Impact of Substrate Glycoside Linkage and Elemental Sulfur on Bioenergetics of and Hydrogen Production by the Hyperthermophilic Archaeon Pyrococcus furiosus

Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905, USA.
Applied and Environmental Microbiology (Impact Factor: 3.67). 12/2007; 73(21):6842-53. DOI: 10.1128/AEM.00597-07
Source: PubMed


Glycoside linkage (cellobiose versus maltose) dramatically influenced bioenergetics to different extents and by different
mechanisms in the hyperthermophilic archaeon Pyrococcus furiosus when it was grown in continuous culture at a dilution rate of 0.45 h−1 at 90°C. In the absence of S0, cellobiose-grown cells generated twice as much protein and had 50%-higher specific H2 generation rates than maltose-grown cultures. Addition of S0 to maltose-grown cultures boosted cell protein production fourfold and shifted gas production completely from H2 to H2S. In contrast, the presence of S0 in cellobiose-grown cells caused only a 1.3-fold increase in protein production and an incomplete shift from H2 to H2S production, with 2.5 times more H2 than H2S formed. Transcriptional response analysis revealed that many genes and operons known to be involved in α- or β-glucan uptake
and processing were up-regulated in an S0-independent manner. Most differentially transcribed open reading frames (ORFs) responding to S0 in cellobiose-grown cells also responded to S0 in maltose-grown cells; these ORFs included ORFs encoding a membrane-bound oxidoreductase complex (MBX) and two hypothetical
proteins (PF2025 and PF2026). However, additional genes (242 genes; 108 genes were up-regulated and 134 genes were down-regulated)
were differentially transcribed when S0 was present in the medium of maltose-grown cells, indicating that there were different cellular responses to the two sugars.
These results indicate that carbohydrate characteristics (e.g., glycoside linkage) have a major impact on S0 metabolism and hydrogen production in P. furiosus. Furthermore, such issues need to be considered in designing and implementing metabolic strategies for production of biofuel
by fermentative anaerobes.

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    • "Calculated end-product yields reveal that the Caldicellulosiruptor, Pyrococcus, Thermococcus, and Thermotoga species surveyed, produced, in most cases, near-maximal H2 yields with concomitant CO2 and acetate production, and little or no ethanol, formate, and lactate [24-40]. It is important to note that while some studies [29-31,34,35,39] report lower overall end-product yields, likely due to a large amount of carbon flux being directed towards biomass production under a given growth condition, H2:ethanol ratios remain high. Cal. "
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    ABSTRACT: Background Fermentative bacteria offer the potential to convert lignocellulosic waste-streams into biofuels such as hydrogen (H2) and ethanol. Current fermentative H2 and ethanol yields, however, are below theoretical maxima, vary greatly among organisms, and depend on the extent of metabolic pathways utilized. For fermentative H2 and/or ethanol production to become practical, biofuel yields must be increased. We performed a comparative meta-analysis of (i) reported end-product yields, and (ii) genes encoding pyruvate metabolism and end-product synthesis pathways to identify suitable biomarkers for screening a microorganism’s potential of H2 and/or ethanol production, and to identify targets for metabolic engineering to improve biofuel yields. Our interest in H2 and/or ethanol optimization restricted our meta-analysis to organisms with sequenced genomes and limited branched end-product pathways. These included members of the Firmicutes, Euryarchaeota, and Thermotogae. Results Bioinformatic analysis revealed that the absence of genes encoding acetaldehyde dehydrogenase and bifunctional acetaldehyde/alcohol dehydrogenase (AdhE) in Caldicellulosiruptor, Thermococcus, Pyrococcus, and Thermotoga species coincide with high H2 yields and low ethanol production. Organisms containing genes (or activities) for both ethanol and H2 synthesis pathways (i.e. Caldanaerobacter subterraneus subsp. tengcongensis, Ethanoligenens harbinense, and Clostridium species) had relatively uniform mixed product patterns. The absence of hydrogenases in Geobacillus and Bacillus species did not confer high ethanol production, but rather high lactate production. Only Thermoanaerobacter pseudethanolicus produced relatively high ethanol and low H2 yields. This may be attributed to the presence of genes encoding proteins that promote NADH production. Lactate dehydrogenase and pyruvate:formate lyase are not conducive for ethanol and/or H2 production. While the type(s) of encoded hydrogenases appear to have little impact on H2 production in organisms that do not encode ethanol producing pathways, they do influence reduced end-product yields in those that do. Conclusions Here we show that composition of genes encoding pathways involved in pyruvate catabolism and end-product synthesis pathways can be used to approximate potential end-product distribution patterns. We have identified a number of genetic biomarkers for streamlining ethanol and H2 producing capabilities. By linking genome content, reaction thermodynamics, and end-product yields, we offer potential targets for optimization of either ethanol or H2 yields through metabolic engineering.
    Full-text · Article · Dec 2012 · BMC Microbiology
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    • "Similarly arguing for the presence of additional functionally-equivalent enzymes, deletion of both of the identified ferredoxin:NADPH oxidoreductases had no detectable effects on growth or H 2 production (Table 2). An experimentally challenging observation is that T. kodakarensis TS1102 (ΔTK1260–61) exhibited no growth defects even though transcription of sipA and sipB is very highly induced by S° addition (Chou et al., 2007). This strongly suggests that SipA and SipB play key role(s) in growth and/or metabolism with S° present (Clarkson et al., 2010) but we saw no evidence for such importance. "
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    ABSTRACT: Hydrogen (H₂) production by Thermococcus kodakarensis compares very favourably with the levels reported for the most productive algal, fungal and bacterial systems. T. kodakarensis can also consume H₂ and is predicted to use several alternative pathways to recycle reduced cofactors, some of which may compete with H₂ production for reductant disposal. To explore the reductant flux and possible competition for H₂ production in vivo, T. kodakarensis TS517 was mutated to precisely delete each of the alternative pathways of reductant disposal, H₂ production and consumption. The results obtained establish that H₂ is generated predominantly by the membrane-bound hydrogenase complex (Mbh), confirm the essential role of the SurR (TK1086p) regulator in vivo, delineate the roles of sulfur (S°) regulon proteins and demonstrate that preventing H₂ consumption results in a substantial net increase in H₂ production. Constitutive expression of TK1086 (surR) from a replicative plasmid restored the ability of T. kodakarensis TS1101 (ΔTK1086) to grow in the absence of S° and stimulated H₂ production, revealing a second mechanism to increase H₂ production. Transformation of T. kodakarensis TS1101 with plasmids that express SurR variants constructed to direct the constitutive synthesis of the Mbh complex and prevent expression of the S° regulon was only possible in the absence of S° and, under these conditions, the transformants exhibited wild-type growth and H₂ production. With S° present, they grew slower but synthesized more H₂ per unit biomass than T. kodakarensis TS517.
    Full-text · Article · Aug 2011 · Molecular Microbiology
    • "Organism Domain Temp. grown (°C) Culturing type Substrate Reported end products H 2 / hexose Reference Thermophiles Thermoanaerobacterium saccharolyticum YS485 B 5 5 Batch Cellobiose Acetate, lactate, ethanol 0.87 [53] Thermoanaerobacterium thermosaccharolyticum PSU-2 B 6 0 Batch Starch Acetate, ethanol, butyrate 2.8 [62] Clostridium thermocellum ATCC 27405 B 6 0 Chemostat α-Cellulose Acetate, lactate, ethanol, formate 1.65 [63] Hyperthermophiles and extreme thermophiles Thermotoga elfii DSM 9442 B 65 Controlled batch Sucrose Acetate 3.3 [20] 65 Batch Glucose Acetate 3.3 [64] Thermotoga neapolitana DSM 4359 B 8 0 Batch Glucose Acetate, lactate 2.4 [65] 80 Controlled batch Glucose/ xylose Acetate, lactate 3.3 [66] 85 Batch Glucose Acetate, lactate 3.8 [67] 77 Batch Glucose Acetate, butyrate 3.2 [68] Thermotoga maritima DSM 3109 B 80 Batch Glucose Acetate 4 [69] Caldicellulosiruptor saccharolyticus DSM 8903 B 7 0 Controlled batch Sucrose Acetate, lactate 3.3 [20] 72 Controlled batch Glucose/ xylose Acetate, lactate 3.4 [66] Thermoanaerobacter tengcongensis JCM11007 B 7 5 Batch Starch Acetate, ethanol 2.8 [23] 75 Batch Glucose Acetate 4 [23] Thermococcus kodakaraensis TSF100 A 8 5 Chemostat Starch Acetate, alanine 3.3 [70] Pyrococcus furiosus DSM 3638 A Chemostat Maltose Acetate, butyrate 2.9 [71] 90 Batch Cellobiose Acetate, alanine 2.8 [30] 90 Batch Maltose Acetate, alanine 3.5 [30] 90 Chemostat Maltose Acetate, alanine 2.6 [72] 90 Chemostat Cellobiose Acetate, alanine, ethanol 3.8 [72] "
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    ABSTRACT: Hydrogen produced from biomass by bacteria and archaea is an attractive renewable energy source. However, to make its application more feasible, microorganisms are needed with high hydrogen productivities. For several reasons, hyperthermophilic and extremely thermophilic bacteria and archaea are promising is this respect. In addition to the high polysaccharide-hydrolysing capacities of many of these organisms, an important advantage is their ability to use most of the reducing equivalents (e.g. NADH, reduced ferredoxin) formed during glycolysis for the production of hydrogen, enabling H2/hexose ratios of between 3.0 and 4.0. So, despite the fact that the hydrogen-yielding reactions, especially the one from NADH, are thermodynamically unfavourable, high hydrogen yields are obtained. In this review we focus on three different mechanisms that are employed by a few model organisms, viz. Caldicellulosiruptor saccharolyticus and Thermoanaerobacter tengcongensis, Thermotoga maritima, and Pyrococcus furiosus, to efficiently produce hydrogen. In addition, recent developments to improve hydrogen production by hyperthermophilic and extremely thermophilic bacteria and archaea are discussed.
    No preview · Article · Jul 2010 · Environmental Technology
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