (90, 86, and 75%), methane yield (12.0, 11.6, and 9.9 mol-CH4/kg-COD) and an increase in total volatile acids concentration (251, 456, and 1393 mg-HAc/L, respectively). At 15 kg-COD/m³/d, R-Meso performed similarly to R-Thermo, and at 20 kg-COD/m3/d, R-Meso outperformed (81% COD removal efficiency, 9.3 mol-CH4/kg-CODrem and 154.5 mol-CH4/m3/d). Temperature greatly influenced the distribution of metabolic pathways, as shown by thermodynamic and kinetic analyses, thus impacting bacterial diversity. At 55 °C, amongst the bacterial genera, Tepidiphilus stood out (>28.2%), followed by Acetomicrobium, Coprothermobacter and Candidatus_Caldatribacterium. The OLRA clearly impacted the archaeal community; Methanothermobacter (77.4%) was favored over Methanosarcina (14.8%). Under thermophilic temperature, it seems that syntrophic acetate oxidation (SAO) bacteria might have competed for substrate with acetoclastic methanogens, while in R-Meso microorganisms responsible for the initial steps of organic matter breakdown, such as members of the Firmicutes and Proteobacteria phyla (at least 67%), were dominant. In summary, R-Meso, characterized by a more uniform distribution of metabolic pathways, as well as a diverse and well-adapted microbial consortium, have exhibited enhanced stability and outperformed R-Thermo at high-loads.

Cobalt-modified digestate-derived biochar enhances kitchen waste anaerobic dry digestion: Performance, microbial mechanisms, and metabolic pathways

To achieve simultaneous in-situ high-value utilization of digestate and efficient resource recovery of kitchen waste (KW). In this work, Co-modified digestate-derived biochar (DDB-Co) was prepared for the first time, the effects of DDB-Co on KW mesophilic dry anaerobic digestion were investigated, and the underlying mechanisms were elucidated. The results showed that the specific surface area of DDB-Co increased to 98.7 m2/g, which was 37.4 % higher than that of the unmodified group (DDB). DDB-Co exhibited a dose-dependent effect on biogas production from the KW dry digestion, with the optimal dose of 4.0 g/L (calculated on a solid content) DDB-Co yielding 205.3 mL/(g·VS) of biogas, representing an 80.9 % increase compared to the CK group. However, higher doses, such as 8.0 g/L DDB-Co, reduced the cumulative biogas production. Mechanistic analysis indicated that 4.0 g/L DDB-Co facilitated the dissolution of particulate organic matter and accelerates the conversion process of volatile fatty acids (VFA) in KW, thereby preventing excessive acidification. Microbial community structure analysis revealed that DDB-Co reduced microbial richness and diversity but selectively enriched microorganisms such as Lactobacillus and Methanothrix, accelerating the direct interspecific electron transfer (DIET) reaction rate and increasing biogas production. Metagenomic analysis demonstrated that DDB-Co primarily upregulated the relative abundances of key enzymes in the acetate decarboxylation methanogenesis pathway, such as acetate kinase (ackA), phosphate acetyltransferase (pta), and acetyl-CoA synthetase (acs), enhancing biogas yield and maintaining system stability. The findings of this work expand the high-value utilization pathways for digestate and provide an alternative solution for the efficient resource recovery of KW.

Anaerobic Degradation of Aromatic and Aliphatic Biodegradable Plastics: Potential Mechanisms and Pathways.

Biodegradable plastics (BDPs) have been widely used as substitutes for traditional plastics, and their environmental fate is a subject of intense research interest. Compared with the aerobic degradation of BDPs, their biodegradability under anaerobic conditions in environmental engineering systems remains poorly understood. This study aimed to investigate the degradability of BDPs composed of poly(butylene adipate-co-terephthalate) (PBAT), poly(lactide acid) (PLA), and their blends, and explore the mechanism underlying their microbial degradation under conditions of anaerobic digestion (AD). The BDPs readily depolymerized under thermophilic conditions but were hydrolyzed at a slow rate under conditions of mesophilic AD. After 45 days of thermophilic AD, a decrease in the molecular weight and significant increase in the production of methane and carbon dioxide production were observed. Network and metagenomics analyses identified AD as reservoirs of plastic-degrading bacteria that produce multiple plastic-degrading enzymes. PETase was identified as the most abundant plastic-degrading enzyme. A potential pathway for the anaerobic biodegradation of BDPs was proposed herein. The polymers of high molecular weight were subjected to abiotic hydrolysis to form oligomers and monomers, enabling subsequent microbial hydrolysis and acetogenesis. Ultimately, complete degradation was achieved predominantly via the pathway involved in the conversion of acetic acid to methane. These findings provide novel insight into the mechanism underlying the anaerobic degradation of BDPs and the microbial resources crucial for the efficient degradation of BDPs.

Unveiling the inactivation mechanisms of different viruses in sludge anaerobic digestion based on factors identification and damage analysis

Despite anaerobic digestion having potential for pathogen reduction in sewage sludge, the behaviors of viruses as the primary health concern are rarely studied. This study investigated the inactivation kinetics and mechanisms of four typical virus surrogates with different structures in mesophilic (MAD) and thermophilic (TAD) anaerobic digestion of sludge. Virus inactivation in MAD was virus-type-dependent correspondingly to different function loss. Temperature drove the faster inactivation proceeding for enveloped Phi6, while temperature and ammonia were the critical inactivation factors for nonenveloped MS2, causing genome degradation and protein functional damage. Interaction with sludge solids played critical role in DNA viruses T4 and Phix174 inactivation via inducing host binding function damage. By comparison, TAD enhanced viral protein denaturation, bringing efficient inactivation with reducing heterogeneity among nonenveloped viruses. These insights into unique virus behaviors in anaerobic digestion systems can provide guidance for developing more effective disinfection protocols and improving sludge biosafety.

A cyclic shift-temperature operation method to train microbial communities of mesophilic anaerobic digestion

Temperature is the critical factor affecting the efficiency and cost of anaerobic digestion (AD). The current work develops a shift-temperature AD (STAD) between 35 °C and 55 °C, intending to optimise microbial community and promote substrate conversion. The experimental results showed that severe inhibition of biogas production occurred when the temperature was firstly increased stepwise from 35 °C to 50 °C, whereas no inhibition was observed at the second warming cycle. When the organic load rate was increased to 6.37 g VS/L/d, the biogas yield of the STAD reached about 400 mL/g VS, nearly double that of the constant-temperature AD (CTAD). STAD promoted the proliferation of Methanosarcina (up to 57.32 %), while severely suppressed hydrogenophilic methanogens. However, when the temperature was shifted to 35 °C, most suppressed species recovered quickly and the excess propionic acid was quickly consumed. Metagenomic analysis showed that STAD also promoted gene enrichment related to pathways metabolism, membrane functions, and methyl-based methanogenesis.

Fate and effect of Polyamide-6 microplastics in mesophilic and thermophilic anaerobic digestion

Research has demonstrated that depending on the type and concentration, microplastics affect anaerobic digestion (AD). Owing to the high abundance of polyamide-6 (PA6) in wastewater treatment plants and limited understanding of its behavior, this study investigates PA6 microplastics' effect in AD. Biochemical methane potential experiments were performed under mesophilic (35 °C) and thermophilic (55 °C) conditions using PA6 at concentrations from 0 to 200 particles/g total solids (TS). Under both conditions, methane production increased in the highest (200 particles/g TS) PA6-dosed reactors, with thermophilic conditions having a statistically significant effect. Methane yield increased from 403.1 ± 5.3 mL/g VS to 436.6 ± 9.2 mL/g VS under thermophilic and from 332.1 ± 1.5 to 340.6 ± 6.6 mL/g VS under mesophilic conditions for the 200 particles/g TS dose, corresponding to increases of 8.3% and 2.6% respectively. PA6 crystallinity decreased from 32.8% to 27.1% and 26.8%, corresponding to decreases of 17.4% in mesophilic and 18.2% in thermophilic reactors compared to pristine control. Similarly, crystallinity decreased in PA6 microplastics collected from abiotic reactors, with thermophilic conditions showing a greater effect. The carbonyl index (CI) values were similar between biotic and abiotic reactors, but PA6 from all reactors had significantly higher CI than pristine PA6, suggesting abiotic factors also affect carbonyl bonds. Additionally, an increase in average PA6 mass was observed for mesophilic and thermophilic conditions by 22.0 % and 23.0 %, respectively. The study shows that temperature and other abiotic factors, like sludge chemistry, significantly influence the fate and effect of PA6 microplastics in digesters. Including abiotic reactors seems crucial for a full understanding of the impact of microbial and non-microbial factors in microplastic studies in the AD process. Studying the effects of microplastics on AD is only one part of the picture, whereas simultaneously examining their fate in digestion is necessary for a complete understanding.

Effect of Biochar Addition on Biogas Production Using Konjac Waste through Mesophilic Two-Phase Anaerobic Digestion

Effect of Biochar Addition on Biogas Production Using Konjac Waste through Mesophilic Two-Phase Anaerobic Digestion

Investigation of start-up strategies and temperature regulation on enhanced methanogenesis in anaerobic digestion of food waste

Temperature is a crucial parameter for anaerobic digestion of food waste. Thermophilic anaerobic digestion (TAD) exhibits high methane production but has a hard initiation. This study compared two start-up strategies, one-step temperature increase and step-wise temperature increase, for achieving rapid TAD initiation. Compared to the step-wise temperature increase, the one-step temperature increase demonstrated stronger stability with slight pH fluctuation. Thermophilic communities were established more rapidly by the one-step temperature increase. Its substrate degradation was more enhanced through improving carbohydrates and lipid metabolisms than step-wise’s. These indicated one-step temperature increase strategy can initiate TAD efficiently. Additionally, a comparative analysis between TAD and mesophilic anaerobic digestion (MAD) was conducted on multilevels. TAD produced 25.29% more methane than MAD. The increased Defluviitoga and Hydrogenispora in TAD promoted hydrogenotrophic methanogenesis. The enriched genes related to the decomposition of glucose and glycerol resulted in higher substrate hydrolysis, acidogenesis, and acetogenesis in TAD than that in MAD. This study elucidated the full stages of TAD, which strengthened methanogenesis.

Mechanisms of biochar-mediated reduction of antibiotic-resistant bacteria and biogas production enhancement in anaerobic digesters

This study examined the impact of different biochar (BC) as an anaerobic digestion (AD) additive on antibiotic-resistant bacteria (ARB) survival and AD performance using dairy cow manure. Bamboo BC and Olive BC with different particle sizes were added into the mesophilic AD at 15 g/L and 30 g/L dosages (Bamboo-15, Bamboo-30, Olive-15, and Olive-30). The study provides a detailed analysis of biogas production, organic metabolism, and ARB and microbial dynamics, elucidating the mechanisms by which BC influences AD. Findings reveal significant reductions in CEZ-resistant bacteria (CEZ-r) across all reactors, ranging from 12.88 % to 76.47 %. Both Bamboo and Olive BC increased CEZ-r removal by 3.08–5.94 times compared to the control. Additionally, BC supplementation prevented the rise in CEZ-r percentage within the total bacteria count observed in the control reactor. Bamboo BC outperformed Olive BC in enhancing biogas yield, with Bamboo-15 and Bamboo-30 showing significant increases of 43.2 % and 48.0 %, respectively, compared to the control. Adding BC in AD regulates ARB by decreasing potential ARG hosts and impeding the transmission of resistance. It also enhances biogas production by improving the efficiency of methanogenic bacteria and optimizing the methanogenic pathway. This research provides insights into how BC can be used to enhance AD performance and mitigate ARB proliferation, offering a sustainable approach to waste management and energy production.

Occurrence of 40 sanitary indicators in French digestates derived from different anaerobic digestion processes and raw organic wastes from agricultural and urban origin.

This study investigated the sanitary quality of digestates resulting from the mesophilic anaerobic digestion (AD) of urban and agricultural organic wastes (OWs). 40 sanitary indicators, including pathogenic bacteria, antimicrobial resistance genes, virulence factor genes, and mobile genetic elements were evaluated using real-time PCR and/or droplet digital PCR. 13 polycyclic aromatic hydrocarbons (PAHs) and 13 pharmaceutical products (PHPs) were also measured. We assessed agricultural OWs from three treatment plants to study the effect of different AD processes (feeding mode, number of stages, pH), and used three laboratory-scale reactors to study the effect of different feed-supplies (inputs). The lab-scale reactors included: Lab1 fed with 97% activated sludge (urban waste) and 3% cow manure; Lab2 fed with 85% sludge-manure mixture supplemented with 15% wheat straw (WS); and Lab3 fed with 81% sludge-manure mixture, 15% WS, and 4% zeolite powder. Activated sludge favored the survival of the food-borne pathogens Clostridium perfringens and Bacillus cereus, carrying the toxin-encoding genes cpe and ces, respectively. Globally, the reactors fed with fecal matter supplemented with straw (Lab2) or with straw and zeolite (Lab3) had a higher hygienization efficiency than the reactor fed uniquely with fecal matter (Lab1). Three pathogenic bacteria (Enterococcus faecalis, Enterococcus faecium, and Mycobacterium tuberculosis complex), a beta-lactam resistance gene (bla TEM), and three mobile genetic elements (intI1, intI2, and IS26) were significantly decreased in Lab2 and Lab3. Moreover, the concentrations of 11 PAHs and 11 PHPs were significantly lower in Lab2 and Lab3 samples than in Lab1 samples. The high concentrations of micropollutants, such as triclosan, found in Lab1, could explain the lower hygienization efficiency of this reactor. Furthermore, the batch-fed reactor had a more efficient hygienization effect than the semi-continuous reactors, with complete removal of the ybtA gene, which is involved in the production of the siderophore yersiniabactin, and significant reduction of intI2 and tetO. These data suggest that it is essential to control the level of chemical pollutants in raw OWs to optimize the sanitary quality of digestates, and that adding co-substrate, such as WS, may overcome the harmful effect of pollutants.

Comparison of mesophilic and thermophilic anaerobic digestion of food waste: Focusing on methanogenic performance and pathogens removal

Food waste (FW) is a valuable and sustainable resource for anaerobic digestion (AD). However, mesophilic AD, commonly used, poses risks associated with pathogenic microorganisms, potentially limiting the safe utilization of digestion by-products. This study aimed to evaluate and compare methane production efficiencies and pathogen reduction capabilities between mesophilic and thermophilic AD processes. Methane production was assessed in batch and continuous operations under thermophilic conditions, resulting in methane yields of 380 and 360 mL/g VS, representing increases of 5.56 % and 22.44 % respectively compared to mesophilic conditions. Pathogen removal efficiency analysis showed that thermophilic AD achieved Escherichia coli elimination rates between 98.9 % and 99.9 %, with higher removal rates for Salmonella ranging from 77.9 % to 99.8 % compared to mesophilic processing. Overall, results from batch and continuous experiments demonstrate that thermophilic AD not only enhances methane production but also significantly improves pathogens reduction, offering a more effective approach for managing FW in AD systems. The findings of this study enhance the scientific understanding of anaerobic digestion technology in food waste treatment applications and provide crucial technical support and decision-making guidance for environmental management and renewable energy production.

Enhanced growth of benthic microalgae by tablet from liquid dairy cattle manure-based anaerobic digestate

An effective strategy for utilizing anaerobic digestates is required to promote biomass power generation. We developed an anaerobic digestate tablet using liquid dairy cattle manure derived from a small mesophilic anaerobic digester installed on a dairy farm. Anaerobic digestate tablets are intended for use in the fertilization of oligotrophic coastal seas to promote primary production. The purpose of this study was to evaluate (1) the dissolution behavior of nutrients from anaerobic digestate tablets and (2) the effect of the application of anaerobic digestate tablets on the growth of benthic microalgae using a culture experiment. Batch experiments were conducted to investigate the dissolution behavior of the nutrients. Cumulative amounts of dissolved inorganic nitrogen and phosphate in the anaerobic digestate tablet ranged from 110 to 28.9 μg g−1 after 28 days. The dissolved inorganic nitrogen in the anaerobic digestate tablet was mainly ammonium nitrogen and accounted for 92.4–96.9%, which is advantageous for the growth of microalgae. The growth curve of the benthic microalga Nitzchia longissima was monitored using f/2 medium added to the anaerobic digestate tablet. The growth of Nitzchia longissima was two orders of magnitude greater than that of the positive control. The enhanced growth of Nitzchia longissima by the anaerobic digestate tablet was considered a concomitant effect of moderate dissolution of ammonium nitrogen and high affinity for benthic microalgae. In conclusion, the anaerobic digestate tablets prepared in this study have the advantage of supplying nitrogen to benthic microalgae. This study proposes a new method for utilizing anaerobic digestates.

Case study: enhancing methane production and polylactic acid decomposition in mesophilic anaerobic digestion system with hydrogen enrichment

Case study: enhancing methane production and polylactic acid decomposition in mesophilic anaerobic digestion system with hydrogen enrichment

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 (24–30 %) 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 337.7–385.6 mL/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.

Valorization of fruit and vegetable waste in a novel three-stage hybrid anaerobic digester for enhanced biogas production: Performance study and microbial community analysis

The study proposes the performance of a three-stage hybrid mesophilic anaerobic digester comprising a suspended-growth hydrolytic reactor, a hybrid (suspended-growth + attached-growth) acidogenic down-flow reactor, and a hybrid methanogenic up-flow reactor, for enhanced biogas production. Commingled fruit and vegetable waste (FVW) – shredded to sizes 1 – 4 mm – plus 2.5 L of tap water served as the feedstock in 8 different batch combinations, where the weight of the waste was increased from 3.5 to 7 kg. Decoupling the three major anaerobic steps ensured desirable working pH ranges for the hydrolytic (4.02 – 6.79), the acidogenic (5.69 – 6.62), and the methanogenic stages (6.94 – 7.7). Triplicate studies of each batch type vis-à-vis all three stages were performed. The highest cumulative soluble chemical oxygen demand percentage removal of 96.3 % was noticed in the case of batch run 5. The maximum biogas yield of 781.8 ml/g CODremoved was corresponding to batch 7, with 6.5 kg FVW, whereas the lowest biogas yield, 735.8 ml/g CODremoved, was corresponding to batch 1. The produced biogas from the methanogenic digester was of high quality (≤ 0.01 % H2S) and constituted 58–62 % CH4 and 6–7 % H2. Microbial community analysis confirmed the presence of pertinent bacterial and archaeal groups, such as Firmicutes, Proteobacteria, Spirochaetes, and Methanothrix, including key homoacetogenic genera, such as Acetobacterium Woodii, Desulfotomaculum, and Desulfitobacterium.

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.

The effects of co-supplemented Fe, Co and Ni on Fe bioavailability and microbial community structure in mesophilic food waste anaerobic digestion by using response surface methodology

Optimized doses and dosing strategies for trace element application in the anaerobic digestion (AD) process still face challenges. In this study, the response surface methodology (RSM) of Box-Behnken was used to investigate the effects of Fe, Co and Ni co-supplementation dosages on the mesophilic AD process of food waste (FW). According to RSM results, the maximum experimental methane yield of 677.9 mL/g VS was obtained under the optimal condition (Fe: 113.1, Co: 0.51, and Ni: 2.44 mg/L), representing a 27.4 % increase compared to the control group without trace element addition. The noticeable decrease in Fe bioavailability in control group indicates a higher demand for Fe during AD of FW. However, while increasing the Fe dose did not enhance its easily bioavailable fraction, that of Co and Ni exhibited a negative effect on Fe bioavailability. In the optimal condition, the syntrophic methanogenesis between syntrophic genera and obligate hydrogenotrophic methanogens improved, along with the increased relative abundance of ATP-binding cassette transporter and two-component systems, which correlated with enhanced Fe bioavailability. These findings provide deeper insights into the interactions of co-supplemented trace elements during AD of FW.

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.

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.