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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Chain elongators, friends, and foes

Bioproduction of medium chain carboxylic acids has recently emerged as an alternative strategy to valorize low-value organic waste and side-streams. Key to this route is chain elongation, an anaerobic microbial process driven by ethanol, lactic acid, or carbohydrates. Additionally, because these technologies use wastes as feedstocks, mixed microbial communities are often considered as biocatalysts. Understanding and steering these microbiomes is key to optimize bioprocess performance. From a meta-analysis of publicly available sequencing data, we (i) explore how the current collection of isolated chain elongators compares to microbiome members, (ii) discuss the main beneficial and antagonistic interactions with community partners, and (iii) identify the key research gaps and needs to help understand chain elongation microbiomes, and design/steer these novel bioproduction processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Nutrient Exposure Alters Microbial Composition, Structure, and Mercury Methylating Activity in Periphyton in a Contaminated Watershed

The conversion of mercury (Hg) to monomethylmercury (MMHg) is a critical area of concern in global Hg cycling. Periphyton biofilms may harbor significant amounts of MMHg but little is known about the Hg-methylating potential of the periphyton microbiome. Therefore, we used high-throughput amplicon sequencing of the 16S rRNA gene, ITS2 region, and Hg methylation gene pair (hgcAB) to characterize the archaea/bacteria, fungi, and Hg-methylating microorganisms in periphyton communities grown in a contaminated watershed in East Tennes(see United States). Furthermore, we examined how nutrient amendments (nitrate and/or phosphate) altered periphyton community structure and function. We found that bacterial/archaeal richness in experimental conditions decreased in summer and increased in autumn relative to control treatments, while fungal diversity generally increased in summer and decreased in autumn relative to control treatments. Interestingly, the Hg-methylating communities were dominated by Proteobacteria followed by Candidatus Atribacteria across both seasons. Surprisingly, Hg methylation potential correlated with numerous bacterial families that do not contain hgcAB, suggesting that the overall microbiome structure of periphyton communities influences rates of Hg transformation within these microbial mats. To further explore these complex community interactions, we performed a microbial network analysis and found that the nitrate-amended treatment resulted in the highest number of hub taxa that also corresponded with enhanced Hg methylation potential. This work provides insight into community interactions within the periphyton microbiome that may contribute to Hg cycling and will inform future research that will focus on establishing mixed microbial consortia to uncover mechanisms driving shifts in Hg cycling within periphyton habitats.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The influence of spaceflight on the astronaut salivary microbiome and the search for a microbiome biomarker for viral reactivation

Background: Spaceflight impacts astronauts in many ways but little is known on how spaceflight affects the salivary microbiome and the consequences of these changes on astronaut health, such as viral reactivation. In order to understand this, the salivary microbiome was analyzed with 16S rRNA gene amplicon sequencing, and saliva viral titers were analyzed with quantitative polymerase chain reaction (qPCR) with primers specific for Epstein-Barr virus (EBV), herpes simplex virus (HSV), and varicella zoster virus (VZV) from 10 astronauts pre-flight, in-flight, and post-flight. Results: Streptococcus was the most abundant organism in the saliva, making up 8% of the total organisms detected, and their diversity decreased during spaceflight. Other organisms that had statistically significant changes were Proteobacteria and Fusobacteria which increased during flight and Actinobacteria which decreased during flight. At the genus level, Catonella , Megasphera , and Actinobacillus were absent in more than half of saliva samples collected pre-flight but were then detected during flight. In those subjects that already had these genera pre-flight, their relative abundances increased during flight. Correlation analyses between the microbiome and viral titers revealed a positive correlation with Gracilibacteria , Absconditabacteria , and Abiotrophia and a negative correlation between Oribacterium , Veillonella , and Haemophilus . There was also a significant positive correlation between microbiome richness and EBV viral titers. Conclusions: This is the first study to look at how the salivary microbiome changes as a result of spaceflight and the search for bacterial biomarkers for viral reactivation. Further studies examining the role of specific organisms that were shown to be correlative and predictive in viral reactivation, a serious problem in astronauts during spaceflight, could lead to mitigation strategies to help prevent disease during both short and long duration space missions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

From soil to sequence: filling the critical gap in genome-resolved metagenomics is essential to the future of soil microbial ecology

Abstract Soil microbiomes are heterogeneous, complex microbial communities. Metagenomic analysis is generating vast amounts of data, creating immense challenges in sequence assembly and analysis. Although advances in technology have resulted in the ability to easily collect large amounts of sequence data, soil samples containing thousands of unique taxa are often poorly characterized. These challenges reduce the usefulness of genome-resolved metagenomic (GRM) analysis seen in other fields of microbiology, such as the creation of high quality metagenomic assembled genomes and the adoption of genome scale modeling approaches. The absence of these resources restricts the scale of future research, limiting hypothesis generation and the predictive modeling of microbial communities. Creating publicly available databases of soil MAGs, similar to databases produced for other microbiomes, has the potential to transform scientific insights about soil microbiomes without requiring the computational resources and domain expertise for assembly and binning.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Identification of shared viral sequences in peat moss metagenomes reveals elements of a possible Sphagnum core virome

Viruses are an understudied component of plant microbiomes. Identifying viruses that are shared between individual plants, or members of the “core virome”, could reveal stable viral populations with the potential to modulate the composition and function of the microbiome. Here, we examined the virome associated with Sphagnum mosses, a keystone species that has direct influence over the fate of peatland carbon stores. We analyzed bulk metagenomes and metatranscriptomes generated from Sphagnum field samples collected over a ten-month period to identify virus-like sequences shared among plants. Individual Sphagnum samples harbored distinct DNA and RNA viromes where only a small percentage (< 1%) of the total number of identified viral contigs were shared among all samples. Based on taxonomic classification, the shared viral contigs represent bacterial viruses, or phage (Caudoviricetes), as well as viruses of eukaryotes, namely nucleocytoplasmic large DNA viruses (Nucleocytoviricota) and RNA viruses (Riboviria). We linked the shared phage-like contigs to viral regions within sequenced genomes of bacterial taxa that are members of the Sphagnum core microbiome, suggesting that these contigs represent temperate phage or degraded prophage. The putative nucleocytoplasmic large DNA viruses and RNA viruses were phylogenetically diverse and showed sequence similarity to viruses associated with a broad range of hosts and environmental sources. The identification of shared viral contigs suggested that, despite the compositional heterogeneity between samples, Sphagnum mosses may harbor a core virome. Future work validating the presence of the core virome is warranted as it may aid in understanding how persistent viruses impact microbiome ecology and symbiont evolution within this climatically relevant keystone species.

Metagenomics↗
DOE OSTI2022

The Epidermal Microbiome Within an Aggregation of Leopard Sharks ( Triakis semifasciata ) Has Taxonomic Flexibility with Gene Functional Stability Across Three Time-points

The epidermis of Chondrichthyan fishes consists of dermal denticles with production of minimal but protein-rich mucus that collectively, influence the attachment and biofilm development of microbes, facilitating a unique epidermal microbiome. Here, we use metagenomics to provide the taxonomic and functional characterization of the epidermal microbiome of the Triakis semifasciata (leopard shark) at three time-points collected across 4 years to identify links between microbial groups and host metabolism. Our aims include (1) describing the variation of microbiome taxa over time and identifying recurrent microbiome members (present across all time-points); (2) investigating the relationship between the recurrent and flexible taxa (those which are not found consistently across time-points); (3) describing the functional compositions of the microbiome which may suggest links with the host metabolism; and (4) identifying whether metabolic processes are shared across microbial genera or are unique to specific taxa. Microbial members of the microbiome showed high similarity between all individuals (Bray–Curtis similarity index = 82.7, where 0 = no overlap, 100 = total overlap) with the relative abundance of those members varying across sampling time-points, suggesting flexibility of taxa in the microbiome. One hundred and eighty-eight genera were identified as recurrent, including Pseudomonas, Erythrobacter, Alcanivorax, Marinobacter, and Sphingopxis being consistently abundant across time-points, while Limnobacter and Xyella exhibited switching patterns with high relative abundance in 2013, Sphingobium and Sphingomona in 2015, and Altermonas, Leeuwenhoekiella, Gramella, and Maribacter in 2017. Of the 188 genera identified as recurrent, the top 19 relatively abundant genera formed three recurrent groups. The microbiome also displayed high functional similarity between individuals (Bray–Curtis similarity index = 97.6) with gene function composition remaining consistent across all time-points. These results show that while the presence of microbial genera exhibits consistency across time-points, their abundances do fluctuate. Microbial functions however remain stable across time-points; thus, we suggest the leopard shark microbiomes exhibit functional redundancy. We show coexistence of microbes hosted in elasmobranch microbiomes that encode genes involved in utilizing nitrogen, but not fixing nitrogen, degrading urea, and resistant to heavy metal.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Response of Total (DNA) and Metabolically Active (RNA) Microbial Communities in Miscanthus × Giganteus Cultivated Soil to Different Nitrogen Fertilization Rates

Miscanthus × giganteus is a promising high-yielding perennial plant to meet growing bioenergy demands; however, the degree to which the soil microbiome affects its nitrogen cycling and subsequently, biomass yield remains unclear. In this study, we hypothesize that contributions of metabolically active soil microbial membership may be underestimated with DNA-based approaches. We assessed the response of the soil microbiome to nitrogen availability in terms of both DNA and RNA soil microbial communities from the Long-term Assessment of Miscanthus Productivity and Sustainability (LAMPS) field trial. DNA and RNA were extracted from 271 samples, and 16S small subunit (SSU) rRNA amplicon sequencing was performed to characterize microbial community structure. Significant differences were observed in the resulting soil microbiomes and were best explained by the sequencing library of origin, either DNA or RNA. Similar numbers of membership were detected in DNA and RNA microbial communities, with more than 90% of membership shared. However, the profile of dominant membership within DNA and RNA differed, with varying proportions of Actinobacteria and Proteobacteria and Firmicutes and Proteobacteria. Only RNA microbial communities showed seasonal responses to nitrogen fertilization, and these differences were associated with nitrogen-cycling bacteria. The relative abundance of bacteria associated with nitrogen cycling was 7-fold higher in RNA than in DNA, and genes associated with denitrifying bacteria were significantly enriched in RNA, suggesting that these bacteria may be underestimated with DNA-only approaches. Our findings indicate that RNA-based SSU characterization can be a significant and complementing resource for understanding the role of soil microbiomes in bioenergy crop production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Assembly of the Populus Microbiome Is Temporally Dynamic and Determined by Selective and Stochastic Factors

Recent work shows that the plant microbiome, particularly the initial assembly of this microbiome, influences plant health, survival, and fitness. Here, we characterize the initial assembly of the Populus microbiome across ten genotypes belonging to two poplar species in a common garden using 16S rRNA gene and ITS2 region amplicon sequencing of the leaf endosphere, leaf surface, root endosphere, and rhizosphere. We sampled these microbiomes three times throughout the first growing season and found that the composition of the microbiome changed dramatically over time across all plant-associated habitats and host genotypes. For archaea and bacteria, these changes were dominated by strong homogenizing selection (accounting for 29 to 62% of pairwise comparisons). However, fungal assembly was generally characterized by multiple ecological assembly processes (i.e., a mix of weak selective and dispersal processes). Interestingly, genotype, while a significant moderator of microbiome composition, generally explained less variation than sample date across plant-associated habitats. We defined a set of core genera that accounted for, on average, 36% of the microbiome. The relative abundance of this core community was consistent over time. Additionally, using source tracking modeling, we determined that new microbial taxa colonize from both aboveground and belowground sources, and combined with our ecological assembly null models, we found that both selective and dispersal processes explained the differences between exo- (i.e., leaf surface and rhizosphere) and endospheric microbiomes. Taken together, our results suggest that the initial assembly of the Populus microbiome is time-, genotype-, and habitat-dependent and is moderated by both selective and stochastic factors.

16S rRNA↗
DOE OSTI2021

Microbial Community Field Surveys Reveal Abundant Pseudomonas Population in Sorghum Rhizosphere Composed of Many Closely Related Phylotypes

While the root-associated microbiome is typically less diverse than the surrounding soil due to both plant selection and microbial competition for plant derived resources, it typically retains considerable complexity, harboring many hundreds of distinct bacterial species. Here, we report a time-dependent deviation from this trend in the rhizospheres of field grown sorghum. In this study, 16S rRNA amplicon sequencing was used to determine the impact of nitrogen fertilization on the development of the root-associated microbiomes of 10 sorghum genotypes grown in eastern Nebraska. We observed that early rhizosphere samples exhibit a significant reduction in overall diversity due to a high abundance of the bacterial genus Pseudomonas that occurred independent of host genotype in both high and low nitrogen fields and was not observed in the surrounding soil or associated root endosphere samples. When clustered at 97% identity, nearly all the Pseudomonas reads in this dataset were assigned to a single operational taxonomic unit (OTU); however, exact sequence variant (ESV)-level resolution demonstrated that this population comprised a large number of distinct Pseudomonas lineages. Furthermore, single-molecule long-read sequencing enabled high-resolution taxonomic profiling revealing further heterogeneity in the Pseudomonas lineages that was further confirmed using shotgun metagenomic sequencing. Finally, field soil enriched with specific carbon compounds recapitulated the increase in Pseudomonas, suggesting a possible connection between the enrichment of these Pseudomonas species and a plant-driven exudate profile.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Lake Arrowhead Microbial Genomics Conference

The Lake Arrowhead Microbial Genomics Conference (formerly the E. coli and Small Genomes Conference, and the Microbial Genomes Conference) was held September 11-15, 2022, at the UCLA Conference Center at Lake Arrowhead, California. This conference is part of a yearly meeting initiated in 1991 to bring together genome sequencers, bioinformatics specialists, biologists, and geneticists, to forge interactions that would result in meaningful functional genomics. The goal of the meeting was to translate the influx of new genome sequencing information into useful biological studies. The Lake Arrowhead 2022 meeting had a major focus on microbial communities, the human microbiome, pathogens, phage therapy, bioinformatic methods of analysis, bioenergenics, and environmental microbiology. The field of genomics has reached the point where deriving the sequence of an organism’s entire genome is now seen as a beginning rather than an endpoint. Moreover, understanding the sequences of whole communities, and particularly those that make up different microbiomes, is now a central point of the field. The 2022 meeting covered microorganisms for which extensive analyses exist, and those for which new biological and technical strategies are being developed. The focus on biodiversity, the human microbiome, pathogenic organisms and methods of countering them, and bioenergetics added special significance to this meeting. This meeting was designed to have a mix of 36 invited presentations and poster sessions with more than 80 posters, and had 147 participants. This meeting has become established as a major annual microbial genomics conference during the last 29 years and has assured status and quality. It offers a major opportunity for young investigators and students to attend, present posters, and give talks. In fact, 50% of the invited speakers were early career stage scientists. An important impact of the conference was the forging of new collaborations between scientists and particularly young scientists with the array of multidisciplinary researchers at the meeting.

Miller, Jeffrey H.↗
DOE OSTI2021

A Survey of Bacterial Microcompartment Distribution in the Human Microbiome

Bacterial microcompartments (BMCs) are protein-based organelles that expand the metabolic potential of many bacteria by sequestering segments of enzymatic pathways in a selectively permeable protein shell. Sixty-eight different types/subtypes of BMCs have been bioinformatically identified based on the encapsulated enzymes and shell proteins encoded in genomic loci. BMCs are found across bacterial phyla. The organisms that contain them, rather than strictly correlating with specific lineages, tend to reflect the metabolic landscape of the environmental niches they occupy. From our recent comprehensive bioinformatic survey of BMCs found in genome sequence data, we find many in members of the human microbiome. Here we survey the distribution of BMCs in the different biotopes of the human body. Given their amenability to be horizontally transferred and bioengineered they hold promise as metabolic modules that could be used to probiotically alter microbiomes or treat dysbiosis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Impact of sanitizer application on Salmonella mitigation and microbiome shift on diced tomato during washing and storage

Here, this study examined the shift in Salmonella and indigenous microbiota on diced tomato after washing with three different sanitizers and during post-wash cold storage. Roma tomatoes were inoculated with a Salmonella cocktail (initial level ~ 5.6 CFU g –1 ), diced and washed along with uninoculated diced tomato in simulated flume wash water with sanitizers, including 10 mg L –1 free chlorine (FC10), 90 mg L –1 peracetic acid (PA90), PA90 in combination with a proprietary acidified surfactant blend (PS90), and unsanitized control (CK). Salmonella, total mesophilic aerobic bacteria (MAB), and yeast and mold (YM) populations on both inoculated and uninoculated samples were measured before and after washing, and during storage at 4 °C. 16S rRNA amplicon sequencing was also performed to determine the shift in bacterial microbiome. Washing with all tested sanitizers, especially PS90 (> 4 log Salmonella reduction), reduced Salmonella and MAB populations on inoculated diced tomatoes. Additionally, application of sanitizers mitigated Salmonella cross-contamination onto uninoculated samples. PS90 treatment inhibited the proliferation of most dominant bacteria on diced tomatoes during storage, including Erwiniaceae, Curtobacterium, Pantoea, Erwinia and Enterobacterales, which may benefit product quality and safety.

16S rRNA amplicon sequencing↗
DOE OSTI2023

Topography of respiratory tract and gut microbiota in mice with influenza A virus infection

Introduction Influenza A virus (IAV)-induced dysbiosis may predispose to severe bacterial superinfections. Most studies have focused on the microbiota of single mucosal surfaces; consequently, the relationships between microbiota at different anatomic sites in IAV-infected mice have not been fully studied. Methods We characterized respiratory and gut microbiota using full-length 16S rRNA gene sequencing by Nanopore sequencers and compared the nasopharyngeal, oropharyngeal, lung and gut microbiomes in healthy and IAV-infected mice. Results The oropharyngeal, lung and gut microbiota of healthy mice were dominated by Lactobacillus spp., while nasopharyngeal microbiota were comprised primarily of Streptococcus spp. However, the oropharyngeal, nasopharyngeal, lung, and gut microbiota of IAV-infected mice were dominated by Pseudomonas, Escherichia , Streptococcus , and Muribaculum spp., respectively. Lactobacillus murinus was identified as a biomarker and was reduced at all sites in IAV-infected mice. The microbiota composition of lung was more similar to that of the nasopharynx than the oropharynx in healthy mice. Discussion These findings suggest that the main source of lung microbiota in mice differs from that of adults. Moreover, the similarity between the nasopharyngeal and lung microbiota was increased in IAV-infected mice. We found that IAV infection reduced the similarity between the gut and oropharyngeal microbiota. L. murinus was identified as a biomarker of IAV infection and may be an important target for intervention in post-influenza bacterial superinfections.

Chen, Qichao↗
DOE OSTI2023

Exposing new taxonomic variation with inflammation — a murine model-specific genome database for gut microbiome researchers

The murine CBA/J mouse model widely supports immunology and enteric pathogen research. This model has illuminated Salmonella interactions with the gut microbiome since pathogen proliferation does not require disruptive pretreatment of the native microbiota, nor does it become systemic, thereby representing an analog to gastroenteritis disease progression in humans. Despite the value to broad research communities, microbiota in CBA/J mice are not represented in current murine microbiome genome catalogs. Here we present the first microbial and viral genomic catalog of the CBA/J murine gut microbiome. Using fecal microbial communities from untreated and Salmonella-infected, highly inflamed mice, we performed genomic reconstruction to determine the impacts on gut microbiome membership and functional potential. From high depth whole community sequencing (~ 42.4 Gbps/sample), we reconstructed 2281 bacterial and 4516 viral draft genomes. Salmonella challenge significantly altered gut membership in CBA/J mice, revealing 30 genera and 98 species that were conditionally rare and unsampled in non-inflamed mice. Additionally, inflamed communities were depleted in microbial genes that modulate host anti-inflammatory pathways and enriched in genes for respiratory energy generation. Our findings suggest decreases in butyrate concentrations during Salmonella infection corresponded to reductions in the relative abundance in members of the Alistipes. Strain-level comparison of CBA/J microbial genomes to prominent murine gut microbiome databases identified newly sampled lineages in this resource, while comparisons to human gut microbiomes extended the host relevance of dominant CBA/J inflammation-resistant strains. This CBA/J microbiome database provides the first genomic sampling of relevant, uncultivated microorganisms within the gut from this widely used laboratory model. Using this resource, we curated a functional, strain-resolved view on how Salmonella remodels intact murine gut communities, advancing pathobiome understanding beyond inferences from prior amplicon-based approaches. Salmonella-induced inflammation suppressed Alistipes and other dominant members, while rarer commensals like Lactobacillus and Enterococcus endure. The rare and novel species sampled across this inflammation gradient advance the utility of this microbiome resource to benefit the broad research needs of the CBA/J scientific community, and those using murine models for understanding the impact of inflammation on the gut microbiome more generally.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Improved Microbial Community Characterization of 16S rRNA via Metagenome Hybridization Capture Enrichment

Environmental microbial diversity is often investigated from a molecular perspective using 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics. While amplicon methods are fast, low-cost, and have curated reference databases, they can suffer from amplification bias and are limited in genomic scope. In contrast, shotgun metagenomic methods sample more genomic regions with fewer sequence acquisition biases, but are much more expensive (even with moderate sequencing depth) and computationally challenging. Here, we develop a set of 16S rRNA sequence capture baits that offer a potential middle ground with the advantages from both approaches for investigating microbial communities. These baits cover the diversity of all 16S rRNA sequences available in the Greengenes (v. 13.5) database, with no sequence having <78% sequence identity to at least one bait for all segments of 16S. The use of our baits provide comparable results to 16S amplicon libraries and shotgun metagenomic libraries when assigning taxonomic units from 16S sequences within the metagenomic reads. We demonstrate that 16S rRNA capture baits can be used on a range of microbial samples (i.e., mock communities and rodent fecal samples) to increase the proportion of 16S rRNA sequences (average > 400-fold) and decrease analysis time to obtain consistent community assessments. Furthermore, our study reveals that bioinformatic methods used to analyze sequencing data may have a greater influence on estimates of community composition than library preparation method used, likely due in part to the extent and curation of the reference databases considered. Thus, enriching existing aliquots of shotgun metagenomic libraries and obtaining modest numbers of reads from them offers an efficient orthogonal method for assessment of bacterial community composition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The microbiome structure of decomposing plant leaves in soil depends on plant species, soil pore sizes, and soil moisture content

Microbial communities are known as the primary decomposers of all the carbon accumulated in the soil. However, how important soil structure and its conventional or organic management, moisture content, and how different plant species impact this process are less understood. To answer these questions, we generated a soil microcosm with decomposing corn and soy leaves, as well as soil adjacent to the leaves, and compared it to control samples. We then used high-throughput amplicon sequencing of the ITS and 16S rDNA regions to characterize these microbiomes. Leaf microbiomes were the least diverse and the most even in terms of OTU richness and abundance compared to near soil and far soil, especially in their bacterial component. Microbial composition was significantly and primarily affected by niche (leaves vs. soil) but also by soil management type and plant species in the fungal microbiome, while moisture content and pore sizes were more important drivers for the bacterial communities. The pore size effect was significantly dependent on moisture content, but only in the organic management type. Overall, our results refine our understanding of the decomposition of carbon residues in the soil and the factors that influence it, which are key for environmental sustainability and for evaluating changes in ecosystem functions.

16S rDNA↗
DOE OSTI2020

Development of a thermophilic coculture for corn fiber conversion to ethanol

Abstract The fiber in corn kernels, currently unutilized in the corn to ethanol process, represents an opportunity for introduction of cellulose conversion technology. We report here that Clostridium thermocellum can solubilize over 90% of the carbohydrate in autoclaved corn fiber, including its hemicellulose component glucuronoarabinoxylan (GAX). However, Thermoanaerobacterium thermosaccharolyticum or several other described hemicellulose-fermenting thermophilic bacteria can only partially utilize this GAX. We describe the isolation of a previously undescribed organism, Herbinix spp . strain LL1355, from a thermophilic microbiome that can consume 85% of the recalcitrant GAX. We sequence its genome, and based on structural analysis of the GAX, identify six enzymes that hydrolyze GAX linkages. Combinations of up to four enzymes are successfully expressed in T. thermosaccharolyticum . Supplementation with these enzymes allows T. thermosaccharolyticum to consume 78% of the GAX compared to 53% by the parent strain and increases ethanol yield from corn fiber by 24%.

09 BIOMASS FUELS↗