Impact of substrate glycoside linkage and elemental sulfur on bioenergetics of and hydrogen production by the hyperthermophilic archaeon Pyrococcus furiosus

Abstract

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 degrees C. In the absence of S(0), cellobiose-grown cells generated twice as much protein and had 50%-higher specific H(2) generation rates than maltose-grown cultures. Addition of S(0) to maltose-grown cultures boosted cell protein production fourfold and shifted gas production completely from H(2) to H(2)S. In contrast, the presence of S(0) in cellobiose-grown cells caused only a 1.3-fold increase in protein production and an incomplete shift from H(2) to H(2)S production, with 2.5 times more H(2) than H(2)S formed. Transcriptional response analysis revealed that many genes and operons known to be involved in alpha- or beta-glucan uptake and processing were up-regulated in an S(0)-independent manner. Most differentially transcribed open reading frames (ORFs) responding to S(0) in cellobiose-grown cells also responded to S(0) 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 S(0) 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 S(0) 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.

FIG. 1.

Venn diagram analysis showing the numbers of genes regulated twofold or more (a) on cellobiose (C) (C and C+S⁰) compared to maltose (M) (M and M+S⁰), (b) on maltose (M and M+S⁰) compared to cellobiose (C and C+S⁰), (c and d) in the presence of S⁰, and (e and f) on tryptone (T) plus S⁰ (T+S⁰). For example, in panel a, 64 genes (in the absence of S⁰) and 110 genes (in the presence of S⁰) were up-regulated twofold or more on cellobiose compared to maltose; 22 genes were common to both comparisons. See Tables 2 to 4 and the supplemental material for the complete lists.

FIG. 2.

Heat plot based on the least-squares means (mixed model analysis) of selected genes involved in energy conservation in P. furiosus grown on cellobiose (C), maltose (M), cellobiose plus S⁰ (C+S⁰), maltose plus S⁰ (M+S⁰), and tryptone plus S⁰ (T+S⁰). Open boxes indicate the highest expression levels, while dark gray boxes indicate the lowest expression levels. FNOR, ferredoxin NADPH oxidoreductase; ADH, alcohol dehydrogenase; AOR, aldehyde oxidoreductase.

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 that the metabolite was not detected. G1P, glucose-1-phosphate; G6P, glucose-6-phosphate.