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Title: Biohydrogenesis in the Thermotogales

The production and consumption of molecular hydrogen drives the physiology and bioenergetics of many microorganisms in hydrothermal environments. As such, the potential of these microorganisms as model systems to probe fundamental issues related to biohydrogen production merits consideration. It is important to understand how carbon/energy sources relate to the disposition of reducing power and, ultimately, the formation of molecular hydrogen by high temperature microorganisms. This project focused on bacteria in the thermophilic order Thermotogales, fermentative anaerobes that produce H2 from simple and complex carbohydrates. The major thrusts of the project are summarized in the Objectives listed below: OBJECTIVE 1: Examine the regulation of substrate catabolic proteins and pathways as this relates to carbon partitioning, disposition of reducing power, and H2 generation in Thermotoga maritima. OBJECTIVE 2: Apply classical genetics and develop molecular genetic tools for Thermotoga species to dissect catabolic and regulatory pathways related to sugar metabolism and H2 evolution. OBJECTIVE 3: Thermotogales biodiversity arises from adaptive specialization that expands on a conserved minimal genome; physiological characterization of selected novel traits will be done to expand understanding of biohydrogenesis. Four species within the genus Thermotoga were examined to understand similarities and differences in the mechanisms by which simple and complexmore » carbohydrates were utilized and converted to molecular hydrogen. Although the core genome of these four species represented 75% of open reading frames (ORFs), there were significant differences in carbohydrate utilization patterns. New ABC transporters were identified within the Thermotogales through genomic and biochemical analysis. Molecular genetics tools were developed to examine Thermotoga maritima physiology. Cell lines were created in which both H2 and acetate levels were elevated on a per cell basis relative to the wild type, while lactate remained undetectable. Genome resequencing indicated that the primary genetic target for these phenotypic changes was the ATP binding component of a maltose ABC transporter. High temperature anaerobic [14C]-maltose transport assays demonstrated maltose uptake was reduced in the H2 overproducing cell lines. This suggested normal rates of maltose transport in the wild type organism lead to a metabolic imbalance that limited H2 synthesis. The microbial ecology of T. maritima was examined through functional genomics experiments. Under low nutrient conditions, T. maritima was observed to produce a range of putative peptides, some of which were related to α-carbon cyclic peptides produced by Bacillus subtilus. Finally, the role of ‘toga’ in these novel microorganisms was shown to involve association with insoluble growth substrates. The ‘toga’ distends from the cytoplasmic membrane-enclosed portion of the cells as they enter the late exponential/stationary phase of growth. Some of the genes encoding toga-associated proteins were up-regulated during this phase of growth and the distension is caused by continued growth of the toga, and not shrinkage of the cytoplasmic aspect of the cells. This increase in cell surface area may have selective value to provide a larger anchor for polysaccharide hydrolytic enzymes during a time of nutritional stress. This project led to many interesting insights about the Thermotogales that have both scientific and technological implications. Ongoing work will leverage these developments to further elucidate many interesting features of these novel microorganisms.« less
  1. North Carolina State University
Publication Date:
OSTI Identifier:
Report Number(s):
DOE-North Carolina State-64687
DOE Contract Number:
Resource Type:
Technical Report
Research Org:
North Carolina State University
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Contributing Orgs:
University of Connecticut, University of Nebraska-Lincoln
Country of Publication:
United States
08 HYDROGEN Hydrogen, Thermotogales, hyperthermophiles, bacteria