Methanogenic Phase Research Articles

Different combinations of operating temperature and solids retention time during two-phase anaerobic digestion impacts removal of antibiotic resistance genes

Two-phase anaerobic digestion (AD) performance is significantly influenced by operating parameters such as temperature and solids retention time (SRT), while their impact on antibiotic resistance genes (ARGs) during the acidogenic (AP) and methanogenic (MP) phases remains unclear. This study assessed the abundance of eight ARGs in full-scale two-phase AD, then operated lab-scale two-phase AD systems to evaluate temperature combinations (thermophilic-thermophilic, thermophilic-mesophilic, mesophilic-thermophilic, and mesophilic-mesophilic) at a constant SRT (AP = 2/MP = 13d) and to further assess different SRTs (AP = 2/MP = 13d and AP = 4/MP = 11d). qPCR results revealed that full-scale two-phase AD reduced total ARGs abundance by 87.70 ± 0.50 %. In lab-scale tests, the thermophilic-thermophilic configuration achieved nearly complete ARGs removal (97.61 ± 0.21 %), while the combination of AP = 4/MP = 11d had the highest removal efficiency (83.39 ± 1.17 %). Network analysis indicated that Firmicutes, Bacteroidota, and Proteobacteria were the primary ARG hosts, with Firmicutes dominant. These findings highlight the optimal operating parameters in two-phase AD for maximizing ARGs removal.

The role of sulfidated zero-valent iron in enhancing anaerobic digestion of waste activated sludge.

Zero-valent iron (ZVI) has been confirmed in enhancing methane production by improving interspecies electron transfer during anaerobic digestion (AD) of waste activated sludge (WAS). In this study, we suppose that sulfidated zero-valent iron (S-ZVI), a semiconductor material, has better property of electron transfer in AD process. Based on two-phase anaerobic digestion process, nitrite and S-ZVI were used separately for improving acidogenic phase and methanogenic phase of anaerobic sludge digestion. Based on XRD and XPS, Fe(Ⅱ)-O and Fe(Ⅲ)-O were substituted by Fe(Ⅱ)-S and Fe(Ⅲ)-S after ZVI sulfidation, which increased the electric conductivity of S-ZVI. Nyquist plot and Tafel corrosion curve showed that the sulfidation treatment can reduce impedance of electronic transmission. The results showed that the addition of S-ZVI increased methane production by 24.22% compared with ZVI alone. The addition of S-ZVI increased the relative abundance of Actinobacteria in R3(Raw+S-ZVI) and R6(NO2-+S-ZVI), enhancing the activity of microbial in anaerobic sludge digestion. The relative abundance of Acidobacteriota, Synergistota, WPS-2, Firmicutes, Caldisericota, and Gampylobactenota have changed markedly, and facilitated the activity of acidogenic microbes to produce more biodegradable organic matter, further boosting methane production. The findings of in-situ formation of S-ZVI in R5 anaerobic digestion saves the economic cost of S-ZVI preparation, which will be helpful for the extensive application of this technology.

Microbial mechanism for improving biogas production performance from boiling acidified pig manure feedstock.

A short heat treatment (HT, 90°C-100°C, 5min) was applied to two-phase anaerobic digestion (TPAD) of pig manure (PM) to investigate its effect on microbial inactivation in the acidified feedstock during the methanogenic phase. The results showed that no differences in biogas production at organic loading rate (OLR) below 4.28g volatile solid (VS)/(L·d). However, as the organic load increased, the TPAD with HT exhibited greater biogas production and volatile organic acids removal than the control. By stabilizing pH, digestion of HT acidified feedstock at 9.56g VS/(L·d) OLR resulted in a biogas yield of 340.1mL/g VS, exceeding the control by 35.6%. HT enriched syntrophic bacteria, Methanothrix and Methanobacterium methanogens, and one-carbon metabolizing genes in the methanogenic phase, enhancing energy storage and conversion. Accordingly, acidification followed by HT could be a potential method to improve microbial community and load tolerance in methanogenic phase of TPAD.

Effect of zero-valent iron particle size on alleviating acid stress in anaerobic digestion of food waste.

This work evaluated the effect of zero-valent iron (ZVI) particle size (150μm-100 nm) on the performance of food waste anaerobic digestion (AD) under various acid stress conditions. The results indicated that ZVI significantly improved the AD performance, ensuring successful CH4 production even under high acid stress. However, the extent of this promoting effect was highly dependent on the particle size. Nano-scale ZVI (nZVI) demonstrated superior performance in the AD process in terms of CH4 yield and CH4 production rate compared to micro-scale ZVI (mZVI). Specifically, the CH4 yield in the nZVI reactor was 8.24-8.68% higher than that in the mZVI reactor. This improvement was attributed to nZVI's more effective up-regulatory effect on hydrolase activity, as the activities of protease and α-amylase in nZVI reactors were 44.55%-48.54% and 14.65%-77.52% higher, respectively, than those in mZVI reactors. Furthermore, the methanogenic lag phase and maximum CH4 production rate in the nZVI reactor were 41.64%-49.69% shorter and 9.71-37.69% higher than those in the mZVI reactor, respectively. This can be attributed to nZVI's enhanced promoting effect on the activity of coenzyme 420, Syntrophomonas and Methanosarcina, thereby accelerating the CH4 production.

Geochemical and GMS – Modeling Approach in Characterization of Anticipated Environmental Threats of Leachate from an Open Dumpsite, Niger Delta, Nigeria

Dumping of solid waste in open dumpsites is widespread in developing countries. A major disadvantage of this waste management system is the generation of leachate; this has been a primary concern to the environment because it can percolate into the groundwater and consequently contaminate it. In this work, leachate and groundwater samples were collected from a dumpsite in Asaba and its adjoining area to ascertain the conceivable effect of leachate percolation on groundwater quality. The results of physicochemical and heavy metals analyses of leachate confirmed that its characteristics were highly variable, with severe contamination by organics, salts, and heavy metals. The BOD5/COD ratio in leachate varied from 0.08 – 0.12 mg/l, suggesting a dumpsite yet to attain final maturation but is in its late stage of the methanogenic phase. The low BOD5/COD ratio value (0.08 mg/l) observed for leachate shows some microbial activity in the decomposing leachate yet to stabilize. The plume model showed leachate infiltrating beneath the dumpsite up to depths greater than 20 m after one year. The low concentrations of significant cations, anions, heavy metals, and COD in groundwater likely indicate that leachate did not significantly affect groundwater quality. However, specific parameters such as Fe exceeded the WHO limits; the source of Fe may be related to the geology of the area. The good protective capacity around the dumpsite and groundwater mounds was a barrier against inflow from contaminated leachate.

Impact of Nanoparticle Addition and Ozone Pre-Treatment on Mesophilic Methanogenesis in Temperature-Phased Anaerobic Digestion

Biodegradable organic waste offers significant opportunities for resource recovery within the frame of the circular economy. In this work, the effects of carbon-encapsulated iron nanoparticles and ozone pre-treatments in the mesophilic methanogenic stage of a temperature-phased an-aerobic digestion have been studied using biochemical methanogenic potential (BMP) tests and modeling simulation. To do that, digestates from a pre-treated thermophilic acidogenic reactor that co-digested sludge and wine vinasse were used. The addition of nanoparticles favored the removal of particulate matter, which increased by 9% and 6% in terms of total solids and volatile solids, respectively. When combined with ozone pre-treatment, these increases were 27% and 24%, respectively, demonstrating enhanced AD efficiency. The dose of iron nanoparticles encapsulated in carbon did not result in a statistically significant increase in methane production when sludge and vinasse were used as feedstock. The combination of nanoparticles with the ozone pre-treatment significantly improved the methanogenic phase of the second stage, increasing the methane production yield by 22% and reducing the lag phase from 10 days to 3 days, according to the modified Gompertz model.

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.

Mass flow and microbial shifts in recirculated two-phase anaerobic digestion for biohythane production: Effect of hydraulic retention time

Biohythane, the promising gaseous biofuel, can be produced by the recirculated two-phase anaerobic digestion (R-TPAD). The effect of hydraulic retention time (HRT) as 30, 20 and 10 days on biohythane production via R-TPAD (recirculation ratio = 0.4) was investigated with co-digestion of food waste and paper waste. The results showed that HRTs of 30 and 20 days maintained stable performance for R-TPAD but HRT of 10 days led to the final decrease in biogas production. The failure in the end was attributed to the wash-out of microbes. The removal efficiencies for COD and carbohydrates were decreased by short HRTs while for VS it remained relatively stable at about 75.7 ± 4.0%. All through the operation, acidogenic phase and methanogenic phase were separated stably and butyrate pathway was the dominant pathway for hydrogen fermentation in the first stage. The microbial community in the first stage reached the highest diversity when total HRT was 20 days, where the hydrogen production rate was the highest. The genera Lactobacillus and Caproiciproducens kept dominating the dark fermentation in the acidogenic phase, and Ruminococcus and Methanosarcina were decreasing in the methanogenic phase. The biohythane yields were 411 mL/g-VSfed, 332 mL/g-VSfed and 253 mL/g-VSfed, respectively, where the H2 contents were 18.4%, 14.7% and 8.8%, respectively.

Metagenomic analysis reveals the mechanisms of biochar supported nano zero-valent iron in two-phase anaerobic digestion of food waste: microbial community, CAZmey, functional genes and antibiotic resistance genes

The mechanisms of biochar supported nano zero-valent iron (BC/nZVI) on two-phase anaerobic digestion of food waste were investigated. Results indicated that the performance of both acidogenic phase and methanogenic phase was effectively facilitated. BC/nZVI with the amount of 120 mg/L increased methane production by 32.21%. In addition, BC/nZVI facilitated direct interspecies electron transfer (DIET) between Geobacter and methanogens. Further analysis showed that BC/nZVI increased the abundance of most CAZymes in acidogenic phase. The study also found that BC/nZVI had positive effects on metabolic pathways and related functional genes. The abundances of acdA and ackA in acidogenic phase were increased by 151.75% and 36.26%, respectively, and the abundances of pilA and TorZ associated with DIET were also increased. Furthermore, BC/nZVI mainly removed IMP-12, CAU-1, cmeB, ErmR, MexW, ErmG, Bla2, vgaD, MuxA, and cpxA from this system, and reduced the antibiotic resistance genes for antibiotic inactivation resistance mechanisms.

Performance, interaction, and metabolic pathway of novel dry–wet anaerobic digestion for treating high-solid agricultural waste

Anaerobic digestion (AD) with high-solid content of agricultural waste often leads to the accumulation of volatile fatty acids (VFAs) due to the imbalance of the functions of hydrolytic acidification bacteria and methanogenic archaea. This study developed a novel dry–wet AD of high-solid content was developed to make acidogenic bacteria and methanogenic archaea having higher collaboration ability. The results showed that the dry–wet AD system achieved a dynamic balance and synergistic effect between hydrolytic acidification and the methane production process, which shortened the lag phase to a minimum of 1.21 days. Additionally, the system exhibited a highest volumetric biogas production rate of 1.76 L L−1 d−1 when CM:CS and the initial pH were 6:4, 8.0, respectively. The total VFA concentration reached a maximum of 4.69 g L−1 d−1 and acetic acid (HAc) and butyric acid (HBu) were dominant VFAs (88.75%–93.27%) in the leachate in hydrolytic acidification phase. Metagenomic analysis indicated Pseudomonadales, Clostridiales, Bacteroidales, Spirochaetales, and Methanosaeta were dominant microorganism in hydrolytic acidification phase and methanogenic phase, respectively. The gene abundance of key enzymes in the acetoclastic methanogenic pathway significantly exceeded that of the hydrotrophic methanogenic pathway. This study provides a novel AD technology and insights into the energy conversion of agricultural waste.

Optimization of sulfate reduction and methanogenesis via phase separation in a two-phase internal circulation reactor for the treatment of high-sulfate organic wastewater

To enhance the performance of the internal circulation (IC) reactor when treating high-sulfate organic wastewater, a laboratory-scale two-phase IC reactor with distinct phase separation capabilities was designed, and the sulfate reduction and methanogenesis processes were optimized by segregating the reactor into two specialized reaction zones. The results demonstrated that the first and second reaction areas of the two-phase IC reactor could be maintained at 4.5–6.0 and 7.5–8.5, respectively, turning them into the specialized phase for sulfate reduction and methanogenesis. Through phase separation, the two-phase IC reactor achieved a COD degradation and sulfate reduction efficiency of more than 80% when the influent sulfate concentration exceeded 5,000 mg/L, which were 32.32% and 16.04% higher than that before phase separation. Functional analyses indicated a greater activity of both the dissimilatory and assimilatory sulfate reduction pathways in the acidogenic phase, largely due to a rise in the relative abundance of the genera Desulfovibrio, Bacteroides, and Lacticaseibacillus, the primary carriers of sulfate reduction functional genes. In contrast, all the acetoclastic, hydrogenotrophic, and methylotrophic methanogenesis pathways were inhibited in the acidogenic phase but thrived in the methanogenic phase, coinciding with shifts in the genus Methanothrix, which harbors the mcrA, mcrB, and mcrG genes essential for the final transformation step of all three methanogenesis pathways.

Effect of the inoculum mixing ratio on the anaerobic digestion of food waste: Reactor performance and microbial community

Anaerobic digestion (AD) of food waste (FW) needs to break the hydrolysis limit and improve efficiency. Mixed inoculants of different sources are often used to enhance AD performance, while the mechanism of interaction between mixed inoculants of different sources is uncertain. The effect of the mixing ratio of two different inoculants, Baijiu pit mud (PM) and anaerobic digested sludge (ADS), on the AD of FW was examined. Specifically, the five ratios of PM:ADS (1:0, 3:1, 1:1, 1:3, and 0:1, based on volatile solids) on the AD performance and microbial community were evaluated. Biogas production was 551.70 % higher under a PM:ADS ratio of 1:3 (408.62 mL/g VS) compared with a ratio of 0:1. The acidification rate in 1:3 was 84.05 % and it was 41.85 % higher than 0:1(42.2 %). The abundance of unclassified_f_Comamonadaceae (degradation of cellulose), Petrimonas (acid and hydrogen production) and Methanobrevibacter were higher following the PM addition. Furthermore, the abundance of key enzymes (F420 hydrogenase) and methane metabolism (ko00680) varied under different ratios of PM:ADS. These results suggest that the addition of PM promotes the acidification and methanogenic phases of the AD of FW.

Biogas generation through anaerobic digestion of orange peel waste and crude glycerol in single‐stage and two‐stage batch system

AbstractOrange peel waste (OPW) and crude glycerol (CG) can be used in anaerobic processes. To avoid inhibition due to D‐limonene from OPW, the process can be carried out in two stages. Thus, this work aimed to evaluate the generation of methane in biogas through the anaerobic digestion of OPW and CG, individually and mixed, in a single‐stage and two‐stage batch system. For this purpose, the biochemical methane potential (BMP) of the wastes, separately and mixed, was determined in a single‐stage system (methanogenic phase) and in a two‐stage system (acidogenic and methanogenic), following the German standard VDI 4630. The results obtained were statistically analyzed using Shapiro−Wilk and non‐parametric tests. For OPW, the BMP value found was 160.5 m3 CH4. tonSV−1. For CG, the result for BMP was 344.8 m3 CH4. tonVS−1. The result of these mixed wastes was equivalent to 501.3 m3 CH4. tonSV−1, for BMP. Through the statistical tests used, it was found that the use of OPW and CG, separately and mixed, in the two‐stage system does not present a statistically significant difference in the production of biogas when using the same wastes in a single‐stage system. However, it was found that the co‐digestion of these wastes is promising for biogas generation and contributes to managing these agro‐industrial wastes.

Mechanism of improving anaerobic fermentation performance of kitchen waste pretreated by ionizing irradiation-part 1: rice.

Ionizing irradiation, as a new pretreatment method for the anaerobic fermentation of organic pollutants, is featured with fast reaction speed, good treatment effect, no need to add any chemical reagents, and no secondary pollution. This study explores the mechanism of improving anaerobic fermentation performance of rice samples pretreated by cobalt-60 gamma irradiation through the influence on fermentation substrate, acidogenic phase and methanogenic phase. The results reveal that the soluble chemical oxygen demand of the irradiated rice sample at an absorbed dose of 9.6 kGy increases by 12.4 times due to the dissolution of small molecules of fat-soluble organic matter. The yield of biogas in the acidogenic phase increases by 22.2% with a slight increase in hydrogen gas content. The yield of biogas and methane gas content in the methanogenic phase increases by 27.3% and 15%, respectively. Microbial genome analysis, performed with MiSeq high-throughput sequencing and metagenomic methods, suggests the microbial abundance and metabolic functions in the anaerobic fermentation process change significantly as a result of the pretreatment by gamma irradiation.

Time-shifted expression of acetoclastic and methylotrophic methanogenesis by a single Methanosarcina genomospecies predominates the methanogen dynamics in Philippine rice field soil

BackgroundThe final step in the anaerobic decomposition of biopolymers is methanogenesis. Rice field soils are a major anthropogenic source of methane, with straw commonly used as a fertilizer in rice farming. Here, we aimed to decipher the structural and functional responses of the methanogenic community to rice straw addition during an extended anoxic incubation (120 days) of Philippine paddy soil. The research combined process measurements, quantitative real-time PCR and RT-PCR of particular biomarkers (16S rRNA, mcrA), and meta-omics (environmental genomics and transcriptomics).ResultsThe analysis methods collectively revealed two major bacterial and methanogenic activity phases: early (days 7 to 21) and late (days 28 to 60) community responses, separated by a significant transient decline in microbial gene and transcript abundances and CH4 production rate. The two methanogenic activity phases corresponded to the greatest rRNA and mRNA abundances of the Methanosarcinaceae but differed in the methanogenic pathways expressed. While three genetically distinct Methanosarcina populations contributed to acetoclastic methanogenesis during the early activity phase, the late activity phase was defined by methylotrophic methanogenesis performed by a single Methanosarcina genomospecies. Closely related to Methanosarcina sp. MSH10X1, mapping of environmental transcripts onto metagenome-assembled genomes (MAGs) and population-specific reference genomes revealed this genomospecies as the key player in acetoclastic and methylotrophic methanogenesis. The anaerobic food web was driven by a complex bacterial community, with Geobacteraceae and Peptococcaceae being putative candidates for a functional interplay with Methanosarcina. Members of the Methanocellaceae were the key players in hydrogenotrophic methanogenesis, while the acetoclastic activity of Methanotrichaceae members was detectable only during the very late community response.ConclusionsThe predominant but time-shifted expression of acetoclastic and methylotrophic methanogenesis by a single Methanosarcina genomospecies represents a novel finding that expands our hitherto knowledge of the methanogenic pathways being highly expressed in paddy soils.7k5euw-hvBknz_jds4sXXfVideo

Two‐phase anaerobic codigestion of a cassava starch‐based biopolymer with papaya waste

AbstractThe objective of this work was to evaluate anaerobic digestion processes of mixtures of cassava starch‐based polymer (CSP) and papaya fruit pulp waste (PPW) at different ratios, with physical division of digestion stages. Acidogenic phase (phase 1) was conducted under temperature of 45°C and hydraulic retention time of 96 h. Monitoring reactors with destructive samples were subjected to the same conditions in order to assess the initial degradation profile of the substrates. The methanogenic phase (phase 2) was conducted under temperature of 37°C and the hydraulic retention time was defined as equal to the time the biogas production ceased. The results showed that the CSP/PPW mixtures presented interesting synergism and high biogas production potential under the conditions studied. The treatment with CSP/PPW at the ratio of 75/25 showed the best digestion performances in the phase 1. The highest CSP to PPW ratios showed satisfactory results for accumulated biogas productions (5955.58 mL) and for maximum biogas production rate (25.64 mL gVS−1 day−1) in the phase 2.

Digestão anaeróbica em duas fases de águas residuais de mandioca com adição de glicerol residual para produção de hidrogênio e metano

Abstract Biogas production through co-digestion of two or more waste products has garnered increasing attention from researchers seeking to optimize this process. Biogas and methane production increase with the addition of glycerol to agro-industrial wastes during anaerobic biodigestion. However, the utilization of a two-phase process focused on hydrogen production has not been widely explored. This work aims to evaluate two-phase anaerobic biodigestion of cassava wastewater by adding residual glycerol and swine wastewater to enhance hydrogen and methane production. A pilot-scale biodigester was used during the acidogenic phase at 38.5°C, containing 4% glycerol. The effluent was submitted to methanogenic treatment, and the influence of temperature (36.0 to 39.0°C) and sodium bicarbonate concentration (2.0 to 6.0 g L-1) were evaluated. The results indicated that the optimum conditions during the methanogenic phase were 39.0°C with a sodium bicarbonate concentration of 5.0 g L-1. The two-phase biodigestion produced 30.8 mL of (H2) RCOD-1 and 104.5 mL of (CH4) RCOD-1. Thus, the substrates and inoculum used were adequate for the anaerobic biodigestion process, increasing the energetic efficiency of the process due to hydrogen production.

Effect of reactor operation modes on mitigating antibiotic resistance genes (ARGs) and methane production from hydrothermally-pretreated pig manure

Numerous efforts have been made to enhance the performance of anaerobic digestion (AD) for accelerating renewable energy generation, however, it remains unclear whether the intensified measures could enhance the proliferation and transmissions of antibiotic resistance genes (ARGs) in the system. This study assessed the impact of an innovative pig manure AD process, which includes hydrothermal pretreatment (HTP) and a two-stage configuration with separated acidogenic and methanogenic phases, on biomethane (CH4) production and ARGs dynamics. Results showed that HTP significantly increase CH4 production from 0.65 to 0.75 L/L/d in conventional single-stage AD to 0.82 and 0.91 L/L/d in two-stage AD. This improvement correlated with a rise in the relative abundance of Methanosarcina, a key methanogenesis microorganism. In the two-stage AD, the methanogenic stage offered an ideal environment for methanogens growth, resulting in substantially faster and higher CH4 production by about 10% compared to single-stage AD. Overall, the combined use of HTP and the two-stage AD configuration enhanced CH4 production by 40% compared to traditional single-stage AD. The abundance and diversity of ARGs were significantly reduced in the acidogenic reactors after HTP. However, the ARGs levels increased by about two times in the following methanogenesis stage and reached similar or higher levels than in single stage AD. The erm(F), erm(G), ant(6)-Ia, tet(W), mef(A) and erm(B) were the six main ARGs with significant differences in relative abundances in various treatments. The two-stage AD mode could better remove sul2, but it also had a rebound which elevated the risk of ARGs to the environment and human health. Network analysis identified pH and TVFAs as critical factors driving microbial communities and ARG proliferation in the new AD process. With the results, this study offers valuable insights into the trade-offs between AD performance enhancement and ARG-related risks, pinpointing essential areas for future research and practical improvements.

Study on the Effect of Two-Phase Anaerobic Co-Digestion of Rice Straw and Rural Sludge on Hydrogen and Methane Production

Hydrogen and methane, as chemical raw materials with broad application prospects in the future market, can be produced by the two-phase anaerobic co-digestion of rice straw and sludge. The study was carried out using a medium-temperature batch experiment with rice straw, a rural crop residue from Sichuan, and residual sludge from a sewage treatment station. The effect of the mixing ratio of rice straw and rural sludge on hydrogen and methane production from anaerobic digestion was investigated with a view to alleviating the energy crisis and efficient resource utilization. The experimental results showed that hydrogen production was most favorable when rice straw/sludge = 5:1, with a cumulative hydrogen yield as high as 38.59 ± 1.12 mL/g VSadded, while methane production was most favorable when 3:1, with a cumulative methane yield as high as 578.21 ± 29.19 mL/g VSadded. By calculating the energy yield, it was determined that 3:1 is more favorable for the two-phase anaerobic digestion capacity of rice straw and sludge, which is as high as 20.88 ± 1.07 kJ/g VSadded, and its conversion of hydrogen and methane is 0.75% and 78.19%, respectively. The hydrogen production pathway was dominated by the butyric acid type, whose hydrogen production phase pH (5.84 ± 0.13) was slightly higher than the optimal pH for hydrogen-producing bacteria, while the methanogenic phase could meet the optimal pH for methanogenic bacteria (6.93 ± 0.17).

Deciphering anaerobic ethanol metabolic pathways shaped by operational modes

Efficient anaerobic digestion requires the syntrophic cooperation among diverse microorganisms with various metabolic pathways. In this study, two operational modes, i.e., the sequencing batch reactor (SBR) and the continuous-flow reactor (CFR), were adopted in ethanol-fed systems with or without the supplement of powdered activated carbon (PAC) to examine their effects on ethanol metabolic pathways. Notably, the operational mode of SBR and the presence of CO2 facilitated ethanol metabolism towards propionate production. This was further evidenced by the dominance of Desulfobulbus, and the increased relative abundances of enzymes (EC: 1.2.7.1 and 1.2.7.11) involved in CO2 metabolism in SBRs. Moreover, SBRs exhibited superior biomass-based rates of ethanol degradation and methanogenesis, surpassing those in CFRs by 53.1% and 22.3%, respectively. Remarkably, CFRs with the extended solids retention time enriched high relative abundances of Geobacter of 71.7% and 70.4% under conditions with and without the addition of PAC, respectively. Although both long-term and short-term PAC additions led to the increased sludge conductivity and a reduced methanogenic lag phase, only the long-term PAC addition resulted in enhanced rates of ethanol degradation and propionate production/degradation. The strategies by adjusting operational mode and PAC addition could be adopted for modulating the anaerobic ethanol metabolic pathway and enriching Geobacter.