Bioenergy recovery and carbon emissions benefits of short-term bio-thermophilic pretreatment on low organic sewage sludge anaerobic digestion: A pilot-scale study.

Sewage sludge in cities of Yangzi River Belt, China, generally exhibits a lower organic content and higher silt contentdue to leakage of drainage system, which caused low bioenergy recovery and carbon emission benefits in conventional anaerobic digestion (CAD). Therefore, this paper is on a pilot scale, a bio-thermophilic pretreatment anaerobic digestion (BTPAD) for low organic sludge (volatile solids (VS) of 4%) was operated with a long-term continuous flow of 200 days. The VS degradation rate and CH4 yield of BTPAD increased by 19.93% and 53.33%, respectively, compared to those of CAD. The analysis of organic compositions in sludge revealed that BTPAD mainly improved the hydrolysis of proteins in sludge. Further analysis of microbial community proportions by high-throughput sequencing revealed that the short-term bio-thermophilic pretreatment was enriched in Clostridiales, Coprothermobacter and Gelria, was capable of hydrolyzing acidified proteins, and provided more volatile fatty acid (VFA) for the subsequent reaction. Biome combined with fluorescence quantitative polymerase chain reaction (PCR) analysis showed that the number of bacteria with high methanogenic capacity in BTPAD was much higher than that in CAD during the medium temperature digestion stage, indicating that short-term bio-thermophilic pretreatment could provide better methanogenic conditions for BTPAD. Furthermore, the greenhouse gas emission footprint analysis showed that short-term bio-thermophilic pretreatment could reduce the carbon emission of sludge anaerobic digestion system by 19.18%.

Impact of methane mitigation strategies on the native ruminant microbiome: A protocol for a systematic review and meta-analysis.

Recently, research has investigated the role of the ruminant native microbiome, and the role microbes play in methane (CH4) production and mitigation. However, the variation across microbiome studies makes implementing impactful strategies difficult. The first objective of this study is to identify, summarize, compile, and discuss the current literature on CH4 mitigation strategies and how they interact with the native ruminant microbiome. The second objective is to perform a meta-analysis on the identified16S rRNA sequencing data. A literature search using Web of Science, Scopus, AGRIS, and Google Scholar will be implemented. Eligible criteria will be defined using PICO (population, intervention, comparator, and outcomes) elements. Two independent reviewers will be utilized for both the literature search and data compilation. Risk of bias will be assessed using the Cochrane Risk Bias 2.0 tool. Publicly available 16S rRNA amplicon gene sequencing data will be downloaded from NCBI Sequence Read Archive, European Nucleotide Archive or similar database using appropriate extraction methods. Data processing will be performed using QIIME2 following a standardized protocol. Meta-analyses will be performed on both alpha and beta diversity as well as taxonomic analyses. Alpha diversity metrics will be tested using a Kruskal-Wallis test with a Benjamini-Hochberg multiple testing correction. Beta diversity will be statistically tested using PERMANOVA testing with multiple test corrections. Hedge's g standardized mean difference statistic will be used to calculate fixed and random effects model estimates using a 95% confidence interval. Heterogeneity between studies will be assessed using the I2 statistic. Potential publication bias will be further assessed using Begg's correlation test and Egger's regression test. The GRADE approach will be used to assess the certainty of evidence. The following protocol will be used to guide future research and meta-analyses for investigating CH4 mitigation strategies and ruminant microbial ecology. The future work could be used to enhance livestock management techniques for GHG control. This protocol is registered in Open Science Framework (https://osf.io/vt56c) and available in the Systematic Reviews for Animals and Food (https://www.syreaf.org/contact).

Effects of two types of Coccomyxa sp. KJ on in vitro ruminal fermentation, methane production, and the rumen microbiota.

Coccomyxa sp. KJ is a unicellular green microalga that accumulates abundant lipids when cultured under nitrogen-deficient conditions (KJ1) and high nitrogen levels when cultured under nitrogen-sufficient conditions (KJ2). Considering the different characteristics between KJ1 and KJ2, they are expected to have different effects on rumen fermentation. This study aimed to determine the effects of KJ1 and KJ2 on in vitro ruminal fermentation, digestibility, CH4 production, and the ruminal microbiome as corn silage substrate condition. Five treatments were evaluated: substrate only (CON) and CON + 0.5% dry matter (DM) KJ1 (KJ1_L), 1.0% DM KJ1 (KJ1_H), 0.5% DM KJ2 (KJ2_L), and 1.0% DM KJ2 (KJ2_H). DM degradability-adjusted CH4 production was inhibited by 48.4 and 40.8% in KJ2_L and KJ2_H, respectively, compared with CON. The proportion of propionate was higher in the KJ1 treatments than the CON treatment and showed further increases in the KJ2 treatments. The abundances of Megasphaera, Succiniclasticum, Selenomonas, and Ruminobacter, which are related to propionate production, were higher in KJ2_H than in CON. The results suggested that the rumen microbiome was modified by the addition of 0.5-1.0% DM KJ1 and KJ2, resulting in increased propionate and reduced CH4 production. In particular, the KJ2 treatments inhibited ruminal CH4 production more than the KJ1 treatments. These findings provide important information for inhibiting ruminal CH4 emissions, which is essential for increasing animal productivity and sustaining livestock production under future population growth.

Feed efficiency and enteric methane emissions indices are inconsistent with the outcomes of the rumen microbiome composition.

The correlation between enteric methane emissions (eME) and feed efficiency (FE) in cattle is linked to the anaerobic fermentation of feedstuffs that occurs in the rumen. Several mathematical indices have been developed to predict feed efficiency and identify low methane emitters in herds. To investigate this, the current study aimed to evaluate the rumen microbial composition in the same group of animals ranked according to six different indices (three indices for FE and three for eME). Thirty-three heifers were ranked into three groups, each consisting of 11 animals, based on FE (feed conversion efficiency - FCE, residual weight gain - RG, and residual feed intake - RFI) and eME indices (production, yield, and intensity). Rumen fluids were collected using a stomach tube and analyzed using 16S rRNA and 18S rRNA, targeting rumen bacteria, archaea, and protozoa. The sequencing analysis revealed that the presence of unique microbial species in the rumen varies across animals ranked by the FE and eME indices. The High RG group harbored 17 unique prokaryotic taxa, while the High FCE group contained only seven. Significant differences existed in the microbial profiles of the animals based on the FE and eME indices. For instance, Raoultibacter was more abundant in the Intermediate RFI group but less so in the Intermediate RG and Intermediate FCE groups. The abundance of Entodinium was higher while Diplodinium was lower in the High FCE group, in contrast to the High RG and High RFI groups. Methanobrevibacter exhibited similar abundances across eME indices. However, the heifers did not demonstrate the same production, yield, and intensity of eME. The present findings underscore the importance of standardizing the FE and eME indices. This standardization is crucial for ensuring consistent and reliable assessments of the composition and function of the rumen microbiome across different herds.

A global metagenomics-based analysis of BPA degradation and its coupling with nitrogen, sulfur, and methane metabolism in landfill leachates.

Microbial metabolism in landfill leachate systems is critically important in driving the degradation reactions of organic pollutants, including the emerging pollutant bisphenol A (BPA). However, little research has addressed the microbial degradation of BPA in landfill leachate and its interactions with nitrogen (N), sulfur (S), and methane (CH4) metabolism on a global scale. To this end, in this study on a global scale, an extremely high concentration of BPA was detected throughout the global landfill leachates. Subsequent reconstructive analyses of metagenomic datasets from 113 sites worldwide revealed that the predominant BPA-degrading microflora included Proteobacteria, Firmicutes, and Bacteroidota. Further metabolic analyses revealed that all four biochemical pathways involved in the degradation of BPA were achieved through biochemical cooperation between different bacterial members of the community. In addition, BPA degraders have also been found to actively collaborate synergistically with non-BPA degraders in the N and S removal as well as CH4 catabolism in landfill leachates. Collectively, this study not only provides insights into the dominant microbial communities and specific types of BPA-degrading microbial members in the community of landfill leachates worldwide, but also reveals the synergistic interactions between BPA mineralization and N, S, and CH4 metabolism. These findings offer valuable and important insights for future comprehensive and in-depth investigations into BPA metabolism in different environments.

Heterologous Biosynthesis of Methanobactin from Methylocystis sp. Strain SB2 in Methylosinus trichosporium OB3b.

Aerobic methanotrophs, or methane-consuming microbes, are strongly dependent on copper for their activity. To satisfy this requirement, some methanotrophs produce a copper-binding compound, or chalkophore, called methanobactin (MB). In addition to playing a critical role in methanotrophy, MB has also been shown to have great promise in treating copper-related human diseases, perhaps most significantly Wilson's disease. In this congenital disorder, copper builds up in the liver, leading to irreversible damage and, in severe cases, complete organ failure. Remarkably, MB has been shown to reverse such damage in animal models, and there is a great deal of interest in upscaling MB production for expanded clinical trials. Such efforts, however, are currently hampered as (1) the natural rate of MB production rate by methanotrophs is low, (2) the use of methane as a substrate for MB production is problematic as it is explosive in air, (3) there is limited understanding of the entire pathway of MB biosynthesis, and (4) the most attractive form of MB is produced by Methylocystis sp. strain SB2, a methanotroph that is genetically intractable. Herein, we report heterologous biosynthesis of MB from Methylocystis sp. strain SB2 in an alternative methanotroph, Methylosinus trichosporium OB3b, not only on methane but also on methanol. As a result, the strategy described herein not only facilitates enhanced MB production but also provides opportunities to construct various mutants to delineate the entire pathway of MB biosynthesis, as well as the creation of modified forms of MB that may have enhanced therapeutic value.

Capturing methane with recombinant soluble methane monooxygenase and recombinant methyl-coenzyme M reductase.

Methane capture via oxidation is considered one of the 'Holy Grails' of catalysis (Tucci and Rosenzweig, 2024). Methane is also a primary greenhouse gas that has to be reduced by 1.2 billion metric tonnes in 10 years to decrease global warming by only 0.23°C (He and Lidstrom, 2024); hence, new technologies are needed to reduce atmospheric methane levels. In Nature, methane is captured aerobically by methanotrophs and anaerobically by anaerobic methanotrophic archaea; however, the anaerobic process dominates. Here, we describe the history and potential of using the two remarkable enzymes that have been cloned with activity for capturing methane: aerobic capture via soluble methane monooxygenase and anaerobic capture via methyl-coenzyme M reductase. We suggest these two enzymes may play a prominent, sustainable role in addressing our current global warming crisis.

Increasing methane production in an anaerobic membrane bioreactor for treating landfill leachate: Impact of organic concentration and HRT.

The anaerobic biological treatment of landfill leachate frequently encounters the souring problems because of the high concentration of organic in landfill leachate. Nonetheless, the performance of anaerobic membrane bioreactor (AnMBR) is commendable in terms of removal of organic compounds. Hence, this study explored the effect of organic concentration and hydraulic retention time(HRT) on the removal performance of actual landfill leachate, additionally, carbon conversion through carbon mass balance analysis was analyzed, in order to determine the optimal treatment potential of AnMBR in treating landfill leachate. For HRT values between 14.5 h and 34.6 h, and the influent COD (Chemical Oxygen Demand) range of 12,773.33-15706.67 mg/L, AnMBR could efficiently treat landfill leachate. As HRT was fixed at 14.5 h and influent COD was around 12,206.7-15,373.33 mg/L, AnMBR achieved a maximum organic removal rate of 18.22 ± 0.51 kg COD/(m3∙d) with methane yield of 0.24 ± 0.01 m3 CH4/kg COD and methane content of 88.26%. Based on carbon mass balance, increasing COD concentration in the influent (less than 16,000 mg/L) boosted the conversion of organic compounds (45.19 ± 4.24%) into CH4; while decreasing HRT (more than 27.0 h) also promoted the conversion of organic compounds into CH4 (38.36-60.93%) resulting in a decreased TOC (Total Organic Carbon) loss by 2.02-7.19% with outflow. AnMBR may efficiently produce methane while treating landfill leachate by assessing the random forest model (RF) and adjusting the balance between HRT and influent COD concentration.

Biogas potential of organosolv pretreated wheat straw as mono and co-substrate: substrate synergy and microbial dynamics.

Anaerobic digestion (AD) technology can potentially address the gap between energy demand and supply playing a crucial role in the production of sustainable energy from utilization of biogenic waste materials as feedstock. The biogas production from anaerobic digestion is primarily influenced by the chemical compositions and biodegradability of the feedstock. Organosolv-steam explosion offers a constructive approach as a promising pretreatment method for the fractionation of lignocellulosic biomasses delivering high cellulose content.This study showed how synergetic co-digestion serves to overcome the challenges of mono-digestion's low efficiency. Particularly, the study evaluated the digestibility of organosolv-steam pretreated wheat straw (WSOSOL) in mono as well as co-digesting substrate with cheese whey (CW) and brewery spent grains (BSG). The highest methane yield was attained with co-digestion of WSOSOL + CW (338 mL/gVS) representing an enhanced biogas output of 1-1.15 times greater than its mono digestion. An ammonium production was favored under co-digestion strategy accounting for 921 mg/L from WSOSOL + BSG. Metagenomic study was conducted to determine the predominant bacteria and archaea, as well as its variations in their populations and their functional contributions during the AD process. The Firmicutes have been identified as playing a significant role in the hydrolysis process and the initial stages of AD. An enrichment of the most prevalent archaea genera enriched were Methanobacterium, Methanothrix, and Methanosarsina. Reactors digesting simpler substrate CW followed the acetoclastic, while digesting more complex substrates like BSG and WSOSOL followed the hydrogenotrophic pathway for biomethane production. To regulate the process for an enhanced AD process to maximize CH4, a comprehensive understanding of microbial communities is beneficial.

The iron nitrogenase reduces carbon dioxide to formate and methane under physiological conditions: A route to feedstock chemicals.

Nitrogenases are the only known enzymes that reduce molecular nitrogen (N2) to ammonia. Recent findings have demonstrated that nitrogenases also reduce the greenhouse gas carbon dioxide (CO2), suggesting CO2 to be a competitor of N2. However, the impact of omnipresent CO2 on N2 fixation has not been investigated to date. Here, we study the competing reduction of CO2 and N2 by the two nitrogenases of Rhodobacter capsulatus, the molybdenum and the iron nitrogenase. The iron nitrogenase is almost threefold more efficient in CO2 reduction and profoundly less selective for N2 than the molybdenum isoform under mixtures of N2 and CO2. Correspondingly, the growth rate of diazotrophically grown R. capsulatus strains relying on the iron nitrogenase notably decreased after adding CO2. The in vivo CO2 activity of the iron nitrogenase facilitates the light-driven extracellular accumulation of formate and methane, one-carbon substrates for other microbes, and feedstock chemicals for a circular economy.

Biokinetic and microbial insights into regulatory mechanisms of long-chain fatty acid degradation during food waste-lipid co-digestion within anaerobic membrane bioreactor.

This study investigated the effects of varying lipid ratios on the anaerobic co-digestion of high-lipid food waste (FW) in a mesophilic anaerobic membrane bioreactor (AnMBR). At a lipid concentration of 5 %, optimal biogas production (3.84 L/L/d) and lipid removal efficiency (78 %) were achieved; however, increasing lipid concentrations resulted in significant accumulations of long-chain fatty acids (LCFAs) and volatile fatty acids (VFAs). Batch tests further demonstrated the impact of various types of LCFAs, with stearic acid showing the slowest microbial growth rate (0.033d-1), confirming its role in the accumulation of acetate-dominated VFAs, potentially limiting the methanogenesis process at elevated lipid levels. Furthermore, at 8 % lipid content, the downregulation of key LCFA degradation enzymes and dominance of hydrogenotrophic methanogens indicated adverse conditions. The importance of the intricate interplay between LCFA degradation kinetics and microbial community for the system efficiency was evidenced, offering insights for optimizing and managing high-lipidic wastes.

Elucidating the role of sub-thermophilic temperature and pre-hydrolyzation for effective upgrading scheme of old swine manure digesters.

Solids concentration, temperature, and digester configuration were subjected to biomethanation study to identify effective retrofitting schemes for old swine waste digesters. Batch assays were commenced to determine an appropriate scenario at 30-55 °C and total solids 1-3 %TS. Sub-thermophilic temperature (45 °C) was found desirable with an additional 11.1 % methane yield, while digestion at higher TS induced ammonium inhibition. Subsequent batch experiments lasted 72 hrs for hydrolytic-acidogenic assessment under various temperatures. Heating control at 45 °C and 55 °C for 24 hrs increased hydrolysis efficiency 4.6-5.7 folds above control but showed no significant difference (α = 0.05) between them. Limited heat supply from biogas engine dictated the continuous digestion study to operate pre-hydrolysis reactor at maximum temperature of 45 °C. The two-stage strategy demonstrated best overall performances at the sub-thermphilic combination, raising methane yield by 35.4 %. Next-Generation Sequencing indicated remarkable shifts in abundance and diversity, especially for hydrolytic organisms, which expanded from 54 to 70.2 % by sub-thermophilic temperature.

Biomethanization of rigid packaging made entirely of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate): Mono- and co-digestion tests and microbial insights.

This study evaluates the anaerobic mesophilic mono- and co-digestion of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) plastic bottles as a proxy for rigid packaging materials. Initial tests showed a 97.3 ± 0.2 % reduction in weight and an observable alteration in the surface (thinning, color fading and pitting) of the PHBH bottles after eight weeks. Subsequent tests showed that PHBH squares (3 × 3 cm) produced 400 NmL-CH4/g-VSfed, at a slower rate compared to powdered PHBH but with similar methane yield. Co-digestion experiments with food waste, swine manure, or sewage sludge showed successful digestion of PHBH alongside organic waste (even at a high bioplastic loading of 20 % volatile solids basis), with methane production comparable to or slightly higher than that observed in mono-digestion. Molecular analyses suggested that the type of co-substrate influenced microbial activity and that methane production was mainly driven by hydrogenotrophic methanogenesis. These results suggest the potential for integrating rigid PHBH packaging into anaerobic digesters.

Degradation of paper-based boxes for food delivery in composting and anaerobic digestion tests.

This study evaluated the fate of food delivery boxes when subjected to biological treatments, reproducing at the lab-scale the conditions of full-scale plants. Four paper-based boxes were composted: two made of paper only, one coupled with polylactic acid (PLA), and one with a barrier coating. One paper only box and the box with PLA were also investigated for their anaerobic degradability with biochemical methane potential (BMP) and semi-continuous tests. During composting, the boxes were not recognisable inside the compost after four (paper only boxes), eight (box with PLA), and twelve (box with barrier coating) weeks. In BMP tests, the paper only box showed a degradability similar to that of food waste (92 %), while the box with PLA degraded only at 76 %. Furthermore, undigested pieces of PLA were found in semi-continuous tests. Accordingly, paper resulted suitable for biological treatments, while the presence of PLA or other barrier coatings can be critical.

Bioaugmentation by enriched hydrogenotrophic methanogens into trickle bed reactors for H2/CO2 conversion.

Biomethanation represents a promising approach for biomethane production, with biofilm-based processes like trickle bed reactors (TBRs) being among the most efficient solutions. However, maintaining stable performance can be challenging, and both pure and mixed culture approaches have been applied to address this. In this study, inocula enriched with hydrogenotrophic methanogens were introduced to to TBRs as bioaugmentation strategy to assess their impacts on the process performance and microbial community dynamics. Metagenomic analysis revealed a metagenome-assembled genome belonging to the hydrogenotrophic genus Methanobacterium, which became dominant during enrichment and successfully colonized the TBR biofilm after bioaugmentation. The TBRs achieved a biogas production with > 96 % methane. The bioaugmented reactor consumed additional H2. This may be due to microbial species utilizing CO2 and H2 via various CO2 reduction pathways. Overall, implementing bioaugmentation in TBRs showed potential for establishing targeted species, although challenges remain in managing H2 consumption and optimizing microbial interactions.

Effect of the dietary supplementation with sunflower oil-enriched bromoform from Asparagopsis taxiformis on lambs' growth, health, and ruminal methane production.

The red seaweed Asparagopsis taxiformis has a potent antimethanogenic effect, which has been proven both in vitro and in vivo. Vegetable oil immersions of this seaweed (hereafter Bromoil) help stabilise the bromoform (CHBr3) responsible for its antimethanogenic effect. We evaluate the effects of increasing the levels of CHBr3 in lamb diets on growth performance, methane (CH4) production, animal health and meat quality. Twenty-four Merino Branco ram lambs were fed a ground complete compound feed, supplemented with 50 mL/kg DM of sunflower oil with different CHBr3 content. The treatments were defined by the CHBr3 doses in the oil: 0 mg (control - B0), 15 mg (B15), 30 mg (B30) and 45 mg (B45) of CHBr3 per kg of feed DM. The feed was prepared daily by mixing Bromoil with the compound feed. At the end of the experiment, the lambs were sacrificed, the ruminal content was collected for in vitro fermentation to evaluate CH4 production and organic matter (OM) degradability, and the rumen mucosa was sampled for histological examination. Meat samples were collected for chemical composition and CHBr3 analysis. The half-life of CHBr3 in the air-exposed feed was 3.98 h making it very difficult to establish the practiced level of CHBr3 supplementation. Lambs-fed treatments B30 and B45 decreased DM intake by up to 28%. Average daily gain was also reduced due to CHBr3 supplementation, with B45 showing results 40% lower than B0. DM feed conversion ratio was similar for all treatments. The degradability of OM, the volume of total gas and of gas without CH4 were unaffected by the experimental treatments, evaluated by the in vitro method. However, the volume of CH4 decreased by up to 75% for treatments above 30 mg/kg DM, while the yield of CH4/g OM degraded was reduced by up to 78% with treatments above 30 mg/kg DM. Meat chemical composition was not affected by Bromoil supplementation and no traces of CHBr3 were found in meat samples. During this experiment, the animals presented normal health and behaviour. However, postslaughter examination of the rumen showed distinct lesions on the ventral region of the rumen mucosa of animals supplemented with Bromoil. These lesions were more severe in the animals receiving treatments B30 and B45. This research determined that although concentrations of CHBr3 in the diet above 30 mg/kg DM helped to reduce CH4 emissions, it negatively affected the performance and rumen wall.

Preparation of coenzyme F430 biosynthetic enzymes and intermediates.

Methyl-coenzyme M reductase (MCR) is the key enzyme in pathways for the formation and anaerobic oxidation of methane. As methane is a potent greenhouse gas and biofuel, investigations of MCR catalysis and maturation are of interest for the development of both methanogenesis inhibitors and natural gas conversion strategies. The activity of MCR is dependent on a unique, nickel-containing coenzyme F430, the most highly reduced tetrapyrrole found in nature. Coenzyme F430 is biosynthesized from sirohydrochlorin in four steps catalyzed by the CfbABCDE enzymes. Here, methods for the expression and purification of the coenzyme F430 biosynthesis enzymes are described along with conditions for the synthesis and purification of biosynthetic intermediates on the milligram scale from commercially available porphobilinogen.

Synergistic effect and microbial community structure of waste-activated sludge and kitchen waste solids residue mesophilic anaerobic co-digestion.

Anaerobic co-digestion was conducted on the solid residues after three-phase separation of kitchen waste (KWS) and waste-activated sludge (WAS), the synergistic effects and process performance were studied during co-digestion at different ratios of KWS to WAS. KWS and WAS mix ratios of 0:1, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 and 1:0 (based on TS). The results showed that a ratio of KWS to WAS of 1:1 got a very high methane recovery with a methane yield of 310.45 ± 30.05 mL/g VSadded. The highest concentration of free ammonia among all reaction systems was only 70.23 ± 5.53 mg/L, which was not enough to produce ammonia inhibition in the anaerobic co-digestion system. However, when the KWS content exceeded 50%, methane inhibition and prolongation of the lag phase were observed due to the accumulation of volatile fatty acids (VFAs), and during the lag phase. Microbial community analysis showed that various bacterial groups involved in acid production and hydrolysis were mainly dominated by phylum Firmicutes, Chloroflexi, Proteobacteria and Bacteroidetes. Hydrogenotrophic methanogen was found to dominate all archaeal communities in the digesters. Co-digestion of KWS with WAS significantly increased the relative abundance of Methanobacterium compared with anaerobic digestion of WAS alone.

Thermophilic Hadarchaeota grow on long-chain alkanes in syntrophy with methanogens.

Methanogenic hydrocarbon degradation can be carried out by archaea that couple alkane oxidation directly to methanogenesis, or by syntrophic associations of bacteria with methanogenic archaea. However, metagenomic analyses of methanogenic environments have revealed other archaea with potential for alkane degradation but apparent inability to form methane, suggesting the existence of other modes of syntrophic hydrocarbon degradation. Here, we provide experimental evidence supporting the existence of a third mode of methanogenic degradation of hydrocarbons, mediated by syntrophic cooperation between archaeal partners. We collected sediment samples from a hot spring sediment in Tengchong, China, and enriched Hadarchaeota under methanogenic conditions at 60 °C, using hexadecane as substrate. We named the enriched archaeon Candidatus Melinoarchaeum fermentans DL9YTT1. We used 13C-substrate incubations, metagenomic, metatranscriptomic and metabolomic analyses to show that Ca. Melinoarchaeum uses alkyl-coenzyme M reductases (ACRs) to activate hexadecane via alkyl-CoM formation. Ca. Melinoarchaeum likely degrades alkanes to carbon dioxide, hydrogen and acetate, which can be used as substrates by hydrogenotrophic and acetoclastic methanogens such as Methanothermobacter and Methanothrix.

Editing microbes to mitigate enteric methane emissions in livestock.

Livestock production significantly contributes to greenhouse gas (GHG) emissions particularly methane (CH4) emissions thereby influencing climate change. To address this issue further, it is crucial to establish strategies that simultaneously increase ruminant productivity while minimizing GHG emissions, particularly from cattle, sheep, and goats. Recent advancements have revealed the potential for modulating the rumen microbial ecosystem through genetic selection to reduce methane (CH4) production, and by microbial genome editing including CRISPR/Cas9, TALENs (Transcription Activator-Like Effector Nucleases), ZFNs (Zinc Finger Nucleases), RNA interference (RNAi), Pime editing, Base editing and double-stranded break-free (DSB-free). These technologies enable precise genetic modifications, offering opportunities to enhance traits that reduce environmental impact and optimize metabolic pathways. Additionally, various nutrition-related measures have shown promise in mitigating methane emissions to varying extents. This review aims to present a future-oriented viewpoint on reducing methane emissions from ruminants by leveraging CRISPR/Cas9 technology to engineer the microbial consortia within the rumen. The ultimate objective is to develop sustainable livestock production methods that effectively decrease methane emissions, while maintaining animal health and productivity.