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At least 1,027 records · Page 57
All sourcesNASA NTRSDOE OSTI
DOE OSTI2019

Subsidized or stressed? Shifts in freshwater benthic microbial metagenomics along a gradient of alkaline coal mine drainage

Chemical pollution mixtures enter aquatic environments and interact with microorganisms in eclectic ways with disparate consequences for microbial ecosystem services. Can using a thermodynamic framework help to determine the net influence of a chemical mixture on the functional capacity of benthic microbial communities? In this study, we examined this question by comparing benthic stream microbial communities exposed to a gradient of neutral-alkaline coalmine effluent. Using a combination of approaches (metagenomics, quantitative polymerase chain reaction [qPCR], and functional assays), we show that functional genes and pathways of microbial communities growing in mine effluent differed in composition, but not diversity. The majority of functional genes and pathways that changed decreased at sites exposed to mine effluent, resulting in lower abundances of nitrogenase and methanogen genes and fermentation pathways. However, selenate reductase gene abundance increased with water and sediment concentration of an ecologically important contaminant at mined sites: selenium. Denitrification genes nosZ and nirK differed between sites: metagenome-based nosZ increased with dissolved nitrate concentration and qPCR-based nirK had a hump-shaped pattern across the mining gradient. Osmoprotectant gene abundance did not change. Extracellular enzyme assays and alkaline phosphatase gene relative abundance suggested that mined stream microbial communities may be constrained by phosphorus bioavailability. Subsidies and stressors related to changes in a set of functional genes and pathways, but differences were not consistently predictable using thermodynamic expectations. Laslty, this suggests that pairing hypotheses for expected subsidies and stressors with post hoc explorations can yield valuable directions for future study of how microbial functional capacity responds to pollutant mixtures.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2018

Rex in Caldicellulosiruptor bescii : Novel regulon members and its effect on the production of ethanol and overflow metabolites

Abstract Rex is a global redox‐sensing transcription factor that senses and responds to the intracellular [ NADH ]/[ NAD + ] ratio to regulate genes for central metabolism, and a variety of metabolic processes in Gram‐positive bacteria. We decipher and validate four new members of the Rex regulon in Caldicellulosiruptor bescii ; a gene encoding a class V aminotransferase, the HydG FeFe Hydrogenase maturation protein, an oxidoreductase, and a gene encoding a hypothetical protein. Structural genes for the NiFe and FeFe hydrogenases, pyruvate:ferredoxin oxidoreductase, as well as the rex gene itself are also members of this regulon, as has been predicted previously in different organisms. A C. bescii rex deletion strain constructed in an ethanol‐producing strain made 54% more ethanol (0.16 mmol/L) than its genetic parent after 36 hr of fermentation, though only under nitrogen limited conditions. Metabolomic interrogation shows this rex‐ deficient ethanol‐producing strain synthesizes other reduced overflow metabolism products likely in response to more reduced intracellular redox conditions and the accumulation of pyruvate. These results suggest ethanol production is strongly dependent on the native intracellular redox state in C. bescii , and highlight the combined promise of using this gene and manipulation of culture conditions to yield strains capable of producing ethanol at higher yields and final titer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2011

Structural and Enzymatic Characterization of NanS (YjhS) a 9-O-Acetyl N-acetylneuraminic Acid Esterase from Escherichia coli O157:H7

There is a high prevalence of sialic acid in a number of different organisms, resulting in there being a myriad of different enzymes that can exploit it as a fermentable carbon source. One such enzyme is NanS, a carbohydrate esterase that we show here deacetylates the 9 position of 9-O-sialic acid so that it can be readily transported into the cell for catabolism. Through structural studies, we show that NanS adopts a SGNH hydrolase fold. Although the backbone of the structure is similar to previously characterized family members, sequence comparisons indicate that this family can be further subdivided into two subfamilies with somewhat different fingerprints. NanS is the founding member of group II. Its catalytic center contains Ser19 and His301 but no Asp/Glu is present to form the classical catalytic triad. The contribution of Ser19 and His301 to catalysis was confirmed by mutagenesis. In addition to structural characterization, we have mapped the specificity of NanS using a battery of substrates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Influence of lure choice and survey duration on scent stations for carnivore surveys

Abstract Noninvasive, camera‐based, scent station surveys have become a common approach to estimate population parameters for mammalian carnivores. Many carnivores are particularly sensitive to olfactory cues; and, as such, the quality and quantity of odor attractant used during surveys can significantly affect the accuracy and precision of the survey. Additionally, survey length is also an important consideration because many mammalian predators are highly mobile and elusive in nature. Using scent station surveys in autumn 2013 and 2014 on the Savannah River Site, Aiken, South Carolina, USA, we tested the effects of 5 lures: fatty acid, fish oil, synthetic fermented egg, beaver castor, and skunk essence on visitation rates, visitation probability, and latency to visitation of multiple predator species. We also estimated occupancy probability for 2 carnivore species using different survey lengths to determine the effect of survey period on the precision of occupancy estimates. We found that skunk essence had greater visitation probability and visits than other lures evaluated. Species‐specific bait‐preference models revealed that raccoons ( Procyon lotor ) were more likely to visit skunk essence, and coyotes ( Canis latrans ) were less likely to visit fish oil as compared with fatty acid. Latency to visitation ranged from 104.7 to 112.1 hours and did not vary among scents. Longer survey durations resulted in more precise estimates of occupancy and detection probability. Our results suggest a wide range of scents commonly used for carnivore capture may perform as well as, or better than, fatty acid for noninvasive carnivore surveys. Further, study designs should carefully consider the duration of scent‐station surveys depending on the level of precision desired in estimates of occupancy and detection probability. © 2019 The Wildlife Society.

Webster, Sarah C.↗
DOE OSTI2022

The Brazilian Amazonian rainforest harbors a high diversity of yeasts associated with rotting wood, including many candidates for new yeast species

This study investigated the diversity of yeast species associated with rotting wood in Brazilian Amazonian rainforests. Herein, a total of 569 yeast strains were isolated from rotting wood samples collected in three Amazonian areas (Universidade Federal do Amazonas-Universidade Federal do Amazonas [UFAM], Piquiá, and Carú) in the municipality of Itacoatiara, Amazon state. The samples were cultured in yeast nitrogen base (YNB)-d-xylose, YNB-xylan, and sugarcane bagasse and corncob hemicellulosic hydrolysates (undiluted and diluted 1:2 and 1:5). Sugiyamaella was the most prevalent genus identified in this work, followed by Kazachstania. The most frequently isolated yeast species were Schwanniomyces polymorphus, Scheffersomyces amazonensis, and Wickerhamomyces sp., respectively. The alpha diversity analyses showed that the dryland forest of UFAM was the most diverse area, while the floodplain forest of Carú was the least. Additionally, the difference in diversity between UFAM and Carú was the highest among the comparisons. Thirty candidates for new yeast species were obtained, representing 36% of the species identified and totaling 101 isolates. Among them were species belonging to the clades Spathaspora, Scheffersomyces, and Sugiyamaella, which are recognized as genera with natural xylose-fermenting yeasts that are often studied for biotechnological and ecological purposes. The results of this work showed that rotting wood collected from the Amazonian rainforest is a tremendous source of diverse yeasts, including candidates for new species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Taxogenomic Analysis of a Novel Yeast Species, Lachancea rosae Sp. Nov. F.A., Isolated From the Wild Rose Rosa californica

A novel Saccharomycotina yeast strain, yHQL494, was isolated from the rose hip of the wild rose Rosa californica from Castle Crags State Park, California, USA. Phylogenetic analyses of both whole genome data and the sequences from the D1/D2 region of the large ribosomal subunit (LSU) rRNA gene placed strain yHQL494 within the genus Lachancea and grouped it into a clade with Lachancea lanzarotensis and Lachancea meyersii. Taxogenomic analyses were conducted on publicly available genome sequences to gain a deeper insight into the carbon and nitrogen gene-trait associations across the Lachancea clade. The results of these analyses were found to be consistent across Lachancea species. Growth assays and microscopic analyses were conducted to determine the physiological characteristics of strain yHQL494, including the presence of hyphae or pseudohyphae, ascospore formation, fermentation abilities, and assimilation of carbon and nitrogen compounds. Based on the phenotypic and genomic characteristics of the strain yHQL494 T (=NRRL Y-64858 T , =CBS 18,574 T ), we propose a new species, Lachancea rosae sp. nov. f.a.

Bajgain, Yakendra [Villanova Univ., PA (United Sta↗
DOE OSTI2020

Chapter 2: Genetics of Unstudied Thermophiles for Industry

Thermophilic organisms hold great potential for industry due to their numerous advantages in biotechnological applications such as higher reaction rate, higher substrate loading, decreased susceptibility to reaction contamination, energy savings in industrial fermentations, and ability to express thermostable proteins that can be utilized in many important industrial processes. Bioprospecting for thermophiles will continue to reveal new enzymatic and metabolic paradigms with industrial applicability. In order to translate these paradigms to production scale, routine methods for microbial genetic engineering are needed, yet remain to be developed in many newly isolated thermophiles. Major challenges and recent developments in the establishment of reliable genetic systems in thermophiles are discussed. Here, we use a hyperthermophilic, cellulolytic bacterium, Caldicellulosiruptor bescii, as a case study to demonstrate the development of a genetic system for an industrially useful thermophile, describing in detail methods for transformation, genetic tool utilization, and chromosomal modification using targeted gene deletion and insertion techniques.

93 SC - Biological and Environmental Research (BER↗
DOE OSTI2021

CRISPR-Cas Genome Editing in the Cellulolytic Bacterium Clostridium thermocellum (C. thermocellum)

Clostridium thermocellum is an anaerobic thermophile that can efficiently degrade lignocellulosic biomass and directly convert it into value-added products such as ethanol. The cellulolytic and ethanologenic capabilities of C. thermocellum make it an excellent candidate for consolidated bioprocessing (CBP) for industrial production, where biomass degradation and fermentation occur simultaneously. In this organism, strain development for effective CBP has traditionally been hindered by the lack of genetic tools. Here, we detail our efficient two-step CRISPR-Cas genome editing protocol for C. thermocellum, using both the native Type I-B CRISPR-Cas system and an exogenous Type-II CRISPR system from Geobacillus stearothermophilus. As recombination is limiting in C. thermocellum genome engineering, we highlight effective thermophilic recombinases necessary to improve genome editing in these systems. We additionally provide design rules for the repair template and synthetic guide RNA (gRNA) for each system. Using these newly developed CRISPR and recombineering tools, targeted C. thermocellum engineering will substantiate efforts toward CBP strain development in industrial applications.

anaerobe↗
DOE OSTI2019

Rhodococcus and Yarrowia-Based Lipid Production Using Lignin-Containing Industrial Residues

Improvement in biorefining technologies coupled with development of novel fermentation strategies and analysis will be paramount in establishing supplementary and sustainable biofuel pathways. Oleaginous microorganisms that are capable of accumulating triacylglycerides (TAGs) and fatty acid methyl esters (FAMEs), such as Rhodococcus and Yarrowia species, can be used to produce second-generation biofuels from non-food competing carbon sources. These “microbiorefineries” provide a pathway to upgrade agricultural and industrial waste streams to fungible fuels or precursors to chemicals and materials. Here we provide a general overview on cultivating Rhodococcus and Yarrowia on agro-waste/industrial biomass pretreatment waste streams to produce single-cell oils/lipids and preparing samples for FAME detection.

Le, Rosemary K.↗
DOE OSTI2021

High-Biomass Sorghums as a Feedstock for Renewable Fuels and Chemicals

High-biomass sorghums are intended for use in biorefineries that convert vegetative biomass into renewable fuels and chemicals. The majority of plant biomass consists of cell walls, a complex matrix of cellulose, hemicellulosic polysaccharides and lignin. In the biorefinery, the biomass is ground and then subjected to a thermo-chemical pretreatment (elevated temperature, high pressure, chemical catalysts) that disrupts the cell wall matrix and solubilizes some of the cell wall polymers, followed by enzymatic saccharification of the cellulose, which produces D-glucose. The glucose and sometimes also the xylose derived from the hydrolysis of the hemicellulosic polysaccharides, are subsequently converted to fuels or other useful chemicals by microbial biocatalysts. The genetic improvement of high-biomass sorghums has as its ultimate goals to maximize the yield of fermentable sugars on a per-hectare basis, to minimize the inputs of fertilizer, irrigation, fungicides and pesticides and to reduce the environmental footprint. Breeding strategies thus need to target biomass yield, biomass composition, maturity, pest and disease resistance and nutrient use efficiency. Furthermore, this chapter reviews the genetic basis of these traits and their potential application in breeding programs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Ionic liquid-tolerant microorganisms and microbial communities for lignocellulose conversion to bioproducts

Chemical and physical pretreatment of biomass is a critical step in the conversion of lignocellulose to biofuels and bioproducts. Ionic liquid (IL) pretreatment has attracted significant attention due to the unique ability of certain ILs to solubilize some or all components of the plant cell wall. However, these ILs inhibit not only the enzyme activities but also the growth and productivity of microorganisms used in downstream hydrolysis and fermentation processes. While pretreated biomass can be washed to remove residual IL and reduce inhibition, extensive washing is costly and not feasible in large-scale processes. IL-tolerant microorganisms and microbial communities have been discovered from environmental samples and studies begun to elucidate mechanisms of IL tolerance. The discovery of IL tolerance in environmental microbial communities and individual microbes has lead to the proposal of molecular mechanisms of resistance. Here, we review recent progress on discovering IL-tolerant microorganisms, identifying metabolic pathways and mechanisms of tolerance, and engineering microorganisms for IL tolerance. Research in these areas will yield new approaches to overcome inhibition in lignocellulosic biomass bioconversion processes and increase opportunities for the use of ILs in biomass pretreatment.

09 BIOMASS FUELS↗
DOE OSTI2019

Development of a shuttle plasmid without host restriction sites for efficient transformation and heterologous gene expression in Clostridium cellulovorans

Clostridium cellulovorans capable of producing large amounts of acetate and butyrate from cellulose is a promising candidate for biofuels and biochemicals production from lignocellulosic biomass. However, the restriction modification (RM) systems of C. cellulovorans hindered the application of existing shuttle plasmids for metabolic engineering of this organism. To overcome the hurdle of plasmid digestion by host, a new shuttle plasmid (pYL001) was developed to remove all restriction sites of two major RM systems of C. cellulovorans , Cce 743 I and Cce 743 II. The pYL001 plasmid remained intact after challenge by C. cellulovorans cell extract. Post-electroporation treatments and culturing conditions were also modified to improve cell growth and colony formation on agar plates. With the improvements, the pYL001 plasmid, without in-vivo methylation, was readily transformed into C. cellulovorans with colonies of recombinant cells formed on agar plates within 24 h. Three pYL001-derived recombinant plasmids free of Cce 743 I/ Cce 743 II restriction sites, after synonymous mutation of the heterologous genes, were constructed and transformed into C. cellulovorans . Functional expression of these genes was confirmed with butanol and ethanol production from glucose in batch fermentations by the transformants. In conclusion, the pYL001 plasmid and improved transformation method can facilitate further metabolic engineering of C. cellulovorans for cellulosic butanol production.

09 BIOMASS FUELS↗
DOE OSTI2021

Bacterial α-diglucoside metabolism: perspectives and potential for biotechnology and biomedicine

In a competitive microbial environment nutrient acquisition is a major contributor to the survival of any individual bacterial species, and the ability to access uncommon energy sources can provide a fitness advantage. One set of soluble carbohydrates that have attracted increased attention for use in biotechnology and biomedicine are the α-diglucosides. Maltose is the most well studied member of this class, however the remaining four less common α-diglucosides (trehalose, kojibiose, nigerose, and isomaltose) are increasingly used in processed food and fermented beverages. The consumption of trehalose has recently been shown to be a contributing factor in gut microbiome disease as certain pathogens are using α-diglucosides to outcompete native gut flora. Kojibiose and nigerose have also been examined potential prebiotics and alternative sweeteners for a variety of foods. Compared to the study of maltose metabolism our understanding of the synthesis and degradation of uncommon α-diglucosides is lacking and several fundamental questions that remain unanswered, particularly in regards to the regulation of bacterial metabolism for α-diglucosides. Therefore this minireview attempts to provide a focused analysis of uncommon α-diglucoside metabolism in bacteria, and suggests some future directions for this research area that could potentially accelerate biotechnology and biomedicine developments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Investigating the role of the transcriptional regulator Ure2 on the metabolism of Saccharomyces cerevisiae: a multi-omics approach

Ure2 regulates nitrogen catabolite repression in Saccharomyces cerevisiae. Deletion of URE2 induces a physiological state mimicking the nitrogen starvation and autophagic responses. Previous work has shown that deletion of URE2 increases the fermentation rate of some wine-producing strains of S. cerevisiae. In this work, we investigated the effect of URE2 deletion (ΔURE2) on the metabolism of S. cerevisiae. During growth on glucose, the ΔURE2 mutant grew less well than the wild type; however, it produced ethanol at higher rates. To better under the behavior of this mutant, we performed transcriptomics and metabolomics. Analysis of the RNA sequencing results and metabolite levels indicate that the mutant strain exhibited characteristics of both nitrogen starvation and autophagy. In addition, many pyruvate decarboxylase and alcohol dehydrogenase isoforms were expressed at higher rates than the wild type. The mutant also accumulated less trehalose and glycogen, and produced more lipids. These results suggest that URE2 may be a promising target for metabolic engineering in S. cerevisiae and potentially other strains of yeast as well for the production of lipid-based fuels and chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Ultrasound-guided percutaneous catheter drainage of various types of ruptured amebic liver abscess: a report of 117 cases from a highly endemic zone of India

Objective: To determine the efficacy and safety ultrasound-guided percutaneous catheter drainage (US-PCD) in management of various types of ruptured amebic liver abscess including free rupture (FR) with diffuse intraperitoneal fluid collections (DIFC). Methods: This study analyzed 117 patients with ruptured ALA who underwent US-PCD. The indication for US-PCD was failure to respond to conservative treatment and/or percutaneous needle aspiration. Results: Majority of patients were locally fermented alcohol abusers (95%), and malnourished (75%). Ninety-eight patients had intraperitoneal rupture including 66 contained rupture (CR) with localized intraperitoneal fluid collection (LIFC) and 32 FR with DIFC. Pleuropulmonary complication was found in 19 patients including 13 pleural and 6 pulmonary. A total of 333 catheters were used to drain 202 abscess cavities and associated fluid collections. US-PCD was technically and clinically successful in all cases. Multiple sessions (median 2; range 2–5) of PCD required with upsizing the catheter (median 14 F; range 14–20 F) and placement of additional catheter in 26 (22%) patients. The patients with FR with DIFC required more number of catheters (p = 0.01) and had longer hospital stay (p = 0.01). No major procedure related complication was observed. Six patients developed secondary bacterial infection; two of them presented with cavito-cutaneous fistula at catheter insertion site, and one with cholangitis due to biliary stricture formation necessitating subsequent endoscopic treatment. Post-procedural death occurred from sepsis in a patient with FR. Conclusion: US-PCD is a safe and effective mode of treatment for ruptured ALA including FR with DIFCs. We recommend PCD as a first-line therapy for ruptured ALA.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2015

Elimination of formate production in Clostridium thermocellum

We study the ability of Clostridium thermocellum to rapidly degrade cellulose and ferment resulting hydrolysis products into ethanol makes it a promising platform organism for cellulosic biofuel production via consolidated bioprocessing. Currently, however, ethanol yield are far below theoretical maximum due to branched product pathways that divert carbon and electrons towards formate, H 2 , lactate, acetate, and secreted amino acids. To redirect carbon and electron flux away from formate, pyruvate:formate lyase (pfl) and respective PFL-activating enzyme were deleted. Formate production in the resulting Δpfl strain was eliminated and acetate production decreased by 50% on both complex and defined medium. Growth rate of Δpfl decreased by 2.9-fold on defined medium and diauxic growth was observed on complex medium. Supplementation of defined medium with 2 mM formate restored Δpfl growth rate to 80% of the parent strain. Finally, we discuss the role of pfl in metabolic engineering strategies and C 1 metabolism.

09 BIOMASS FUELS↗
DOE OSTI2017

Heterologous expression of a β- d -glucosidase in Caldicellulosiruptor bescii has a surprisingly modest effect on the activity of the exoproteome and growth on crystalline cellulose

Abstract Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria so far described and are capable of efficiently utilizing complex lignocellulosic biomass without conventional pretreatment. Previous studies have shown that accumulation of high concentrations of cellobiose and, to a lesser extent, cellotriose, inhibits cellulase activity both in vivo and in vitro and high concentrations of cellobiose are present in C. bescii fermentations after 90 h of incubation. For some cellulolytic microorganisms, β-d-glucosidase is essential for the efficient utilization of cellobiose as a carbon source and is an essential enzyme in commercial preparations for efficient deconstruction of plant biomass. In spite of its ability to grow efficiently on crystalline cellulose, no extracellular β-d-glucosidase or its GH1 catalytic domain could be identified in the C. bescii genome. To investigate whether the addition of a secreted β-d-glucosidase would improve growth and cellulose utilization by C. bescii, we cloned and expressed a thermostable β-d-glucosidase from Acidothermus cellulolyticus (Acel_0133) in C. bescii using the CelA signal sequence for protein export. The effect of this addition was modest, suggesting that β-d-glucosidase is not rate limiting for cellulose deconstruction and utilization by C. bescii.

09 BIOMASS FUELS↗