Hydrogen Production and Enzyme Activities in the Hyperthermophile Thermococcus paralvinellae Grown on Maltose, Tryptone, and Agricultural Waste

Abstract

Thermococcus may be an important alternative source of H2 in the hot subseafloor in otherwise low H2 environments such as some hydrothermal vents and oil reservoirs. It may also be useful in industry for rapid agricultural waste treatment and concomitant H2 production. Thermococcus paralvinellae grown at 82°C without sulfur produced up to 5 mmol of H2 L(-1) at rates of 5-36 fmol H2 cell(-1) h(-1) on 0.5% (wt vol(-1)) maltose, 0.5% (wt vol(-1)) tryptone, and 0.5% maltose + 0.05% tryptone media. Two potentially inhibiting conditions, the presence of 10 mM acetate and low pH (pH 5) in maltose-only medium, did not significantly affect growth or H2 production. Growth rates, H2 production rates, and cell yields based on H2 production were the same as those for Pyrococcus furiosus grown at 95°C on the same media for comparison. Acetate, butyrate, succinate, isovalerate, and formate were also detected as end products. After 100 h, T. paralvinellae produced up to 5 mmol of H2 L(-1) of medium when grown on up to 70% (vol vol(-1)) waste milk from cows undergoing treatment for mastitis with the bacterial antibiotic Ceftiofur and from untreated cows. The amount of H2 produced by T. paralvinellae increased with increasing waste concentrations, but decreased in P. furiosus cultures supplemented with waste milk above 1% concentration. All mesophilic bacteria from the waste milk that grew on Luria Bertani, Sheep's Blood (selective for Staphylococcus, the typical cause of mastitis), and MacConkey (selective for Gram-negative enteric bacteria) agar plates were killed by heat during incubation at 82°C. Ceftiofur, which is heat labile, was below the detection limit following incubation at 82°C. T. paralvinellae also produced up to 6 mmol of H2 L(-1) of medium when grown on 0.1-10% (wt vol(-1)) spent brewery grain while P. furiosus produced < 1 mmol of H2 L(-1). Twelve of 13 enzyme activities in T. paralvinellae showed significant (p < 0.05) differences across six different growth conditions; however, methyl viologen-dependent membrane hydrogenase activity remained constant across all media types. The results demonstrate the potential of at least some Thermococcus species to produce H2 if protein and α-glucosides are present as substrates.

Keywords: Thermococcus; bioenergy; hydrogenase; hyperthermophile; waste remediation.

Figure 1

General metabolic pathways for Thermococcus paralvinellae. The enzymes are membrane-bound hydrogenases: Fd-dependent (Mbh), formate-dependent (Mfh), and CO-dependent (Mch); cytoplasmic hydrogenases: NADH-dependent (Sulf) and F₄₂₀-dependent (Frh); NADH:Fd oxidoreductase (FNOR); membrane-bound ATP synthase (ATPase); glutamate dehydrogenase (GDH); alanine aminotransferase (AlaAT); general aminotransferases (AT); pyruvate:Fd oxidoreductase (POR); isovalerate:Fd oxidoreductase (VOR); indolepyruvate:Fd oxidoreductase (IOR); ADP-forming acetyl-CoA/acyl-CoA synthetase (ACS); aldehyde:Fd oxidoreductase (AOR); formaldehyde:Fd oxidoreductase (FOR); and alcohol dehydrogenase (ADH). Fd, electron carrier ferredoxin; F₄₂₀, electron carrier coenzyme F₄₂₀; §, modified Embden-Meyerhof pathway. The activities for most of the enzymes listed are shown in Table 4.

Figure 2

H₂ production by T. paralvinellae (A) and P. furiosus (B) in 0.5% maltose (○), 0.5% tryptone (), 0.5% maltose + 0.05% tryptone (●), 0.5% maltose + 10 mM acetate (), and 0.5% maltose at pH 5.0 (). Error bars represent the standard error.

Figure 3

H₂ production by T. paralvinellae (filled symbols) and P. furiosus (open symbols) when grown on waste milk from cows treated with Ceftiofur (A) and from untreated cows (B). The concentrations of waste milk used were 10 (○, ●), 1 (Δ, ▲), and 0.1% (▽, ▼). Error bars represent the standard error.

Figure 4

H₂ production by T. paralvinellae (filled symbols) and P. furiosus (open symbols) when grown on spent brewery grain. The concentrations spent grain used were 10 (○, ●), 1 (Δ, ▲), and 0.1% (▽, ▼). Error bars represent the standard error.

Figure 5

Degradation of waste milk by T. paralvinellae during 100 h of incubation at 82°C. (A) Clarification of media containing 1% waste milk from untreated cows following 100 h incubation. The bottle on the left was an uninoculated control. Concentrations of protein (B) and reducing sugars (C) in 1% waste milk from untreated cows. The data shown are from inoculated bottles (●) and from uninoculated controls (○) as well as H₂ produced from inoculated bottles (▲). There was no H₂ production in uninoculated bottles. Error bars represent the standard error.

Figure 6

Growth of T. paralvinellae (A) and P. furiosus (B) in the 20-l bioreactor in 0.5% maltose (●), 0.5% tryptone (), 0.5% maltose + 0.05% tryptone (○), 0.5% maltose + 10 mM acetate (), 0.5% maltose at pH 5.0 (), and 0.1% waste milk (). Data are examples from individual bioreactor reactions.