Functional Genomics Analysis of Biohydrogen Production by Hyperthermophilic Microorganisms

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Title: Functional Genomics Analysis of Biohydrogen Production by Hyperthermophilic Microorganisms
Author: Chou, Chungjung
Advisors: Todd R. Klaenhammer, Committee Member
Jason M. Haugh, Committee Member
Robert M. Kelly, Committee Chair
Amy M. Grunden, Committee Member
Abstract: The tightening of fossil fuel supplies has generated interest in alternative energy sources in recent years. One primary focus is the conversion of biological feedstocks into biofuels, such as ethanol and hydrogen using anaerobic microorganisms. Efficient bioprocesses require insights into fermentative metabolism that can be facilitated by functional genomics. The arrival of the genomics era and advancement in system biology tools, such as DNA microarrays, has facilitated analysis of the transcriptomes of model microorganisms that could be used for bioenergy processes. In this work, the potential of using hyperthermophilic microorganisms, Pyrococcus furiosus and Thermotoga maritima, to produce biohydrogen and the underlying metabolic mechanisms that are used to accomplish this were investigated. Previous functional genomics efforts on global transcriptomics in P. furiosus and T. maritima focused on batch growth. However, the sensitivity of transcriptional response analysis makes it difficult to identify distinguish between key metabolic features and various secondary effects attributed to indirect impact on the growth status of the microorganism. To address this, an evaluation of the effects of growth phase, growth rate and cultivation method was undertaken for P. furiosus. Transcriptional data revealed excellent reproducibility between continuous cultures. Changes in growth phase in batch culture and dilution rate in continuous culture resulted in profound differences in aspects of cellular metabolism. Direct comparison between batch and continuous culture revealed differences between transcription of substrate utilization and stress response genes, such as heat shock proteins and anti-oxidative processes. Also examined were the effects of syntrophy between P. furiosus and the methanogenic hyperthermophile Methanococcus jannaschii growing in a chemostat setting. After evaluation of these basal transcriptome in P. furiosus, the effects of glucan linkage and the bioenergetics of elemental sulfur reduction were studied using continuous culture. The production rate of hydrogen and key fermentative products were measured and compared to the transcriptomes for various growth conditions. Interestingly, the utilization of different glucan substrates (α-linked maltose vs. β-linked cellobiose) not only affected the corresponding substrate transporters but also specific protein production, transcription of genes encoding membrane-bound hydrogenases, and trend toward H2S production in continuous culture. Bioenergetics parameters could be correlated to the transcriptional data which showed that the re-distribution of reductant flow was caused by glucan-regulated genes, such as alcohol dehydrogenases (PF0074-PF0075). Fianlly, continuous culture system was further utilized to study fermentative hydrogen production from xylose, glucose and xylose:glucose mixtures by hyperthermophilic bacterium T. maritima. Tryptone-supplemented xylose, glucose and xylose/glucose media were tested for hydrogen production in light of the corresponding global transcriptional profile. The results indicated that xylose-grown culture had higher protein production rate, while glucose grown culture tended to produce hydrogen. Surprisingly, the mix of both substrates increased the overall carbon intake and produces more hydrogen than what would be expected by linear extrapolation from data obtained in the pure substrate scenario. The transcriptional analysis revealed that the genes encoding enzymes in non-oxidative pentose phosphate pathway and the xylose transporter was the basis for this difference. The unexpected increase in the H2 production may be correlated to both the interaction between the pentose and hexose assimilation pathways and the efficiency of the carbohydrate-specific transporters.
Date: 2008-05-09
Degree: PhD
Discipline: Chemical Engineering

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