Interpreting an Archaean paleoenvironment through 3D imagery of microbialites.

While stromatolites, and to a lesser extent thrombolites, have been extensively studied in order to unravel Precambrian (>539 Ma) biological evolution, studies of clastic-dominated microbially induced sedimentary structures (MISS) are relatively scarce. The lack of a consolidated record of clastic microbialites creates questions about how much (and what) information on depositional and taphonomic settings can be gleaned from these fossils. We used µCT scanning, a non-destructive X-ray-based 3D imaging method, to reconstruct morphologies of ancient MISS and mat textures in two previously described coastal Archaean samples from the ~3.48 Ga Dresser Formation, Pilbara, Western Australia. The aim of this study was to test the ability of µCT scanning to visualize and make 3D measurements that can be used to interpret the biotic-environmental interactions. Fossil MISS including mat laminae with carpet-like textures in one sample and mat rip-up chips in the second sample were investigated. Compiled δ13C and δ34S analyses of specimens from the Dresser Fm. are consistent with a taxonomically diverse community that could be capable of forming such MISS. 3D measurements of fossil microbial mat chips indicate significant biostabilization and suggest formation in flow velocities >25 cm s-1. Given the stratigraphic location of these chips in a low-flow lagoonal layer, we conclude that these chips formed due to tidal influence, as these assumed velocities are consistent with recent modeling of Archaean tides. The success of µCT scanning in documenting these microbialite features validates this technique both as a first step analysis for rare samples prior to the use of more destructive techniques and as a valuable tool for gaining insight into microbialite taphonomy.

Differences in archaeal diversity and potential ecological functions between saline and hypersaline lakes on Qinghai-Tibet Plateau were driven by multiple environmental and non-environmental factors beyond the salinity.

BACKGROUND: Saline lakes are home to various archaea that play special and crucial roles in the global biogeochemical cycle. The Qinghai-Tibet Plateau hosts a large number of lakes with diverse salinity ranging from 0.1 to over 400 g/L, harboring complex and diverse archaea. To the best of our knowledge, the formation mechanisms and potential ecological roles of archaea in Qinghai-Tibetan Plateau saline lakes remain largely unknown. RESULTS: Using High-throughput Illumina sequencing, we uncovered the vastly distinct archaea communities between two typical saline lakes with significant salinity differences on the Qinghai Tibet Plateau (Qinghai saline lake and Chaka hypersaline lake) and suggested archaea played different important roles in methanogenesis-related and nitrate reduction-related functions of these two lakes, respectively. Rather than the individual effect of salinity, the composite effect of salinity with diverse environmental parameters (e.g., temperature, chlorophyll a, total nitrogen, and total phosphorus) dominated the explanation of the variations in archaeal community structure in different habitats. Based on the network analysis, we further found the correlations between dominant archaeal OTUs were tight but significantly different between the two habitats, implying that archaeal interactions may also largely determine the shape of archaeal communities. CONCLUSION: The present study improved our understanding of the structure and function of archaea in different saline lakes on the Qinghai-Tibet Plateau and provided a new perspective on the mechanisms underlying shaping their communities.

Origin and modern microbial ecology of secondary mineral deposits in Lehman Caves, Great Basin National Park, NV, USA.

Lehman Caves is an extensively decorated high desert cave that represents one of the main tourist attractions in Great Basin National Park, Nevada. Although traditionally considered a water table cave, recent studies identified abundant speleogenetic features consistent with a hypogenic and, potentially, sulfuric acid origin. Here, we characterized white mineral deposits in the Gypsum Annex (GA) passage to determine whether these secondary deposits represent biogenic minerals formed during sulfuric acid corrosion and explored microbial communities associated with these and other mineral deposits throughout the cave. Powder X-ray diffraction (pXRD), scanning electron microscopy with electron dispersive spectroscopy (SEM-EDS), and electron microprobe analyses (EPMA) showed that, while most white mineral deposits from the GA contain gypsum, they also contain abundant calcite, silica, and other phases. Gypsum and carbonate-associated sulfate isotopic values of these deposits are variable, with δ34SV-CDT between +9.7‰ and +26.1‰, and do not reflect depleted values typically associated with replacement gypsum formed during sulfuric acid speleogenesis. Petrographic observations show that the sulfates likely co-precipitated with carbonate and SiO2 phases. Taken together, these data suggest that the deposits resulted from later-stage meteoric events and not during an initial episode of sulfuric acid speleogenesis. Most sedimentary and mineral deposits in Lehman Caves have very low microbial biomass, with the exception of select areas along the main tour route that have been impacted by tourist traffic. High-throughput 16S rRNA gene amplicon sequencing showed that microbial communities in GA sediments are distinct from those in other parts of the cave. The microbial communities that inhabit these oligotrophic secondary mineral deposits include OTUs related to known ammonia-oxidizing Nitrosococcales and Thaumarchaeota, as well as common soil taxa such as Acidobacteriota and Proteobacteria. This study reveals microbial and mineralogical diversity in a previously understudied cave and expands our understanding of the geomicrobiology of desert hypogene cave systems.

Dynamic evolution of the mTHF gene family associated with primary metabolism across life.

BACKGROUND: The folate cycle of one-carbon (C1) metabolism, which plays a central role in the biosynthesis of nucleotides and amino acids, demonstrates the significance of metabolic adaptation. We investigated the evolutionary history of the methylenetetrahydrofolate dehydrogenase (mTHF) gene family, one of the main drivers of the folate cycle, across life. RESULTS: Through comparative genomic and phylogenetic analyses, we found that several lineages of Archaea lacked domains vital for folate cycle function such as the mTHF catalytic and NAD(P)-binding domains of FolD. Within eukaryotes, the mTHF gene family diversified rapidly. For example, several duplications have been observed in lineages including the Amoebozoa, Opisthokonta, and Viridiplantae. In a common ancestor of Opisthokonta, FolD and FTHFS underwent fusion giving rise to the gene MTHFD1, possessing the domains of both genes. CONCLUSIONS: Our evolutionary reconstruction of the mTHF gene family associated with a primary metabolic pathway reveals dynamic evolution, including gene birth-and-death, gene fusion, and potential horizontal gene transfer events and/or amino acid convergence.

Mechanisms of secondary biogenic coalbed methane formation in bituminous coal seams: a joint experimental and multi-omics study.

Coal seam microbes, as endogenous drivers of secondary biogenic gas production in coal seams, might be related to methane production in coal seams. In this study, we carried out anaerobic indoor culture experiments of microorganisms from three different depths of bituminous coal seams in Huainan mining area, and revealed the secondary biogas generation mechanism of bituminous coal seams by using the combined analysis of macro-genome and metabolism multi-omics. The results showed that the cumulative mass molar concentrations (Molality) of biomethane production increased with the increase of the coal seam depth in two consecutive cycles. At the genus level, there were significant differences in the bacterial and archaeal community structures corresponding to the three coal seams 1#, 6#, and 9#(p < 0.05). The volatile matter of air-dry basis (Vad) of coal was significantly correlated with differences in genus-level composition of bacteria and archaea, with correlations of R bacterial = 0.368 and R archaeal = 0.463, respectively. Functional gene analysis showed that the relative abundance of methanogenesis increased by 42% before and after anaerobic fermentation cultivation. Meanwhile, a total of 11 classes of carbon metabolism homologues closely related to methanogenesis were detected in the liquid metabolites of coal bed microbes after 60 days of incubation. Finally, the fatty acid, amino acid and carbohydrate synergistic methanogenic metabolic pathway was reconstructed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The expression level of mcrA gene within the metabolic pathway of the 1# deep coal sample was significantly higher than that of the other two groups (p < 0.05 for significance), and the efficient expression of mcrA gene at the end of the methanogenic pathway promoted the conversion of bituminous coal organic matter to methane. Therefore, coal matrix compositions may be the key factors causing diversity in microbial community and metabolic function, which might be related to the different methane content in different coal seams.

Extremophiles and their expanding biotechnological applications.

Microbial life is not restricted to any particular setting. Over the past several decades, it has been evident that microbial populations can exist in a wide range of environments, including those with extremes in temperature, pressure, salinity, and pH. Bacteria and Archaea are the two most reported types of microbes that can sustain in extreme environments, such as hot springs, ice caves, acid drainage, and salt marshes. Some can even grow in toxic waste, organic solvents, and heavy metals. These microbes are called extremophiles. There exist certain microorganisms that are found capable of thriving in two or more extreme physiological conditions simultaneously, and are regarded as polyextremophiles. Extremophiles possess several physiological and molecular adaptations including production of extremolytes, ice nucleating proteins, pigments, extremozymes and exopolysaccharides. These metabolites are used in many biotechnological industries for making biofuels, developing new medicines, food additives, cryoprotective agents etc. Further, the study of extremophiles holds great significance in astrobiology. The current review summarizes the diversity of microorganisms inhabiting challenging environments and the biotechnological and therapeutic applications of the active metabolites obtained as a response to stress conditions. Bioprospection of extremophiles provides a progressive direction with significant enhancement in economy. Moreover, the introduction to omics approach including whole genome sequencing, single cell genomics, proteomics, metagenomics etc., has made it possible to find many unique microbial communities that could be otherwise difficult to cultivate using traditional methods. These findings might be capable enough to state that discovery of extremophiles can bring evolution to biotechnology.

Archaeal community composition as key driver of H2 consumption rates at the start-up of the biomethanation process.

The performance of hydrogen consumption by various inocula derived from mesophilic anaerobic digestion plants was evaluated under ex situ biomethanation. A panel of 11 mesophilic inocula was operated at a concentration of 15 gVS.L-1 at a temperature of 35 °C in batch system with two successive injections of H2:CO2 (4:1 mol:mol). Hydrogen consumption and methane production rates were monitored from 44 h to 72 h. Hydrogen consumption kinetics varies significantly based on the inoculum origin, with no accumulation of volatile fatty acids. Microbial community analyses revealed that microbial indicators such as the increase in Methanosarcina sp. abundance and the increase of the Archaea/Bacteria ratio were associated to high initial hydrogen consumption rates. The improvement in the hydrogen consumption rate between the two injections was correlated with the enrichment in hydrogenotrophic methanogens. This work provides new insights into the early response of microbial communities to hydrogen injection and on the microbial structures that may favor their adaptation to the biomethanation process.

Long-term chemical and organic fertilization induces distinct variations of microbial associations but unanimous elevation of soil multifunctionality.

Intricate microbial associations contribute greatly to the multiple functions (multifunctionality) of natural ecosystems. However, the relationship between microbial associations and soil multifunctionality (SMF) in artificial ecosystems, particularly in agricultural ecosystem with frequent fertilization, remains unclear. In this study, based on a 28-year paddy field experiment, high-throughput sequencing and networks analysis was performed to investigate changes in soil microbial (archaea, bacteria, fungi, and protists) associations and how these changes correlate with SMF under long-term fertilization. Compared to no fertilization (CK), both chemical fertilization with N, P, and K (CF) and chemical fertilization plus rice straw retention (CFR) treatments showed significantly higher soil nutrient content, grain yield, microbial abundance, and SMF. With the exception of archaeal diversity, the CF treatment exhibited the lowest bacterial, fungal, and protist diversity, and the simplest microbial co-occurrence network. In contrast, the CFR treatment had the lowest archaeal diversity, but the highest bacterial, fungal, and protist diversity. Moreover, the CFR treatment exhibited the most complex microbial co-occurrence network with the highest number of nodes, edges, and interkingdom edges. These results highlight that both chemical fertilization with and without straw retention caused high ecosystem multifunctionality while changing microbial association oppositely. Furthermore, these results indicate that rice straw retention contributes to the development of the soil microbiome and ensures the sustainability of high-level ecosystem multifunctionality.

Analysis of nearly 3000 archaeal genomes from terrestrial geothermal springs sheds light on interconnected biogeochemical processes.

Terrestrial geothermal springs are physicochemically diverse and host abundant populations of Archaea. However, the diversity, functionality, and geological influences of these Archaea are not well understood. Here we explore the genomic diversity of Archaea in 152 metagenomes from 48 geothermal springs in Tengchong, China, collected from 2016 to 2021. Our dataset is comprised of 2949 archaeal metagenome-assembled genomes spanning 12 phyla and 392 newly identified species, which increases the known species diversity of Archaea by ~48.6%. The structures and potential functions of the archaeal communities are strongly influenced by temperature and pH, with high-temperature acidic and alkaline springs favoring archaeal abundance over Bacteria. Genome-resolved metagenomics and metatranscriptomics provide insights into the potential ecological niches of these Archaea and their potential roles in carbon, sulfur, nitrogen, and hydrogen metabolism. Furthermore, our findings illustrate the interplay of competition and cooperation among Archaea in biogeochemical cycles, possibly arising from overlapping functional niches and metabolic handoffs. Taken together, our study expands the genomic diversity of Archaea inhabiting geothermal springs and provides a foundation for more incisive study of biogeochemical processes mediated by Archaea in geothermal ecosystems.

PrimerEvalPy: a tool for in-silico evaluation of primers for targeting the microbiome.

BACKGROUND: The selection of primer pairs in sequencing-based research can greatly influence the results, highlighting the need for a tool capable of analysing their performance in-silico prior to the sequencing process. We therefore propose PrimerEvalPy, a Python-based package designed to test the performance of any primer or primer pair against any sequencing database. The package calculates a coverage metric and returns the amplicon sequences found, along with information such as their average start and end positions. It also allows the analysis of coverage for different taxonomic levels. RESULTS: As a case study, PrimerEvalPy was used to test the most commonly used primers in the literature against two oral 16S rRNA gene databases containing bacteria and archaea. The results showed that the most commonly used primer pairs in the oral cavity did not match those with the highest coverage. The best performing primer pairs were found for the detection of oral bacteria and archaea. CONCLUSIONS: This demonstrates the importance of a coverage analysis tool such as PrimerEvalPy to find the best primer pairs for specific niches. The software is available under the MIT licence at https://gitlab.citius.usc.es/lara.vazquez/PrimerEvalPy .

AnnoView enables large-scale analysis, comparison, and visualization of microbial gene neighborhoods.

The analysis and comparison of gene neighborhoods is a powerful approach for exploring microbial genome structure, function, and evolution. Although numerous tools exist for genome visualization and comparison, genome exploration across large genomic databases or user-generated datasets remains a challenge. Here, we introduce AnnoView, a web server designed for interactive exploration of gene neighborhoods across the bacterial and archaeal tree of life. Our server offers users the ability to identify, compare, and visualize gene neighborhoods of interest from 30 238 bacterial genomes and 1672 archaeal genomes, through integration with the comprehensive Genome Taxonomy Database and AnnoTree databases. Identified gene neighborhoods can be visualized using pre-computed functional annotations from different sources such as KEGG, Pfam and TIGRFAM, or clustered based on similarity. Alternatively, users can upload and explore their own custom genomic datasets in GBK, GFF or CSV format, or use AnnoView as a genome browser for relatively small genomes (e.g. viruses and plasmids). Ultimately, we anticipate that AnnoView will catalyze biological discovery by enabling user-friendly search, comparison, and visualization of genomic data. AnnoView is available at http://annoview.uwaterloo.ca.

Impact of diversified cropping systems and fertilization strategies on soil microbial abundance and functional potentials for nitrogen cycling.

Diversified cropping systems and fertilization strategies were proposed to enhance the abundance and diversity of the soil microbiome, thereby stabilizing their beneficial services for maintaining soil fertility and supporting plant growth. Here, we assessed across three different long-term field experiments in Europe (Netherlands, Belgium, Northern Germany) whether diversified cropping systems and fertilization strategies also affect their functional gene abundance. Soil DNA was analyzed by quantitative PCR for quantifying bacteria, archaea and fungi as well as functional genes related to nitrogen (N) transformations; including bacterial and archaeal nitrification (amoA-bac,arch), three steps of the denitrification process (nirK, nirS and nosZ-cladeI,II) and N2 assimilation (nifH), respectively. Crop diversification and fertilization strategies generally enhanced soil total carbon (C), N and microbial abundance, but with variation between sites. Overall effects of diversified cropping systems and fertilization strategies on functional genes were much stronger than on the abundance of bacteria, archaea and fungi. The legume-based cropping systems showed great potential not only in stimulating the growth of N-fixing microorganisms but also in boosting downstream functional potentials for N cycling. The sorghum-based intercropping system suppressed soil ammonia oxidizing prokaryotes. N fertilization reduced the abundance of nitrifiers and denitrifiers except for ammonia-oxidizing bacteria, while the application of the synthetic nitrification inhibitor DMPP combined with mineral N reduced growth of both ammonia-oxidizing bacteria and archaea. In conclusion, this study demonstrates a strong impact of diversified agricultural practices on the soil microbiome and their functional potentials mediating N transformations.

Simultaneous methane mitigation and nitrogen removal by denitrifying anaerobic methane oxidation in lake sediments.

Methane (CH4) is a potent greenhouse gas, with lake ecosystems significantly contributing to its global emissions. Denitrifying anaerobic methane oxidation (DAMO) process, mediated by NC10 bacteria and ANME-2d archaea, links global carbon and nitrogen cycles. However, their potential roles in mitigating methane emissions and removing nitrogen from lake ecosystems remain unclear. This study explored the spatial variations in activities of nitrite- and nitrate-DAMO and their functional microbes in Changdanghu Lake sediments (Jiangsu Province, China). The results showed that although the average abundance of ANME-2d archaea (5.0 × 106 copies g-1) was significantly higher than that of NC10 bacteria (2.1 × 106 copies g-1), the average potential rates of nitrite-DAMO (4.59 nmol 13CO2 g-1 d-1) and nitrate-DAMO (5.01 nmol 13CO2 g-1 d-1) showed no significant difference across all sampling sites. It is estimated that nitrite- and nitrate-DAMO consumed approximately 6.46 and 7.05 mg CH4 m-2 d-1, respectively, which accordingly achieved 15.07-24.95 mg m-2 d-1 nitrogen removal from the studied lake sediments. Statistical analyses found that nitrite- and nitrate-DAMO activities were both significantly related to sediment nitrate contents and ANME-2d archaeal abundance. In addition, NC10 bacterial and ANME-2d archaeal community compositions showed significant correlations with sediment organic carbon content and water depth. Overall, this study underscores the dual roles of nitrite- and nitrate-DAMO processes in CH4 mitigation and nitrogen elimination and their key environmental impact factors (sediment organic carbon and inorganic nitrogen contents, and water depth) in shallow lake, enhancing the understanding of carbon and nitrogen cycles in freshwater aquatic ecosystems.

Regulatory sequence-based discovery of anti-defense genes in archaeal viruses.

In silico identification of viral anti-CRISPR proteins (Acrs) has relied largely on the guilt-by-association method using known Acrs or anti-CRISPR associated proteins (Acas) as the bait. However, the low number and limited spread of the characterized archaeal Acrs and Aca hinders our ability to identify Acrs using guilt-by-association. Here, based on the observation that the few characterized archaeal Acrs and Aca are transcribed immediately post viral infection, we hypothesize that these genes, and many other unidentified anti-defense genes (ADG), are under the control of conserved regulatory sequences including a strong promoter, which can be used to predict anti-defense genes in archaeal viruses. Using this consensus sequence based method, we identify 354 potential ADGs in 57 archaeal viruses and 6 metagenome-assembled genomes. Experimental validation identified a CRISPR subtype I-A inhibitor and the first virally encoded inhibitor of an archaeal toxin-antitoxin based immune system. We also identify regulatory proteins potentially akin to Acas that can facilitate further identification of ADGs combined with the guilt-by-association approach. These results demonstrate the potential of regulatory sequence analysis for extensive identification of ADGs in viruses of archaea and bacteria.

Interplay of intracellular and trans-cellular DNA methylation in natural archaeal consortia.

DNA methylation serves a variety of functions across all life domains. In this study, we investigated archaeal methylomics within a tripartite xylanolytic halophilic consortium. This consortium includes Haloferax lucertense SVX82, Halorhabdus sp. SVX81, and an ectosymbiotic Candidatus Nanohalococcus occultus SVXNc, a nano-sized archaeon from the DPANN superphylum. We utilized PacBio SMRT and Illumina cDNA sequencing to analyse samples from consortia of different compositions for methylomics and transcriptomics. Endogenous cTAG methylation, typical of Haloferax, was accompanied in this strain by methylation at four other motifs, including GDGcHC methylation, which is specific to the ectosymbiont. Our analysis of the distribution of methylated and unmethylated motifs suggests that autochthonous cTAG methylation may influence gene regulation. The frequency of GRAGAaG methylation increased in highly expressed genes, while CcTTG and GTCGaGG methylation could be linked to restriction-modification (RM) activity. Generally, the RM activity might have been reduced during the evolution of this archaeon to balance the protection of cells from intruders, the reduction of DNA damage due to self-restriction in stressful environments, and the benefits of DNA exchange under extreme conditions. Our methylomics, transcriptomics and complementary electron cryotomography (cryo-ET) data suggest that the nanohaloarchaeon exports its methyltransferase to methylate the Haloferax genome, unveiling a new aspect of the interaction between the symbiont and its host.

A fast comparative genome browser for diverse bacteria and archaea.

Genome sequencing has revealed an incredible diversity of bacteria and archaea, but there are no fast and convenient tools for browsing across these genomes. It is cumbersome to view the prevalence of homologs for a protein of interest, or the gene neighborhoods of those homologs, across the diversity of the prokaryotes. We developed a web-based tool, fast.genomics, that uses two strategies to support fast browsing across the diversity of prokaryotes. First, the database of genomes is split up. The main database contains one representative from each of the 6,377 genera that have a high-quality genome, and additional databases for each taxonomic order contain up to 10 representatives of each species. Second, homologs of proteins of interest are identified quickly by using accelerated searches, usually in a few seconds. Once homologs are identified, fast.genomics can quickly show their prevalence across taxa, view their neighboring genes, or compare the prevalence of two different proteins. Fast.genomics is available at https://fast.genomics.lbl.gov.

Physiological versatility of ANME-1 and Bathyarchaeotoa-8 archaea evidenced by inverse stable isotope labeling.

BACKGROUND: The trophic strategy is one key principle to categorize microbial lifestyles, by broadly classifying microorganisms based on the combination of their preferred carbon sources, electron sources, and electron sinks. Recently, a novel trophic strategy, i.e., chemoorganoautotrophy-the utilization of organic carbon as energy source but inorganic carbon as sole carbon source-has been specifically proposed for anaerobic methane oxidizing archaea (ANME-1) and Bathyarchaeota subgroup 8 (Bathy-8). RESULTS: To further explore chemoorganoautotrophy, we employed stable isotope probing (SIP) of nucleic acids (rRNA or DNA) using unlabeled organic carbon and 13C-labeled dissolved inorganic carbon (DIC), i.e., inverse stable isotope labeling, in combination with metagenomics. We found that ANME-1 archaea actively incorporated 13C-DIC into RNA in the presence of methane and lepidocrocite when sulfate was absent, but assimilated organic carbon when cellulose was added to incubations without methane additions. Bathy-8 archaea assimilated 13C-DIC when lignin was amended; however, their DNA was derived from both inorganic and organic carbon sources rather than from inorganic carbon alone. Based on SIP results and supported by metagenomics, carbon transfer between catabolic and anabolic branches of metabolism is possible in these archaeal groups, indicating their anabolic versatility. CONCLUSION: We provide evidence for the incorporation of the mixed organic and inorganic carbon by ANME-1 and Bathy-8 archaea in the environment. Video Abstract.

Associations between the rumen microbiota and carcass merit and meat quality in beef cattle.

The rumen microbiota is important for energy and nutrient acquisition in cattle, and therefore its composition may also affect carcass merit and meat quality attributes. In this study, we examined the associations between archaeal and bacterial taxa in the rumen microbiota of beef cattle and 12 different attributes, including hot carcass weight (HCW), dressing percentage, ribeye area (REA), intramuscular fat content, marbling score, fat thickness, yield grade, moisture content, purge loss, and shear force. There were significant correlations between the relative abundance of certain archaeal and bacterial genera and these attributes. Notably, Selenomonas spp. were positively correlated with live weight and HCW, while also being negatively correlated with purge loss. Members of the Christensenellaceae R-7, Moryella, and Prevotella genera exhibited positive and significant correlations with various attributes, such as dressing percentage and intramuscular fat content. Ruminococcaceae UCG-001 was negatively correlated with live weight, HCW, and dressing percentage, while Acidaminococcus and Succinivibrionaceae UCG-001 were negatively correlated with intramuscular fat content, moisture content, and marbling score. Overall, our findings suggest that specific changes in the rumen microbiota could be a valuable tool to improve beef carcass merit and meat quality attributes. Additional research is required to better understand the relationship between the rumen microbiota and these attributes, with the potential to develop microbiome-targeted strategies for enhancing beef production. KEY POINTS: • Certain rumen bacteria were associated with carcass merit and meat quality • Moryella was positively correlated with intramuscular fat in beef carcasses • Acidaminococcus spp. was negatively correlated with marbling and intramuscular fat.