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Despite the increasing interest in developing antimethanogenic additives to reduce enteric methane (CH 4 ) emissions and the extensive research conducted over the last decades, the global livestock industry has a very limited number of antimethanogenic feed additives (AMFA) available that can deliver substantial reduction, and they have generally not reached the market yet. This work provides technical recommendations and guidelines for conducting tests intended to screen the potential to reduce, directly or indirectly, enteric CH 4 of compounds before they can be further assessed in in vivo conditions. The steps involved in this work cover the discovery, isolation, and identification of compounds capable of affecting CH 4 production by rumen microbes, followed by in vitro laboratory testing of potential candidates. The finding of new bioactive compounds as AMFA can be based on 2 approaches: empirical and mechanistic. The empirical approach involves obtaining and screening compounds present in databases and repositories that potentially possess the desired effect but have not yet been tested, screening natural sources of secondary compounds such as plants, fungi, and algae for their antimethanogenic effects, or examining compounds with antimethanogenic effect on microbes in other research domains outside the rumen. In contrast, the mechanistic approach is the theoretical process of discovery new bioactive compounds based on existing knowledge of a biological target or process. The in vitro methodologies reviewed include examining effects at the subcellular level, in single pure cultures of methanogens and examining in more complex mixed rumen microbial populations. Simple in vitro methodologies (subcellular assessments and batch culture) allow testing a large number of compounds, whereas more complex systems simulating the rumen microbial ecosystem can test a limited number of candidates but provide better insight about the antimethanogenic efficacy. This work collated the main advantages, limitations, and technical recommendations associated with each step and methodology use during the identification and screening of AMFA candidates., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The biochar-enabled advanced reduction process (ARP) was developed for enhanced sorption (by biochar) and destruction of PFAS (by ARP) in water. First, the biochar (BC) was functionalized by iron oxide (Fe 3 O 4 ), zero valent iron (ZVI), and chitosan (chi) to produce four biochars (BC, Fe 3 O 4 -BC, ZVI-chi-BC, and chi-BC) with improved physicochemical properties (e.g., specific surface area, pore structure, hydrophobicity, and surface functional groups). Batch sorption experimental results revealed that compared to unmodified biochar, all modified biochars showed greater sorption efficiency, and the chi-BC performed the best for PFAS sorption. The chi-BC was then selected to facilitate reductive destruction and defluorination of PFAS in water by ARP in the UV-sulfite system. Adding chi-BC in UV-sulfite ARP system significantly enhanced both degradation and defluorination efficiencies of PFAS (up to ∼100% degradation and ∼85% defluorination efficiencies). Radical analysis using electron paramagnetic resonance (EPR) spectroscopy showed that sulfite radicals dominated at neutral pH (7.0), while hydrated electrons (e aq - ) were abundant at higher pH (11) for the efficient destruction of PFAS in the ARP system. Our findings elucidate the synergies of biochar and ARP in enhancing PFAS sorption and degradation, providing new insights into PFAS reductive destruction and defluorination by different reducing radical species at varying pH conditions., Competing Interests: Declaration of competing interest The authors declare they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Methyl-coenzyme M reductase (MCR) is a multi-subunit (α 2 β 2 γ 2 ) enzyme responsible for methane formation via its unique F 430 cofactor. The genes responsible for producing MCR (mcrA, mcrB and mcrG) are typically colocated with two other highly conserved genes mcrC and mcrD. We present here the high-resolution crystal structure for McrD from a human gut methanogen Methanomassiliicoccus luminyensis strain B10. The structure reveals that McrD comprises a ferredoxin-like domain assembled into an α + β barrel-like dimer with conformational flexibility exhibited by a functional loop. The description of the M. luminyensis McrD crystal structure contributes to our understanding of this key conserved methanogen protein typically responsible for promoting MCR activity and the production of methane, a greenhouse gas., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The UV/sulfite-based advanced reduction process (ARP) emerges as an effective strategy to combat per- and polyfluoroalkyl substances (PFAS) pollution in water. Yet, the UV/sulfite-ARP typically operates at highly alkaline conditions (e.g., pH > 9 or even higher) since the generated reductive radicals for PFAS degradation can be quickly sequestered by protons (H + ). To overcome the associated challenges, we prototyped a biochar-surfactant-system (BSS) to synergistically enhance PFAS sorption and degradation by UV/sulfite-ARP. The degradation and defluorination efficiencies of perfluorooctanoic acid (PFOA) depended on solution pH, and concentrations of surfactant (cetyltrimethylammonium bromide; CTAB), sulfite, and biochar. At high pH (8-10), adding biochar and BSS showed no or even small inhibitory effect on PFOA degradation, since the degradation efficiencies were already high enough that cannot be differentiated. However, at acidic and neutral pH (6-7), an evident enhancement of PFOA degradation and defluorination efficiencies occurred. This is due to the synergies between biochar and CTAB that create favorable microenvironments for enhanced PFOA sorption and deeper destruction by prolonging the longevity of reductive radicals (e.g., SO 3 •- ), which is less affected by ambient pH conditions. The performance of UV/sulfite/BSS was further optimized and used for the degradation of four PFAS. At the optimal experimental condition, the UV/sulfite/BSS system can completely degrade PFOA with >30% defluorination efficiency for up to five continuous cycles (n = 5). Overall, our BSS provides a cost-effective and sustainable technique to effectively degrade PFAS in water under environmentally relevant pH conditions. The BSS-enabled ARP technique can be easily tied into PFAS treatment train technology (e.g., advanced oxidation process) for more efficient and deeper defluorination of various PFAS in water., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

( E )-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase, or IspH (formerly known as LytB), catalyzes the terminal step of the bacterial methylerythritol phosphate (MEP) pathway for isoprene synthesis. This step converts ( E )-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into one of two possible isomeric products, either isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). This reaction involves the removal of the C4 hydroxyl group of HMBPP and addition of two electrons. IspH contains a [4Fe-4S] cluster in its active site, and multiple cluster-based paramagnetic species of uncertain redox and ligation states can be detected after incubation with reductant, addition of a ligand, or during catalysis. To characterize the clusters in these species, 57 Fe-labeled samples of IspH were prepared and studied by electron paramagnetic resonance (EPR), 57 Fe electron-nuclear double resonance (ENDOR), and Mössbauer spectroscopies. Notably, this ENDOR study provides a rarely reported, complete determination of the 57 Fe hyperfine tensors for all four Fe ions in a [4Fe-4S] cluster. The resting state of the enzyme ( Ox ) has a diamagnetic [4Fe-4S] 2+ cluster. Reduction generates [4Fe-4S] + ( Red ) with both S = 1/2 and S = 3/2 spin ground states. When the reduced enzyme is incubated with substrate, a transient paramagnetic reaction intermediate is detected ( Int ) which is thought to contain a cluster-bound substrate-derived species. The EPR properties of Int are indicative of a 3+ iron-sulfur cluster oxidation state, and the Mössbauer spectra presented here confirm this. Incubation of reduced enzyme with the product IPP induced yet another paramagnetic [4Fe-4S] + species ( Red+P ) with S = 1/2. However, the g -tensor of this state is commonly associated with a 3+ oxidation state, while Mössbauer parameters show features typical for 2+ clusters. Implications of these complicated results are discussed.

Acetyl-CoA synthase (ACS) is a central enzyme in the carbon and energy metabolism of certain anaerobic species of bacteria and archaea that catalyzes the direct synthesis and cleavage of the acetyl CC bond of acetyl-CoA by an unusual enzymatic mechanism of special interest for its use of organonickel intermediates. An Fe 4 S 4 cluster associated with a proximal, reactive Ni p and distal spectator Ni d comprise the active site metal complex, known as the A cluster. Experimental and theoretical methods have uncovered much about the ACS mechanism, but have also opened new unanswered questions about the structure and reactivity of the A cluster in various intermediate forms. Here we report a method for large scale isolation of ACS with its A cluster in the acetylated state. Isolated acetyl-ACS and the two-electron reduced ACS, produced by acetyl-ACS reaction with CoA, were characterized by UV-visible and EPR spectroscopy. Reactivity with electron acceptors provided an assessment of the apparent E m for two-electron reduction of the A cluster. The results help to distinguish between alternative electronic states of the reduced cluster, provide evidence for a role of the Fe/S cluster in catalysis, and offer an explanation of why one-electron reductive activation is observed for a reaction cycle involving 2-electron chemistry., Competing Interests: Declaration of Competing Interest The authors declare no competing financial conflict of interest with the contents of this article. Any opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the view of the Department of Defense or the Uniformed Services University of the Health Sciences., (Published by Elsevier Inc.)

Methanogens and anaerobic methane-oxidizing archaea (ANME) are important players in the global carbon cycle. Methyl-coenzyme M reductase (MCR) is a key enzyme in methane metabolism, catalyzing the last step in methanogenesis and the first step in anaerobic methane oxidation. Divergent mcr and mcr-like genes have recently been identified in uncultured archaeal lineages. However, the assembly and biochemistry of MCRs from uncultured archaea remain largely unknown. Here we present an approach to study MCRs from uncultured archaea by heterologous expression in a methanogen, Methanococcus maripaludis. Promoter, operon structure, and temperature were important determinants for MCR production. Both recombinant methanococcal and ANME-2 MCR assembled with the host MCR forming hybrid complexes, whereas tested ANME-1 MCR and ethyl-coenzyme M reductase only formed homogenous complexes. Together with structural modeling, this suggests that ANME-2 and methanogen MCRs are structurally similar and their reaction directions are likely regulated by thermodynamics rather than intrinsic structural differences., (© 2022. The Author(s).)