Search Results (42)

In this study, thermophilic anaerobic digestion (TAD, 60 °C) was evaluated as a downstream waste-to-energy step within a cascading thermophilic biorefinery using four interrelated substrates—primary cafeteria wastes (pCFWs) and primary corn stover wastes (pCSWs) were evaluated without physicochemical pretreatment. Their corresponding secondary substrates, secondary cafeteria waste (sCFW) and secondary corn stover wastes (sCSWs), were residual solids generated after thermophilic bioethanol production and exopolysaccharide production, respectively. To our knowledge, this is the first study to demonstrate sequential thermophilic valorization in which primary wastes are untreated and the remaining biotreated secondary residues are subsequently converted into biomethane, adding each step to bioeconomy. Biomethane potential was quantified to determine how substrate composition and upstream bioprocessing influence methane yield and biodegradability. In the batch, pCFW achieved the highest biodegradability (84% VS reduction) but suffered rapid acidification at higher loadings, whereas pCSW was hydrolysis-limited by lignocellulosic recalcitrance. Upstream bioprocessing (biological pretreatment) improved digestibility, with sCSW exhibiting a 1.8-fold increase in methane yield (300 L CH₄ kg⁻¹ VS) relative to pCSW. All co-digestion treatments outperformed monodigestion, with the best-performing (among the tested) sCFW:sCSW ratio of 3:1 delivering the highest methane yield (413 L CH₄ kg⁻¹ VS) and VS reduction (95.8%). Scale-up in a 40 L fed-batch reactor achieved methane productivities of 49–142 L CH₄ kg⁻¹ VS per feeding cycle, reaching stable operation after two cycles, with cumulative methane production of ~800 L CH₄ kg⁻¹ VS for secondary wastes compared to ~550 L CH₄ kg⁻¹ VS for primary wastes. Microbial analysis revealed dominance of syntrophic acetate-oxidizing bacteria (Acetomicrobium, 22.6%) and hydrogenotrophic methanogens (Methanothermobacter, 72.3%). Therefore, biologically pretreated wastes enabled higher methane recovery and improved solids destruction under thermophilic conditions and demonstrates circular conversion of wastes into renewable biomethane. Full article

Over the course of Martian history, the presence of atmospheric carbon dioxide and potential subsurface molecular hydrogen (H₂), in addition to potential surface and subsurface liquid water, suggests that the Martian subsurface, at minimum, may once have been habitable, particularly to autotrophic chemosynthetic microorganisms. In addition, the widespread nature of clays and other minerals on Mars could have provided sufficient nutrients to support microbial life. Here we tested four methanogenic species (Methanosarcina barkeri, Methanobacterium formicicum, Methanothermobacter wolfeii, and Methanococcus maripaludis) in the presence of illite, nontronite, and Mojave Mars Simulant (MMS), in their standard growth medium. We aimed to determine whether the presence of certain Mars simulants inhibited, promoted, or had no effect on methane (CH₄) production by these microorganisms. The same methanogens were also tested in the presence of montmorillonite, H₂, sodium sulfide (Na₂S), and bicarbonate buffer to determine if this clay could support biotic CH₄ production. Three of the four methanogens tested (M. barkeri, M. formicicum, and M. wolfeii) were capable of CH₄ production in the presence of both clay minerals and MMS, as well as in cultures containing only 10% (w/v) montmorillonite, H₂, Na₂S, and bicarbonate buffer. Conversely, M. maripaludis, a halophile, showed the greatest sensitivity of the four methanogens tested; however, the presence of 5% (w/v) montmorillonite enabled greater CH₄ production under certain circumstances compared to cultures containing the organism’s standard growth medium alone. Overall, these results suggest that the presence of clay minerals on Mars does not preclude the survivability and growth of methanogens in a potential subsurface habitat. In fact, these geological components may provide sufficient nutrients to support microbial growth and survivability. Full article

In 2024, China generated approximately 130 million tons of food waste. This study focuses on food wastewater characterized by exceptionally high organic strength (chemical oxygen demand (COD) > 80 g·L⁻¹, total suspended solids (TSS) > 20 g·L⁻¹) content. Conventional continuous stirred tank reactors (CSTRs) inherently couple hydraulic retention time (HRT) and sludge retention time (SRT), making them prone to microbial washout under high organic loading. To overcome this limitation, this study employed two anaerobic sequencing batch reactors (ASBRs) for treating such high-strength food wastewater. This study systematically evaluated the impacts of temperature (mesophilic: 37 °C and thermophilic: 55 °C) and organic loading rate (OLR) on fermentation performance. Under stable operation (OLR = 5.6 kg_COD·m⁻³·d⁻¹; HRT = 16 days), the mesophilic ASBR achieved a specific methane yield of 307 mL CH₄·g_CODremoved⁻¹, an average COD removal efficiency of 81%, and a volatile fatty acids-to-total alkalinity (VFA/TA) ratio of 0.2, indicating robust process stability. In contrast, the thermophilic ASBR exhibited a VFA/TA ratio of 0.5, signaling incipient acidification. Microbial community analysis revealed significantly higher bacterial and archaeal alpha diversity in the mesophilic system. Notably, Methanothrix—a versatile acetoclastic methanogen—dominated the mesophilic archaeal community (66.65%), conferring functional redundancy and resilience against organic shock loads. By contrast, the thermophilic system was overwhelmingly dominated by the hydrogenotrophic Methanothermobacter (99.28%), resulting in low functional diversity and structural fragility. Compared with a benchmark mesophilic CSTR (specific methane yield: 276 mL CH₄·g_CODremoved⁻¹; COD removal efficiency: 70.6%), the mesophilic ASBR improved methane yield by 11%, COD removal efficiency by 15%, and operational stability (VFA/TA = 0.2 vs. 0.6). This work addresses a gap in ASBR applications for high-strength food wastewater treatment and provides experimental validation of the performance, stability, and scalability of mesophilic ASBRs. The proposed process represents a technically feasible, resource-efficient, and operationally robust solution for the valorization of organic wastewater with COD > 80 g·L⁻¹ and TSS > 20 g·L⁻¹. Full article

Steroidal pharmaceutical wastewater, such as stock liquid and cell lysate, is conventionally treated at a high cost due to its complex composition and high organic content. To treat steroidal pharmaceutical wastewater, make it harmless, and utilize it as a resource, engineering exploration of large-scale biogas engineering was carried out based on its anaerobic digestion characteristics, and the microbial population in the digestion process was analyzed. The results showed that, at a medium temperature of 35 °C and a total solid percentage of 6.5% ± 0.5%, both stock liquid and cell lysate wastewater could be anaerobically fermented normally, with the potential for anaerobic digestion treatment. The cumulative biogas production of lysate gas from the supernatant could reach 758 mL/gVS, which was significantly better than that of traditional raw materials such as straw and feces. The methane content reached 78.9%, and the total VFAs reached 10,204 mg/L on the ninth day. Moreover, we found that co-digestion of steroidal pharmaceutical wastewater with corn straw (CS) significantly enhanced system stability and biogas production efficiency, with synergistic improvement reaching up to 42%. This approach effectively shortened the lag phase observed in the mono-digestion of steroidal pharmaceutical wastewater. Actual treatment in a large-scale biogas project revealed that, after the addition of two kinds of wastewater, the main and auxiliary reactors presented serious acidification problems. Of these, the total volatile fatty acids in the main reactor reached up to 21,000 mg/L, and the methane content in the biogas production decreased to 25%. Additionally, 16S rRNA high-throughput sequencing analysis showed that, after the addition of steroidal pharmaceutical wastewater, the archaea community in the anaerobic reactor changed significantly due to the stress of changes in the fermentation environment. Euryarchaeota became the absolute dominant bacteria, and the methanogenic pathway also changed to the hydrogen trophic methanogenic pathway with Methanothermobacter as the absolute dominant bacterium. This is the first successful industrial-scale application of biogas engineering for treating steroid wastewater, demonstrating its technical feasibility and energy recovery potential. These research outcomes provide critical engineering parameters and practical experience for large-scale resource recovery from similar wastewater streams, offering important reference values for advancing pharmaceutical wastewater treatment from compliance discharge to energy utilization. Full article

Two-stage meso- and thermophilic anaerobic digestion (TSMTAD) of food waste was examined and its microbiological structure was investigated. The first stage was designed for the primary storage of perishable food waste and the second stage for central biogas production. Mesophilic storage with initial neutralization and inoculation of lactic acid bacteria (LAB) resulted in an accumulation of lactic acid (21–23 g/L) with a decreased pH, in which bacterial members in facultative hetero-fermentation-type LAB dominated. Repeated fed-batch storage showed stable accumulation of lactic acid, retaining 89.3% (av.) carbon and preventing the growth of exogenous food pathogens. When the second stage of TSMTAD was compared with direct single-stage anaerobic digestion (SSAD) at 55 °C, the amount of methane accumulated was 1.48-fold higher (896 NmL/g-vs.). The methane yield of the original food refuse was 6.9% higher in the case of TSMTAD. The microbial community structures of both cases were similar, consisting of a sole thermophilic hydrogen-assimilating methanogen, Methanothermobacter thermautotrophicus. However, the abundance of bacteria belonging to two functional groups, H₂ CO₂ and acetic acid producer, and syntrophic acetate-oxidizing bacteria increased in TSMTAD. This may change the metabolic pathway, contributing to the stimulation of methane productivity. Full article

This study compared the behavior of the biomass in two fixed-film anaerobic reactors operated under equivalent organic loading rates but at different temperatures, i.e., 30 °C (RMM) and 55 °C (RMT). The reactors were fed with sugarcane vinasse and molasses (both fermented) in a simulation of sequential periods of season and off-season. The dynamics of biomass growth and retention, as well as the microbial composition, were assessed throughout 171 days of continuous operation, coupled with an additional 10-day test assessing the microbial activity in the bed region. Despite the different inoculum sources used (mesophilic granular vs. thermophilic flocculent sludge types), the biomass growth yield was identical (0.036–0.038 g VSS g⁻¹COD) in both systems. The retention rates (higher in RMT) were regulated according to the initial amount of biomass provided in the inoculation, resulting in similar amounts of total retained biomass (46.8 vs. 43.3 g VSS in RMT and RMM) and biomass distribution patterns (30–35% in the feeding zone) at the end of the operation. Meanwhile, hydrogenotrophic methanogenesis mediated by Methanothermobacter coupled to syntrophic acetate oxidation prevailed in RMT, while the Methanosaeta-mediated acetoclastic pathway occurred in RMM. The results show that different anaerobic consortia can behave similarly in quantitative terms when subjected to equivalent organic loads, regardless of the prevailing methane-producing pathway. The community grows and reaches a balance (or a given cell activity level) defined by the amount of substrate available for conversion. In other words, while the metabolic pathway may differ, the endpoint (the amount of biomass) remains the same if operational stability is maintained. Full article

With the increasing use of renewable energy resources for the power grid, the need for long-term storage technologies, such as power-to-gas systems, is growing. Biomethanation provides the opportunity to store energy in the form of the natural gas-equivalent biomethane. This study investigates a novel plug flow reactor that employs a helical static mixer for the biological methanation of hydrogen and carbon dioxide. In tests, the reactor achieved an average methane production rate of 2.5 $\frac{L_{{CH}_4}}{L_R\ast \mathrm{d}}$ (methane production [L_CH4] per liter of reactor volume [L_R] per day [d]) with a maximum methane content of 94%. It demonstrated good flexibilization properties, as repeated 12 h downtimes did not negatively impact the process. The genera Methanothermobacter and Methanobacterium were predominant during the initial phase, along with volatile organic acid-producing, hydrogenotrophic, and proteolytic bacteria. The average ratio of volatile organic acid to total inorganic carbon increased to 0.52 ± 0.04, while the pH remained stable at an average of pH 8.1 ± 0.25 from day 32 to 98, spanning stable and flexible operation modes. This study contributes to the development of efficient flexible biological methanation systems for sustainable energy storage and management. Full article

Biological methanation is driven by anaerobic methanogenic archaea, cultivated in different media, which consist of multiple macro and micro nutrients. In addition, a reducing agent is needed to lower the oxidation–reduction potential (ORP) and enable the growth of oxygen-sensitive organisms. Until now, sodium sulfide (Na₂S) has been used mainly for this purpose based on earlier published articles at the beginning of anaerobic microbiology research. In a continuation of earlier investigations, in this study, the usage of alternative reducing agents like sodium dithionite (Na₂S₂O₄) and L-Cysteine-HCl shows that similar results can be obtained with fewer environmental and hazardous impacts. Therefore, a newly developed comparison method was used for the cultivation of Methanothermobacter marburgensis. The median methane evolution rate (MER) for the alternatives was similar compared to Na₂S at different concentrations (0.5, 0.25 and 0.1 g/L). However, the use of 0.25 g/L Na₂S₂O₄ or 0.1 g/L L-Cys-HCl led to stable MER values over consecutive batches compared to Na₂S. It was also shown that a lower concentration of reducing agent leads to a higher MER. In conclusion, Na₂S₂O₄ or L-Cys-HCl can be used as a non-corrosive and non-toxic reducing agent for ex situ biological methanation. Economically, Na₂S₂O₄ is cheaper, which is particularly interesting for scale-up purposes. Full article

The current focus on renewable energy in global policy highlights the importance of methane production from biomass through anaerobic digestion (AD). To improve biomass digestion while ensuring overall process stability, microbiome-based management strategies become more important. In this study, metagenomes and metaproteomes were used for metagenomically assembled genome (MAG)-centric analyses to investigate a full-scale biogas plant consisting of three differentially operated digesters. Microbial communities were analyzed regarding their taxonomic composition, functional potential, as well as functions expressed on the proteome level. Different abundances of genes and enzymes related to the biogas process could be mostly attributed to different process parameters. Individual MAGs exhibiting different abundances in the digesters were studied in detail, and their roles in the hydrolysis, acidogenesis and acetogenesis steps of anaerobic digestion could be assigned. Methanoculleus thermohydrogenotrophicum was an active hydrogenotrophic methanogen in all three digesters, whereas Methanothermobacter wolfeii was more prevalent at higher process temperatures. Further analysis focused on MAGs, which were abundant in all digesters, indicating their potential to ensure biogas process stability. The most prevalent MAG belonged to the class Limnochordia; this MAG was ubiquitous in all three digesters and exhibited activity in numerous pathways related to different steps of AD. Full article

High temperatures exacerbate the ammonia inhibition of anaerobic digestion coupled with methanogenesis. The inhibition of methane production by ammonia has been observed in other studies. However, the underlying mechanism is not well understood and requires further investigation. This study explored the effect of ammonia stress on archaeal 16S rRNA transcripts in thermophilic anaerobic digester sludge. Different ammonium concentrations were checked for their influence on the methanogenic rate and hydrogen accumulation. Quantitative PCR was used to compare the changes in total archaeal 16S rRNA expression. A Monte Carlo permutation test within redundancy analysis (RDA) was adopted for exploring the relationship between environmental variables and archaeal 16S rRNA and their transcripts. The results showed that with the increase in ammonium concentration, the methanogenic rate decreased and hydrogen accumulation occurred. The total archaeal 16S rRNA genes and transcripts copy numbers decreased significantly in treatments with higher ammonium concentrations (7 and 10 g NH₄⁺-N/L), but did not change much at lower ammonia concentrations (3 g NH₄⁺-N/L) compared with the 0 g NH₄⁺-N/L treatment. The RDA analysis further revealed that most environmental variables, including ammonia and methane, except for formate, were significantly correlated with the community structure activity of archaeal 16S rRNA transcripts rather than the community structure of their genes. The composition of archaeal 16S rRNA transcripts showed that the hydrogenotrophic methanogen Methanothermobacter dominated the methanogenic community activity in all incubations. It exhibited sensitivity to ammonia stress and should be responsible for the methanogenic inhibition under thermophilic conditions. Our findings suggested that archaeal 16S rRNA transcripts, rather than 16S rRNA genes, are key indicators of ammonia stress and methanogenic activity. Full article

Short- and medium-chain acyl-CoA synthetases catalyze similar two-step reactions in which acyl substrate and ATP bind to form an enzyme-bound acyl-adenylate, then CoA binds for formation of the acyl-CoA product. We investigated the roles of active site residues in CoA binding in acetyl-CoA synthetase (Acs) and a medium-chain acyl-CoA synthetase (Macs) that uses 2-methylbutyryl-CoA. Three highly conserved residues, Arg¹⁹³, Arg⁵²⁸, and Arg⁵⁸⁶ of Methanothermobacter thermautotrophicus Acs (Acs_Mt), are predicted to form important interactions with the 5′- and 3′-phosphate groups of CoA. Kinetic characterization of Acs_Mt variants altered at each of these positions indicates these Arg residues play a critical role in CoA binding and catalysis. The predicted CoA binding site of Methanosarcina acetivorans Macs (Macs_Ma) is structurally more closely related to that of 4-chlorobenzoate:coenzyme A ligase (CBAL) than Acs. Alteration of Macs_Ma residues Tyr⁴⁶⁰, Arg⁴⁹⁰, Tyr⁵²⁵, and Tyr⁵²⁷, which correspond to CoA binding pocket residues in CBAL, strongly affected CoA binding and catalysis without substantially affecting acyl-adenylate formation. Both enzymes discriminate between 3′-dephospho-CoA and CoA, indicating interaction between the enzyme and the 3′-phosphate group is important. Alteration of Macs_Ma residues Lys⁴⁶¹ and Lys⁵¹⁹, located at positions equivalent to Acs_Mt Arg⁵²⁸ and Arg⁵⁸⁶, respectively, had only a moderate effect on CoA binding and catalysis. Overall, our results indicate the active site architecture in Acs_Mt and Macs_Ma differs even though these enzymes catalyze mechanistically similar reactions. The significance of this study is that we have delineated the active site architecture with respect to CoA binding and catalysis in this important enzyme superfamily. Full article

In order to utilize a wider range of low-grade syngas, the syngas biomethanation was studied in this work with respect to the gas–liquid mass transfer and the reactor start-up strategy. Two reactors, a continuous stirred tank (CSTR) and a bubble column with gas recirculation (BCR-C), were used in the experiment by feeding an artificial syngas of 20% H₂, 50% CO, and 30% CO₂ into the reactors at 55 °C. The results showed that the CH₄ productivity was slightly increased by reducing the gas retention time (GRT), but was significantly improved by increasing the stirring speed in the CSTR and the gas circulation rate in the BCR-C. The best syngas biomethanation performance of the CSTR with a CH₄ productivity of 22.20 mmol·L_r⁻¹·day⁻¹ and a yield of 49.01% was achieved at a GRT of 0.833 h and a stirring speed of 300 rpm, while for the BCR-C, the best performance with a CH₄ productivity of 61.96 mmol·L_r⁻¹·day⁻¹ and a yield of 87.57% was achieved at a GRT of 0.625 h and a gas circulation rate of 40 L·L_r⁻¹·h⁻¹. The gas–liquid mass transfer capability provided by gas circulation is far superior to mechanical stirring, leading to a much better performance of low-grade syngas biomethanation in the BCR-C. Feeding H₂/CO₂ during the startup stage of the reactor can effectively stimulate the growth and metabolism of microorganisms, and create a better metabolic environment for subsequent low-grade syngas biomethanation. In addition, during the thermophilic biomethanation of syngas, Methanothermobacter is the dominant genus. Full article

In anaerobic digestion (AD), butyrate is degraded by syntrophic consortium, but can accumulate in highly loaded AD systems. The effect of butyrate on the AD process attracts much less attention than propionate or acetate. In this work, an enrichment culture of the thermophilic butyrate-oxidizing syntrophic consortium was obtained by gradually increasing the initial butyrate concentration from 20 to 170 mM. Surprisingly, even the highest butyrate concentration did not significantly inhibit the methanogenic community, and the stage of acetate degradation was the limiting overall rate of the process. At 170 mM butyrate, the bacterial community changed towards the dominance of syntrophic acetate-oxidizing (SAO) bacteria related to Syntrophaceticus (42.9%), Syntrophomonas (26.2%) and Firmicutes (26.2%), while the archaeal community experienced a sharp decrease in the abundance of Methanosarcina thermophila (from 86.0 to 25.0%) and increase in Methanothermobacter thermautotrophicus (from 3.2 to 53.1%) and Methanomassiliicoccus (from 3.2 to 21.9%). Thus, the shift from acetoclastic methanogenesis to SAO coupled to hydrogenotrophic methanogenesis occurred as an adaptive strategy to overcome high acetate (~200 mM) build-up. Bioaugmentation with the obtained enrichment culture was effective in mitigating the butyrate-dominated VFA build-up during the AD of readily biodegradable waste, increasing the methane production rate, methane yield and volatile solids removal by more than 3.5, 6.2 and 2.9 times, respectively. Our study revealed that the thermophilic butyrate-oxidizing consortia as bioaugmented culture could be the potential strategy to alleviate the high organic load and VFA stress of AD. Full article

Dimethylallyl diphosphate (DMAPP) is a key intermediate metabolite in the synthesis of isoprenoids and is also the prenyl donor for biosynthesizing prenylated flavonoids. However, it is difficult to prepare DMAPP via chemical and enzymatic methods. In this study, three promiscuous kinases from Shigella flexneri (SfPK), Escherichia coli (EcPK), and Saccharomyces cerevisiae (ScPK) and three isopentenyl phosphate kinases from Methanolobus tindarius (MtIPK), Methanothermobacter thermautotrophicus str. Delta H (MthIPK), and Arabidopsis thaliana (AtIPK) were cloned and expressed in Escherichia coli. The enzymatic properties of recombinant enzymes were determined. The Kcat/Km value of SfPK for DMA was 6875 s⁻¹ M⁻¹, which was significantly higher than those of EcPK and ScPK. The Kcat/Km value of MtIPK for DMAP was 402.9 s⁻¹ M⁻¹, which was ~400% of that of MthIPK. SfPK was stable at pH 7.0–9.5 and had a 1 h half-life at 65 °C. MtIPK was stable at pH 6.0–8.5 and had a 1 h half-life at 50 °C. The stability of SfPK and MtIPK was better than that of the other enzymes. Thus, SfPK and MtIPK were chosen to develop a one-pot enzymatic cascade for producing DMAPP from DMA because of their catalytic efficiency and stability. The optimal ratio between SfPK and MtIPK was 1:8. The optimal pH and temperature for the one-pot enzymatic cascade were 7.0 and 35 °C, respectively. The optimal concentrations of ATP and DMA were 10 and 80 mM, respectively. Finally, maximum DMAPP production reached 1.23 mM at 1 h under optimal conditions. Therefore, the enzymatic method described herein for the biosynthesis of DMAPP from DMA can be widely used for the synthesis of isoprenoids and prenylated flavonoids. Full article

Methanogens can produce methane in anaerobic environments via the methanogenesis pathway, and are regarded as one of the most ancient life forms on Earth. They are ubiquitously distributed across distinct ecosystems and are considered to have a thermophilic origin. In this study, we isolated, pure cultured, and completely sequenced a single methanogen strain DL9LZB001, from a hot spring at Tengchong in Southwest China. DL9LZB001 is a thermophilic and hydrogenotrophic methanogen with an optimum growth temperature of 65 °C. It is a putative novel species, which has been named Methanothermobacter tengchongensis—a Class I methanogen belonging to the class Methanobacteria. Comparative genomic and ancestral analyses indicate that the class Methanobacteria originated in a hyperthermal environment and then evolved to adapt to ambient temperatures. This study extends the understanding of methanogens living in geothermal niches, as well as the origin and evolutionary history of these organisms in ecosystems with different temperatures. Full article