Converting corn grains into bioethanol is an expanding practice for sustainable fuel production, but this is accompanied by the production of large quantities of by-products such as whole stillage. In the present study, the influence of advanced wet oxidation and steam explosion (AWOEx) pretreatment on biogas production and lignocellulose decomposition of corn whole stillage (CWS) was evaluated using semi-continuous thermophilic reactors. The digestion of the CWS was shown to be feasible with an organic loading rate (OLR) of 1.12 ± 0.03 kg VS/m³ day and a hydraulic retention time (HRT) of 30 days, achieving a methane yield of 0.75 ± 0.05 L CH₄/g VS_fed for untreated stillage and 0.86 ± 0.04 L CH₄/g VS_fed for pretreated stillage, corresponding with an increase in methane yield of about 15%. However, the reactors showed unstable performance with the highest investigated OLRs and shortest HRTs. Under optimal conditions, the conversion efficiencies of COD, cellulose, hemicellulose, and lignin were 88, 95, 97, and 59% for pretreated CWS, and 86, 94, 95, and 51% for untreated CWS, respectively. Microbial community analysis showed that Proteiniphilum, MBA03, and Acetomicrobium were the dominant genera in the digestate and were likely responsible for the conversion of proteins and volatile fatty acids in CWS.

A new variant of Methanothermobacter wolfeii was isolated from an anaerobic digester using enrichment cultivation in anaerobic conditions. The new isolate was taxonomically identified via 16S rRNA gene sequencing and tagged as M. wolfeii BSEL. The whole genome of the new variant was sequenced and de novo assembled. Genomic variations between the BSEL strain and the type strain were discovered, suggesting evolutionary adaptations of the BSEL strain that conferred advantages while growing under a low concentration of nutrients. M. wolfeii BSEL displayed the highest specific growth rate ever reported for the wolfeii species (0.27 ± 0.03 h ⁻¹ ) using carbon dioxide (CO ₂ ) as unique carbon source and hydrogen (H ₂ ) as electron donor. M. wolfeii BSEL grew at this rate in an environment with ammonium (NH ₄ ⁺ ) as sole nitrogen source. The minerals content required to cultivate the BSEL strain was relatively low and resembled the ionic background of tap water without mineral supplements. Optimum growth rate for the new isolate was observed at 64°C and pH 8.3. In this work, it was shown that wastewater from a wastewater treatment facility can be used as a low-cost alternative medium to cultivate M. wolfeii BSEL. Continuous gas fermentation fed with a synthetic biogas mimic along with H ₂ in a bubble column bioreactor using M. wolfeii BSEL as biocatalyst resulted in a CO ₂ conversion efficiency of 97% and a final methane (CH ₄ ) titer of 98.5% _v , demonstrating the ability of the new strain for upgrading biogas to renewable natural gas. IMPORTANCE: As a methanogenic archaeon, Methanothermobacter wolfeii uses CO ₂ as electron acceptor, producing CH ₄ as final product. The metabolism of M. wolfeii can be harnessed to capture CO ₂ from industrial emissions, besides producing a drop-in renewable biofuel to substitute fossil natural gas. If used as biocatalyst in new-generation CO ₂ sequestration processes, M. wolfeii has the potential to accelerate the decarbonization of the energy generation sector, which is the biggest contributor of CO ₂ emissions worldwide. Nonetheless, the development of CO ₂ sequestration archaeal-based biotechnology is still limited by an uncertainty in the requirements to cultivate methanogenic archaea and the unknown longevity of archaeal cultures. In this study, we report the adaptation, isolation, and phenotypic characterization of a novel variant of M. wolfeii , which is capable of maximum growth with minimal nutrients input. Our findings demonstrate the potential of this variant for the production of renewable natural gas, paving the way for the development of more efficient and sustainable CO ₂ sequestration processes.

Enzymatic hydrolysis of complex components of residual materials, such as food waste, is a rate-limiting step that conditionates the production rate of biofuels. Research into the anaerobic degradation of cellulose and starch, which are abundant components in organic waste, could contribute to optimize biofuels production processes. In this work, a lab-scale anaerobic semi-continuous hydrolytic reactor was operated for 171 days using food waste as feedstock; the effect of Ba²⁺ dosage over the activity of five hydrolytic enzymes was also evaluated. No significant effects were observed on the global performance of the hydrolytic process during the steady-state of the operation of the reactor, nevertheless, it was detected that Ba²⁺ promoted β-amylases activity by 76%, inhibited endoglucanases and α-amylases activity by 39 and 20%, respectively, and had no effect on β-glucosidases and glucoamylases activity. The mechanisms that rule the observed enzymatic activity changes remain unknown; however, the discussion in this paper provides hypothetical explanations for further research.

We examined the microbial populations present in fecal samples of macropods capable of utilizing a mixture of hydrogen and carbon dioxide (70:30) percent. The feces samples were cultured under anaerobic conditions, and production of methane or acetic acids characteristic for methanogenesis and homoacetogenesis was measured. While the feces of adult macropods mainly produced methane from the substrate, the sample from a 2-month-old juvenile kangaroo only produced acetic acid and no methane. The stable highly enriched culture of the joey kangaroo was sequenced to examine the V3 and V4 regions of the 16S rRNA gene. The results showed that over 70% of gene copies belonged to the Clostridia class, with Paraclostridium and Blautia as the most predominant genera. The culture further showed the presence of Actinomyces spp., a genus which has only been identified in the GI tract of macropods in a few studies, and where none, to our knowledge, have been classified as homoacetogenic. The joey kangaroo mixed culture showed a doubling time of 3.54 h and a specific growth rate of 0.199/h, faster than what has been observed for homoacetogenic bacteria in general. IMPORTANCE: Enteric methane emissions from cattle are a significant contributor to greenhouse gas emissions worldwide. Methane emissions not only contribute to climate change but also represent a loss of energy from the animal's diet. However, methanogens play an important role as hydrogen sink to rumen systems; without it, the performance of hydrolytic organisms diminishes. Therefore, effective strategies of methanogen inhibition would be enhanced in conjunction with the addition of alternative hydrogen sinks to the rumen. The significance of our research is to identify homoacetogens present in the GI tract of kangaroos and to present their performance in vitro , demonstrating their capability to serve as alternatives to rumen methanogens.

• Quantified microbial robustness of Methanothermobacter wolfeii BSEL under bioprocess-relevant perturbations using a Fano-factor metric. • Proteomics revealed stress-specific adaptations involving ribosomal proteins, trehalose synthesis, chaperones, and redox regulators. • Oxidative stress response was linked to PAS-domain and HK-domain proteins with sensory and signaling pathways. • Temperature and redox perturbations exerted the strongest impact on growth rate, methane yield, and productivity compared to nitrogen depletion or hydrogen starvation. • Protein-level evidence for hypothetical coding genomic regions expands functional understanding of methanogens, enabling targeted genetic engineering strategies for enabling biomanufacturing of renewable natural gas.