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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Multiple microbial guilds mediate soil methane cycling along a wetland salinity gradient

ABSTRACT Estuarine wetlands harbor considerable carbon stocks, but rising sea levels could affect their ability to sequester soil carbon as well as their potential to emit methane (CH 4 ). While sulfate loading from seawater intrusion may reduce CH 4 production due to the higher energy yield of microbial sulfate reduction, existing studies suggest other factors are likely at play. Our study of 11 wetland complexes spanning a natural salinity and productivity gradient across the San Francisco Bay and Delta found that while CH 4 fluxes generally declined with salinity, they were highest in oligohaline wetlands (ca. 3-ppt salinity). Methanogens and methanogenesis genes were weakly correlated with CH 4 fluxes but alone did not explain the highest rates observed. Taxonomic and functional gene data suggested that other microbial guilds that influence carbon and nitrogen cycling need to be accounted for to better predict CH 4 fluxes at landscape scales. Higher methane production occurring near the freshwater boundary with slight salinization (and sulfate incursion) might result from increased sulfate-reducing fermenter and syntrophic populations, which can produce substrates used by methanogens. Moreover, higher salinities can solubilize ionically bound ammonium abundant in the lower salinity wetland soils examined here, which could inhibit methanotrophs and potentially contribute to greater CH 4 fluxes observed in oligohaline sediments. IMPORTANCE Low-level salinity intrusion could increase CH4 flux in tidal freshwater wetlands, while higher levels of salinization might instead decrease CH4 fluxes. High CH4 emissions in oligohaline sites are concerning because seawater intrusion will cause tidal freshwater wetlands to become oligohaline. Methanogenesis genes alone did not account for landscape patterns of CH4 fluxes, suggesting mechanisms altering methanogenesis, methanotrophy, nitrogen cycling, and ammonium release, and increasing decomposition and syntrophic bacterial populations could contribute to increases in net CH4 flux at oligohaline salinities. Improved understanding of these influences on net CH4 emissions could improve restoration efforts and accounting of carbon sequestration in estuarine wetlands. More pristine reference sites may have older and more abundant organic matter with higher carbon:nitrogen compared to wetlands impacted by agricultural activity and may present different interactions between salinity and CH4. This distinction might be critical for modeling efforts to scale up biogeochemical process interactions in estuarine wetlands.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Functional insights of novel Bathyarchaeia reveal metabolic versatility in their role in peatlands of the Peruvian Amazon

ABSTRACT The decomposition of soil organic carbon within tropical peatlands is influenced by the functional composition of the microbial community. In this study, building upon our previous work, we recovered a total of 28 metagenome-assembled genomes (MAGs) classified as Bathyarchaeia from the tropical peatlands of the Pastaza-Marañón Foreland Basin (PMFB) in the Amazon. Using phylogenomic analyses, we identified nine genus-level clades to have representatives from the PMFB, with four forming a putative novel family (“CandidatusPaludivitaceae”) endemic to peatlands. We focus on theCa. Paludivitaceae MAGs due to the novelty of this group and the limited understanding of their role within tropical peatlands. Functional analysis of these MAGs reveals that this putative family comprises facultative anaerobes, possessing the genetic potential for oxygen, sulfide, or nitrogen oxidation. This metabolic versatility can be coupled to the fermentation of acetoin, propanol, or proline. The other clades outsideCa. Paludivitaceae are putatively capable of acetogenesis andde novoamino acid biosynthesis and encode a high amount of Fe 3+ transporters. Crucially, theCa. Paludivitaceae are predicted to be carboxydotrophic, capable of utilizing CO for energy generation or biomass production. Through this metabolism, they could detoxify the environment from CO, a byproduct of methanogenesis, or produce methanogenic substrates like CO 2 and H 2 . Overall, our results show the complex metabolism and various lineages of Bathyarchaeia within tropical peatlands pointing to the need to further evaluate their role in these ecosystems. IMPORTANCE With the expansion of theCandidatusPaludivitaceae family by the assembly of 28 new metagenome assembled genomes, this study provides novel insights into their metabolic diversity and ecological significance in peatland ecosystems. From a comprehensive phylogenic and functional analysis, we have elucidated their putative unique facultative anaerobic capabilities and CO detoxification potential. This research highlights their crucial role in carbon cycling and greenhouse gas regulation. These findings are essential for resolving the microbial processes affecting peat soil stability, offering new perspectives on the ecological roles of previously underexplored and underrepresented archaeal populations.

Microbiology↗
DOE OSTI2025

Physiological and metabolic responses of Zymomonas mobilis to lignocellulosic hydrolysate

Zymomonas mobilis is a promising biocatalyst for the sustainable conversion of lignocellulosic sugars into biofuels and bioproducts, yet its response to lignocellulosic hydrolysates remains poorly understood. Here, we investigate the physiological response of Z. mobilis to ammonia fiber expansion (AFEX)-pretreated switchgrass hydrolysate using a systems-level approach integrating LC–MS/MS-based lipidomics and shotgun proteomics. Growth on hydrolysate induced substantial shifts in fatty acid and membrane phospholipid composition, alongside broad proteomic remodeling. Notably, Z. mobilis exhibited a stress response characterized by the upregulation of heat shock proteins and efflux transporters and the downregulation of cell motility proteins. Unexpectedly, hydrolysate exposure also led to a robust upregulation of the Entner–Doudoroff pathway, the ethanol fermentation pathway, and other central carbon metabolism enzymes, indicating a substantial cellular investment potentially driven by additional nutrient availability in hydrolysate. These findings provide new insights into the metabolic adaptations of Z. mobilis to lignocellulosic hydrolysates, informing strategies to enhance its biofuel production capabilities.

Zymomonas mobilis↗
DOE OSTI2015

The short chain fatty acid receptor, FFA2, contributes to gestational glucose homeostasis

The structure of the human gastrointestinal microbiota can change during pregnancy, which may influence gestational metabolism; however, a mechanism of action remains unclear. Here we observed that in wild-type (WT) mice the relative abundance of Actinobacteria and Bacteroidetes increased during pregnancy. Along with these changes, short-chain fatty acids (SCFAs), which are mainly produced through gut microbiota fermentation, significantly changed in both the cecum and peripheral blood throughout gestation in these mice. SCFAs are recognized by G protein-coupled receptors (GPCRs) such as free fatty acid receptor-2 (FFA2), and we have previously demonstrated that the fatty acid receptor-2 gene ( Ffar2 ) expression is higher in pancreatic islets during pregnancy. Using female Ffar2 -/- mice, we explored the physiological relevance of signaling through this GPCR and found that Ffar2-deficient female mice developed fasting hyperglycemia and impaired glucose tolerance in the setting of impaired insulin secretion compared with WT mice during, but not before, pregnancy. Insulin tolerance tests were similar in Ffar2 -/- and WT mice before and during pregnancy. Next, we examined the role of FFA2 in gestational β-cell mass, observing that Ffar2 -/- mice had diminished gestational expansion of β-cells during pregnancy. Interestingly, mouse genotype had no significant impact on the composition of the gut microbiome, but did affect the observed SCFA profiles, suggesting a functional difference in the microbiota. Altogether, these results suggest a potential link between increased Ffar2 expression in islets and the alteration of circulating SCFA levels, possibly explaining how changes in the gut microbiome contribute to gestational glucose homeostasis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Screening of industrial wastewaters as feedstock for the microbial production of oils for biodiesel production and high-quality pigments

The production of biodiesel has notably increased over the past decade. Currently, plant oil is the main feedstock for biodiesel production, but, due to concerns related to the competition with food production, alternative oil feedstocks have to be found. Oleaginous yeasts are known to produce high amounts of lipids, but no integrated process from microbial fermentation to final biodiesel production has reached commercial realization yet due to economic constraints. Therefore, growth and lipid production of red yeast Rhodotorula glutinis was tested on low-cost substrates, namely, wastewaters from potato, fruit juice, and lettuce processing. Additionally, the production of carotenoids as high-value by-products was examined. All evaluated wastewaters met the general criteria for microbial lipid production. However, no significant increase in lipid content was observed, probably due to lack of available carbon in wastewaters from fruit juice and lettuce processing, and excess of available nitrogen in potato processing wastewater, respectively. During growth on wastewaters from fruit juice and lettuce processing the carotenoid content increased significantly in the first 48 hours. The relations between carbon content, nitrogen content, and carotenoid production need to be further assessed. For economic viability, lipid and carotenoid production needs to be increased significantly. Lastly, the screening of feedstocks should be extended to other wastewaters.

09 BIOMASS FUELS↗
DOE OSTI2012

Analysis of Casein Biopolymers Adsorption to Lignocellulosic Biomass as a Potential Cellulase Stabilizer

Although lignocellulosic materials have a good potential to substitute current feedstocks used for ethanol production, conversion of these materials to fermentable sugars is still not economical through enzymatic hydrolysis. High cost of cellulase has prompted research to explore techniques that can prevent from enzyme deactivation. Colloidal proteins of casein can form monolayers on hydrophobic surfaces that alleviate the de-activation of protein of interest. Scanning electron microscope (SEM), fourier transform infrared spectroscopy (FT-IR), capillary electrophoresis (CE), and Kjeldahl and BSA protein assays were used to investigate the unknown mechanism of action of induced cellulase activity during hydrolysis of casein-treated biomass. Adsorption of casein to biomass was observed with all of the analytical techniques used and varied depending on the pretreatment techniques of biomass. FT-IR analysis of amides I and II suggested that the substructure of protein from casein or skim milk were deformed at the time of contact with biomass. With no additive, the majority of one of the cellulase mono-component, 97.1 ± 1.1, was adsorbed to CS within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein reduced the adsorption to 32.9% ± 6.0 and 82.8% ± 6.0, respectively.

Eckard, Anahita Dehkhoda↗
DOE OSTI2014

Characterization of Ten Heterotetrameric NDP-Dependent Acyl-CoA Synthetases of the Hyperthermophilic Archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus grows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of ATP. ACS1 and ACS2 were previously purified from P. furiosus and have α 2 β 2 structures but the genome contains genes encoding three additional α -subunits. The ten possible combinations of α and β genes were expressed in E. coli and each resulted in stable and active α 2 β 2 isoenzymes. The α -subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids. The β -subunit determined preference for adenine or guanine nucleotides. The GTP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GTP for GTP-dependent phosphoenolpyruvate carboxykinase and for other GTP-dependent processes. Transcriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both ATP and GTP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs of P. furiosus and other members of the Thermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Current Challenges in Commercially Producing Biofuels from Lignocellulosic Biomass

Biofuels that are produced from biobased materials are a good alternative to petroleum based fuels. They offer several benefits to society and the environment. Producing second generation biofuels is even more challenging than producing first generation biofuels due the complexity of the biomass and issues related to producing, harvesting, and transporting less dense biomass to centralized biorefineries. In addition to this logistic challenge, other challenges with respect to processing steps in converting biomass to liquid transportation fuel like pretreatment, hydrolysis, microbial fermentation, and fuel separation still exist and are discussed in this review. The possible coproducts that could be produced in the biorefinery and their importance to reduce the processing cost of biofuel are discussed. About $1 billion was spent in the year 2012 by the government agencies in US to meet the mandate to replace 30% existing liquid transportation fuels by 2022 which is 36 billion gallons/year. Other countries in the world have set their own targets to replace petroleum fuel by biofuels. Because of the challenges listed in this review and lack of government policies to create the demand for biofuels, it may take more time for the lignocellulosic biofuels to hit the market place than previously projected.

09 BIOMASS FUELS↗
DOE OSTI2023

A Landing Pad System for Multicopy Gene Integration in Issatchenkia orientalis

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user’s choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

Fatma, Zia↗
DOE OSTI2024

raogroupuiuc/yl_variantcalling

NGS analysis for mutation analysis in Yarrowia lipolytica evolved strains. Yarrowia lipolytica, an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.

Deewan, Anshu↗
DOE OSTI2013

Characterizing acetogenic metabolism using a genome-scale metabolic reconstruction of Clostridium ljungdahlii

Background: The metabolic capabilities of acetogens to ferment a wide range of sugars, to grow autotrophically on H-2/CO2, and more importantly on synthesis gas (H-2/CO/CO2) make them very attractive candidates as production hosts for biofuels and biocommodities. Acetogenic metabolism is considered one of the earliest modes of bacterial metabolism. A thorough understanding of various factors governing the metabolism, in particular energy conservation mechanisms, is critical for metabolic engineering of acetogens for targeted production of desired chemicals. Results: Here, we present the genome-scale metabolic network of Clostridium ljungdahlii, the first such model for an acetogen. This genome-scale model (iHN637) consisting of 637 genes, 785 reactions, and 698 metabolites captures all the major central metabolic and biosynthetic pathways, in particular pathways involved in carbon fixation and energy conservation. A combination of metabolic modeling, with physiological and transcriptomic data provided insights into autotrophic metabolism as well as aided the characterization of a nitrate reduction pathway in C. ljungdahlii. Analysis of the iHN637 metabolic model revealed that flavin based electron bifurcation played a key role in energy conservation during autotrophic growth and helped identify genes for some of the critical steps in this mechanism. Conclusions: iHN637 represents a predictive model that recapitulates experimental data, and provides valuable insights into the metabolic response of C. ljungdahlii to genetic perturbations under various growth conditions. Thus, the model will be instrumental in guiding metabolic engineering of C. ljungdahlii for the industrial production of biocommodities and biofuels.

Nagarajan, H↗
DOE OSTI2012

In-silico identification of phenotype-biased functional modules

Phenotypes exhibited by microorganisms can be useful for several purposes, e.g., ethanol as an alternate fuel. Sometimes, the target phenotype maybe required in combination with other phenotypes, in order to be useful, for e.g., an industrial process may require that the organism survive in an anaerobic, alcohol rich environment and be able to feed on both hexose and pentose sugars to produce ethanol. This combination of traits may not be available in any existing organism or if they do exist, the mechanisms involved in the phenotype-expression may not be efficient enough to be useful. Thus, it may be required to genetically modify microorganisms. However, before any genetic modification can take place, it is important to identify the underlying cellular subsystems responsible for the expression of the target phenotype. In this paper, we develop a method to identify statistically significant and phenotypically-biased functional modules. The method can compare the organismal network information from hundreds of phenotype expressing and phenotype non-expressing organisms to identify cellular subsystems that are more prone to occur in phenotype-expressing organisms than in phenotype non-expressing organisms. We have provided literature evidence that the phenotype-biased modules identified for phenotypes such as hydrogen production (dark and light fermentation), respiration, gram-positive, gram-negative and motility, are indeed phenotype-related. Thus we have proposed a methodology to identify phenotype-biased cellular subsystems. We have shown the effectiveness of our methodology by applying it to several target phenotypes. The code and all supplemental files can be downloaded from (http://freescience.org/cs/phenotype-biased-biclusters/).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Rapid construction of metabolic models for a family of Cyanobacteria using a multiple source annotation workflow

Cyanobacteria are photoautotrophic prokaryotes that exhibit robust growth under diverse environmental conditions with minimal nutritional requirements. They can use solar energy to convert CO 2 and other reduced carbon sources into biofuels and chemical products. The genus Cyanothece includes unicellular nitrogen-fixing cyanobacteria that have been shown to offer high levels of hydrogen production and nitrogen fixation. The reconstruction of quality genome-scale metabolic models for organisms with limited annotation resources remains a challenging task. Here we reconstruct and subsequently analyze and compare the metabolism of five Cyanothece strains, namely Cyanothece sp. PCC 7424, 7425, 7822, 8801 and 8802, as the genome-scale metabolic reconstructions iCyc792, iCyn731, iCyj826, iCyp752, and iCyh755 respectively. We compare these phylogenetically related Cyanothece strains to assess their bio-production potential. A systematic workflow is introduced for integrating and prioritizing annotation information from the Universal Protein Resource (Uniprot), NCBI Protein Clusters, and the Rapid Annotations using Subsystems Technology (RAST) method. The genome-scale metabolic models include fully traced photosynthesis reactions and respiratory chains, as well as balanced reactions and GPR associations. Metabolic differences between the organisms are highlighted such as the non-fermentative pathway for alcohol production found in only Cyanothece 7424, 8801, and 8802. Our development workflow provides a path for constructing models using information from curated models of related organisms and reviewed gene annotations. This effort lays the foundation for the expedient construction of curated metabolic models for organisms that, while not being the target of comprehensive research, have a sequenced genome and are related to an organism with a curated metabolic model. Organism-specific models, such as the five presented in this paper, can be used to identify optimal genetic manipulations for targeted metabolite overproduction as well as to investigate the biology of diverse organisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Comparison of sugar content for ionic liquid pretreated Douglas-fir woodchips and forestry residues

The development of affordable woody biomass feedstocks represents a significant opportunity in the development of cellulosic biofuels. Primary woodchips produced by forest mills are considered an ideal feedstock, but the prices they command on the market are currently too expensive for biorefineries. In comparison, forestry residues represent a potential low-cost input but are considered a more challenging feedstock for sugar production due to complexities in composition and potential contamination arising from soil that may be present. We compare the sugar yields, changes in composition in Douglas-fir woodchips and forestry residues after pretreatment using ionic liquids and enzymatic saccharification in order to determine if this approach can efficiently liberate fermentable sugars. : These samples were either mechanically milled through a 2 mm mesh or pretreated as received with the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate [C 2 mim][OAc] at 120°C and 160°C. IL pretreatment of Douglas-fir woodchips and forestry residues resulted in approximately 71-92% glucose yields after enzymatic saccharification. X-ray diffraction (XRD) showed that the pretreated cellulose was less crystalline after IL pretreatment as compared to untreated control samples. Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) revealed changes in lignin and hemicellulose structure and composition as a function of pretreatment. Mass balances of sugar and lignin streams for both the Douglas-fir woodchips and forestry residues throughout the pretreatment and enzymatic saccharification processes are presented. While the highest sugar yields were observed with the Douglas-fir woodchips, reasonably high sugar yields were obtained from forestry residues after ionic liquid pretreatment. Structural changes to lignin, cellulose and hemicellulose in the woodchips and forestry residues of Douglas-fir after [C 2 mim][OAc] pretreatment are analyzed by XRD and 2D-NMR, and indicate that significant changes occurred. Irrespective of the particle sizes used in this study, ionic liquid pretreatment successfully allowed high glucose yields after enzymatic saccharification. These results indicate that forestry residues may be a more viable feedstock than previously thought for the production of biofuels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Metabolic engineering of Caldicellulosiruptor bescii yields increased hydrogen production from lignocellulosic biomass

Background: Members of the anaerobic thermophilic bacterial genus Caldicellulosiruptor are emerging candidates for consolidated bioprocessing (CBP) because they are capable of efficiently growing on biomass without conventional pretreatment. C. bescii produces primarily lactate, acetate and hydrogen as fermentation products, and while some Caldicellulosiruptor strains produce small amounts of ethanol C. bescii does not, making it an attractive background to examine the effects of metabolic engineering. The recent development of methods for genetic manipulation has set the stage for rational engineering of this genus for improved biofuel production. Here, we report the first targeted gene deletion, the gene encoding lactate dehydrogenase (ldh), for metabolic engineering of a member of this genus. Results: A deletion of the C. bescii L-lactate dehydrogenase gene (ldh) was constructed on a non-replicating plasmid and introduced into the C. bescii chromosome by marker replacement. The resulting strain failed to produce detectable levels of lactate from cellobiose and maltose, instead increasing production of acetate and H2 by 21-34% relative to the wild type and pyrFA parent strains. The same phenotype was observed on a real-world substrate switchgrass (Panicum virgatum). Furthermore, the ldh deletion strain grew to a higher maximum optical density than the wild type on maltose and cellobiose, consistent with the prediction that the mutant would gain additional ATP with increased acetate production. Conclusions: Deletion of ldh in C. bescii is the first use of recently developed genetic methods for metabolic engineering of these bacteria. This deletion resulted in a redirection of electron flow from production of lactate to acetate and hydrogen. New capabilities in metabolic engineering combined with intrinsic utilization of lignocellulosic materials position these organisms to provide a new paradigm for consolidated bioprocessing of fuels and other products from biomass.

Biohydrogen↗
DOE OSTI2014

Improvement of cellulose catabolism in Clostridium cellulolyticum by sporulation abolishment and carbon alleviation

Background Clostridium cellulolyticum can degrade lignocellulosic biomass, and ferment the soluble sugars to produce valuable chemicals such as lactate, acetate, ethanol and hydrogen. However, the cellulose utilization efficiency of C. cellulolyticum still remains very low, impeding its application in consolidated bioprocessing for biofuels production. In this study, two metabolic engineering strategies were exploited to improve cellulose utilization efficiency, including sporulation abolishment and carbon overload alleviation. Results The spo0A gene at locus Ccel_1894, which encodes a master sporulation regulator was inactivated. The spo0A mutant abolished the sporulation ability. In a high concentration of cellulose (50 g/l), the performance of the spo0A mutant increased dramatically in terms of maximum growth, final concentrations of three major metabolic products, and cellulose catabolism. The microarray and gas chromatography mass spectrometry (GC-MS) analyses showed that the valine, leucine and isoleucine biosynthesis pathways were up-regulated in the spo0A mutant. Based on this information, a partial isobutanol producing pathway modified from valine biosynthesis was introduced into C. cellulolyticum strains to further increase cellulose consumption by alleviating excessive carbon load. The introduction of this synthetic pathway to the wild-type strain improved cellulose consumption from 17.6 g/l to 28.7 g/l with a production of 0.42 g/l isobutanol in the 50 g/l cellulose medium. However, the spo0A mutant strain did not appreciably benefit from introduction of this synthetic pathway and the cellulose utilization efficiency did not further increase. A technical highlight in this study was that an in vivo promoter strength evaluation protocol was developed using anaerobic fluorescent protein and flow cytometry for C. cellulolyticum. Conclusions In this study, we inactivated the spo0A gene and introduced a heterologous synthetic pathway to manipulate the stress response to heavy carbon load and accumulation of metabolic products. These findings provide new perspectives to enhance the ability of cellulolytic bacteria to produce biofuels and biocommodities with high efficiency and at low cost directly from lignocellulosic biomass.

cellulose catabolism↗
DOE OSTI2013

Community genomic analyses constrain the distribution of metabolic traits across the Chloroflexi phylum and indicate roles in sediment carbon cycling

Background: Sediments are massive reservoirs of carbon compounds and host a large fraction of microbial life. Microorganisms within terrestrial aquifer sediments control buried organic carbon turnover, degrade organic contaminants, and impact drinking water quality. Recent 16S rRNA gene profiling indicates that members of the bacterial phylum Chloroflexi are common in sediment. Only the role of the class Dehalococcoidia, which degrade halogenated solvents, is well understood. Genomic sampling is available for only six of the approximate 30 Chloroflexi classes, so little is known about the phylogenetic distribution of reductive dehalogenation or about the broader metabolic characteristics of Chloroflexi in sediment. Results: We used metagenomics to directly evaluate the metabolic potential and diversity of Chloroflexi in aquifer sediments. We sampled genomic sequence from 86 Chloroflexi representing 15 distinct lineages, including members of eight classes previously characterized only by 16S rRNA sequences. Unlike in the Dehalococcoidia, genes for organohalide respiration are rare within the Chloroflexi genomes sampled here. Near-complete genomes were reconstructed for three Chloroflexi. One, a member of an unsequenced lineage in the Anaerolinea, is an aerobe with the potential for respiring diverse carbon compounds. The others represent two genomically unsampled classes sibling to the Dehalococcoidia, and are anaerobes likely involved in sugar and plant-derived-compound degradation to acetate. Both fix CO 2 via the Wood-Ljungdahl pathway, a pathway not previously documented in Chloroflexi. The genomes each encode unique traits apparently acquired from Archaea, including mechanisms of motility and ATP synthesis. Conclusions: Chloroflexi in the aquifer sediments are abundant and highly diverse. Genomic analyses provide new evolutionary boundaries for obligate organohalide respiration. We expand the potential roles of Chloroflexi in sediment carbon cycling beyond organohalide respiration to include respiration of sugars, fermentation, CO 2 fixation, and acetogenesis with ATP formation by substrate-level phosphorylation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Multiphasic analysis of the temporal development of the distal gut microbiota in patients following ileal pouch anal anastomosis

The indigenous gut microbiota are thought to play a crucial role in the development and maintenance of the abnormal inflammatory responses that are the hallmark of inflammatory bowel disease. Direct tests of the role of the gut microbiome in these disorders are typically limited by the fact that sampling of the microbiota generally occurs once disease has become manifest. This limitation could potentially be circumvented by studying patients who undergo total proctocolectomy with ileal pouch anal anastomosis (IPAA) for the definitive treatment of ulcerative colitis. A subset of patients who undergo IPAA develops an inflammatory condition known as pouchitis, which is thought to mirror the pathogenesis of ulcerative colitis. Following the development of the microbiome of the pouch would allow characterization of the microbial community that predates the development of overt disease. We monitored the development of the pouch microbiota in four patients who underwent IPAA. Mucosal and luminal samples were obtained prior to takedown of the diverting ileostomy and compared to samples obtained 2, 4 and 8 weeks after intestinal continuity had been restored. Through the combined analysis of 16S rRNA-encoding gene amplicons, targeted 16S amplification and microbial cultivation, we observed major changes in structure and function of the pouch microbiota following ileostomy. There is a relative increase in anaerobic microorganisms with the capacity for fermentation of complex carbohydrates, which corresponds to the physical stasis of intestinal contents in the ileal pouch. Compared to the microbiome structure encountered in the colonic mucosa of healthy individuals, the pouch microbial community in three of the four individuals was quite distinct. In the fourth patient, a community that was much like that seen in a healthy colon was established, and this patient also had the most benign clinical course of the four patients, without the development of pouchitis 2 years after IPAA. The microbiota that inhabit the ileal-anal pouch of patients who undergo IPAA for treatment of ulcerative colitis demonstrate significant structural and functional changes related to the restoration of fecal flow. Our preliminary results suggest once the pouch has assumed the physiologic role previously played by the intact colon, the precise structure and function of the pouch microbiome, relative to a normal colonic microbiota, will determine if there is establishment of a stable, healthy mucosal environment or the reinitiation of the pathogenic cascade that results in intestinal inflammation.

59 BASIC BIOLOGICAL SCIENCES↗