To achieve the goal of optimizing hyperthermophilic H2 production and designing metabolic engineering strategies, it is crucial to understand the physiology and regulation of hydrogenesis at high temperatures, especially as this relates to processing carbon and energy sources. Here, an anaerobic chemostat was used to obtain bioenergetics parameters and profiles of key metabolites related to the transcriptome of P. furiosus. This information is summarized in Fig. .
FIG. 3. Proposed metabolic pathways for P. furiosus grown on maltose and cellobiose in the presence and absence of S0. The thickness of lines outlining boxes and of arrows reflects the significance of the product in the metabolic scheme. A dashed line indicates (more ...)
It was interesting that in the absence of S0
, growth on maltose generated much less H2
than growth on cellobiose, despite comparable specific sugar consumption rates on the two sugars. In P. furiosus
, maltose can enter the glycolytic pathway via both ADP-glucokinase (PF0312) and glucanophosphorylase (PF1535). The maltose-only transcriptome showed that relevant hydrolases and transporters were induced by this sugar substrate, although genes involved in downstream hydrogenesis pathway were not. This suggests that maltose-only cultures may have a bottleneck, leading to lower protein production and H2
ratios and greater production of alanine instead of acetate, reduced ferredoxin, and subsequently H2
). Thus, it appears that growth on maltose is somewhat limited because reducing power generated from substrate degradation was not involved in an H2
-producing, energy-conserving process (45
). In contrast, growth on cellobiose triggered transcription of two alcohol dehydrogenases implicated in ethanol production from acetaldehyde, generated from pyruvate by POR (32
). It has been proposed that this reaction removes the bottleneck in energy metabolism and detoxifies the cytoplasm by removing accumulating acetaldehyde, thereby facilitating pyruvate decarboxylation (33
). Transcriptional analysis also suggested that capsular polysaccharide formation is increased in cellobiose-grown cultures, indicating another possible outlet for reducing equivalents during growth on this substrate. Alteration of metabolite profiles depending on carbon and energy sources has been noted in mesophilic fermentative bacteria (14
). For example, inhibition of Clostridium cellulolyticum
growth was relieved when cellobiose and yeast extract were replaced by cellulose and defined nitrogen sources (15
). This effect was attributed to an imbalance in NADH/NAD ratios arising from higher carbon fluxes in addition to reduced demand on biosynthesis in the presence of complex substrates (22
). Whether a similar situation exists for P. furiosus
remains to be seen.
The addition of S0 boosted biomass yields for both maltose- and cellobiose-grown cultures, albeit to a much greater extent for the maltose-grown cultures. This was reflected in the considerably higher number of differentially transcribed genes involved in anabolism and cellular redox management when S0 was added to maltose. The close correlation between H2S generation, biomass (protein) yield, and transcriptional levels of MBX and SipA/SipB suggests that S0 reduction is an energy-conserving process in P. furiosus and not just a mechanism for alleviating H2 inhibition. While all H2 production ceased in cultures grown on maltose plus S0, with corresponding down-regulation of MBH and SH1, only about one-third of the H2 production was replaced by H2S generation in cultures grown on cellobiose plus S0. This very surprising result shows that in cellobiose-grown cultures regulation of the expression of MBH and MBX is not the on/off mechanism that appears to be present in maltose-grown cells, where the addition of S0 causes hydrogen production and expression of the genes encoding the three hydrogenases to cease within minutes (Schut et al., unpublished data). At this point, it is not clear why P. furiosus metabolizes the two sugars so differently.
Both processes appear to use the same pathway from phosphorylated hexoses to the end products acetate, CO2, and H2 (plus H2S) since the relative amounts of these compounds (per unit of sugar utilized) are the same (Table ). However, not only do the H2/H2S ratios differ, but there is also a dramatic difference in carbon flow. For example, on cellobiose alone, about 75% of the sugar is converted to acetate and the gaseous products (sugar/acetate/H2 ratio, ~1:1.5:3.8), whereas only 50% of the maltose is converted (sugar/acetate/H2 ratio, 1:1:2.6). How the “extra” carbon from maltose is used is not known, but in bioenergy terms, if H2 is the required product, then cellobiose should be the carbon source rather than maltose. Conversely, in the presence of S0, the bioenergetics and end product yields and ratios are very similar (Table ), indicating that the same pathways are utilized independent of the glycoside type. The one caveat is the production of H2 by cellobiose-grown cells. In the presence of S0, the metabolism of cellobiose in terms of end products (per unit of sugar) appears to be comparable to the metabolism of maltose (Table ). Consequently, S0 dramatically impacts the carbon flux from sugar into acetate in cellobiose-grown cells but has less impact in maltose-grown cells. Thus, S0 affects carbon flux, in addition to its role as a reductant sink. At present, the transcriptional analyses do not provide insight into how S0 achieves this in cellobiose-grown cells.
It is possible that the profound effect of the glycoside type on the S0
-dependent bioenergetics of P. furiosus
extends to other hyperthermophiles, including bacteria. A homologous MBX operon can be identified in the genome of the facultative S0
-reducing hyperthermophilic bacterium Thermotoga maritima
, probably as a result of lateral gene transfer (7
). However, the transcriptional levels of this operon were relatively low on maltose and cellobiose and were not affected by S0
(S. R. Gray and R. M. Kelly, unpublished data). A similar response was observed for two Sip homologs in T. maritima
. It has been reported that S0
stimulates T. maritima
growth by removing inhibitory H2
but does not impact energy conservation pathways (47
An important outcome of this study is the realization that microbial processes aimed at high levels of H2 production must take into account the impact that substrates and other environmental influences have on cellular bioenergetics. Given the anticipated heterogeneity of biomass feedstocks that will be used for bioenergy conversion processes, a comprehensive understanding of how transcriptional regulation and metabolite production relate to the substrate pool is highly desirable. By combining traditional approaches (chemostat culture for determining bioenergetic parameters) with functional genomics tools (transcriptional response analysis), insights can be obtained and ultimately provide the basis for metabolic engineering strategies. The information provided here for P. furiosus should prove to be useful in efforts to exploit H2 production in this archaeon and other archaea for the production of biofuels.