Production of hydrogen from α-1,4- and β-1,4-linked saccharides by marine hyperthermophilic Archaea

Abstract

Nineteen hyperthermophilic heterotrophs from deep-sea hydrothermal vents, plus the control organism Pyrococcus furiosus, were examined for their ability to grow and produce H₂ on maltose, cellobiose, and peptides and for the presence of the genes encoding proteins that hydrolyze starch and cellulose. All of the strains grew on these disaccharides and peptides and converted maltose and peptides to H₂ even when elemental sulfur was present as a terminal electron acceptor. Half of the strains had at least one gene for an extracellular starch hydrolase, but only P. furiosus had a gene for an extracellular β-1,4-endoglucanase. P. furiosus was serially adapted for growth on CF11 cellulose and H₂ production, which is the first reported instance of hyperthermophilic growth on cellulose, with a doubling time of 64 min. Cell-specific H₂ production rates were 29 fmol, 37 fmol, and 54 fmol of H₂ produced cell⁻¹ doubling⁻¹ on α-1,4-linked sugars, β-1,4-linked sugars, and peptides, respectively. The highest total community H₂ production rate came from growth on starch (2.6 mM H₂ produced h⁻¹). Hyperthermophilic heterotrophs may serve as an important alternate source of H₂ for hydrogenotrophic microorganisms in low-H₂ hydrothermal environments, and some are candidates for H₂ bioenergy production in bioreactors.

Fig. 1.

Proposed model of starch and cellulose degradation, H₂ production, and ATP synthesis in Pyrococcus and Thermococcus (based partly on references , , and 28). The redox balance and ATP produced is per glucose molecule. The proteins are as follows: 1, β-1,4-endoglucanase; 2, cellobiose ABC transporter; 3, β-endoglucanase; 4, β-glucosidase; 5, amylopullulanase; 6, maltose ABC transporter; 7, cyclomaltodextrin glucanotransferase; 8, cyclomaltodextrin ABC transporter; 9, cyclomaltodextrinase; 10, α-amylase; 11, α-glucosidase; 12, α-glucan phosphorylase; 13, ADP-dependent hexokinase; 14, modified Embden-Meyerhof pathway; 15, pyruvate oxidoreductase; 16, acetyl-CoA synthetase; 17, hydrogenase; 18, Na⁺-translocating ATP synthase. Fd, electron carrier ferredoxin.

Fig. 2.

(A) Growth of P. furiosus on 0.5% CF11 cellulose plus 0.01% yeast extract (•) and on 0.01% yeast extract only (+). (B) H₂ produced versus cell concentration for P. furiosus growth on 0.5% cellulose (•), 0.5% cellobiose (○), 0.5% starch (▴), 0.5% maltose (▵), 0.5% casein hydrolysate (×), and 0.01% yeast extract only (+). Each medium was supplemented with 0.01% yeast extract.