Theo A. Hansen

METABOLISM OF SULFATE-REDUCING PROKARYOTES

Dissimilatory sulfate reduction is carried out by a heterogeneous group of bacteria and archaea that occur in environments with temperatures up to 105 °C. As a group together they have the capacity to metabolize a wide variety of compounds ranging from hydrogen via typical organic fermentation products to hexadecane, toluene, and several types of substituted aromatics. Without exception all sulfate reducers activate sulfate to APS; the natural electron donor(s) for the ensuing APS reductase reaction is not known. The same is true for the reduction of the product bisulfite; in addition there is still some uncertainty as to whether the pathway to sulfide is a direct six-electron reduction of bisulfite or whether it involves trithionate and thiosulfate as intermediates. The study of the degradation pathways of organic substrates by sulfate-reducing prokaryotes has led to the discovery of novel non-cyclic pathways for the oxidation of the acetyl moiety of acetyl-CoA to CO2. The most detailed knowledge is available on the metabolism ofDesulfovibrio strains, both on the pathways and enzymes involved in substrate degradation and on electron transfer components and terminal reductases. Problems encountered in elucidating the flow of reducing equivalents and energy transduction are the cytoplasmic localization of the terminal reductases and uncertainties about the electron donors for the reactions catalyzed by these enzymes. New developments in the study of the metabolism of sulfate-reducing bacteria and archaea are reviewed.

FERMENTATION OF GLUTAMATE AND OTHER COMPOUNDS BY ACIDAMINOBACTER-HYDROGENOFORMANS GEN-NOV SP-NOV, AN OBLIGATE ANAEROBE ISOLATED FROM BLACK MUD - STUDIES WITH PURE CULTURES AND MIXED CULTURES WITH SULFATE-REDUCING AND METHANOGENIC BACTERIA

From mud from the Ems-Dollard estuary (The Netherlands) an L-glutamate-fermenting bacterium was isolated. The isolated strain glu 65 is Gram-negative, rodshaped, obligately anaerobic, non-sporeforming and does not contain cytochromes. The G+C content of its DNA is 48 mol percent.Pure cultures of strain glu 65 grew slowly on glutamate (μmax 0.06 h-1) and formed acetate, CO2, formate and hydrogen, and minor amounts of propionate. A more rapid fermentation of glutamate was achieved in mixed cultures with sulfate-reducing bacteria (Desulfovibrio HL21 or Desulfobulbus propionicus) or methanogens (Methanospirillum hungatei or Methanobrevibacter arboriphilicus AZ). In mixed culture with Desulfovibrio HL21 a μmax of 0.10 h-1 was observed. With Desulfovibrio or the methanogens propionate was a major product (up to 0.47 mol per mol glutamate) in addition to acetate.Extracts of glutamate-grown cells possessed high activities of 3-methylaspartase, a key enzyme of the mesaconate pathway leading to acetate, and very high activities of NAD+-dependent glutamate dehydrogenase, an enzyme most likely involved in the pathway to propionate.The following other substrates allowed reasonable to good growth in pure culture: histidine, α-ketoglutarate, serine, cysteine, glycine, adenine, pyruvate, oxaloacetate and citrate. Utilization in mixed cultures was demonstrated for: glutamine, arginine, ornithine, threonine, lysine, alanine, valine, leucine and isoleucine (with Desulfovibrio HL21) and malate (with Methanospirillum).The shift in the fermentation of glutamate and the syntrophic utilization of the above substrates are explained in terms of interspecies hydrogen transfer.Strain glu 65 is described as the type strain of Acidaminobacter hydrogenoformans gen. nov. sp. nov.

Sulfide utilization by purple nonsulfur bacteria.

SummaryThe purple nonsulfur bacteria Rhodospirillum rubrum SMG 107, Rhodopseudomonas capsulata SMG 155, Rps. sphaeroides SMG 158 and Rps. palustris SMG 124 were tested for a possible utilization of sulfide. The first three strains were found to oxidize sulfide to extracellular elemental sulfur only, whereas Rps. palustris SMG 124 converted sulfide into sulfate without intermediate accumulation of elemental sulfur. Growth ceased at lower sulfide concentrations than usually found with purple sulfur bacteria. In consequence of the low sulfide tolerance information on the specific growth rates obtainable with sulfide as photosynthetic electron donor could not be provided by cultivation in batch cultures. Sulfide-limited chemostat cultures of Rps. capsulata SMG 155 showed that the maximum specific growth rate was close to 0.14 h-1 (doubling time 5 h). Sulfide was converted into extracellular elemental sulfur at all dilution rates tested. The maximum specific growth rate of Rps. palustris SMG 124 was found to be much lower (less than 0.03 h-1). Sulfate was the only product of the conversion of sulfide.These data show that at least some purple nonsulfur bacteria may play a role in the dissimilatory sulfur cycle in nature. Taxonomic implications of our results are discussed.

Rhodopseudomonas sulfidophila, nov. spec., a new species of the purple nonsulfur bacteria.

SummaryFrom marine mud flats a new type of photosynthetic purple bacterium was isolated. This type is described as a new species of the Rhodospirillaceae and is named Rhodopseudomonas sulfidophila. The cells are rod-shaped, 0.6 to 0.9 μ wide and 0.9 to 2.0 μ long, and motile by means of polar flagella. Cell division occurs by binary fission. The photosynthetic membrane system is of the vesicular type. The pigments consist of bacteriochlorophyll a and of carotenoids, most probably of the spheroidene group. A wide range of organic compounds can be utilized anaerobically in the light. Growth on organic compounds aerobically in the dark is also possible. Niacin, thiamin, biotin and p-aminobenzoic acid are required as growth factors. The new species needs 2.5% (w/v) sodium chloride for optimal growth. All strains show excellent photolithotrophic growth on hydrogen, hydrogen sulfide, and thiosulfate. They show a remarkably high sulfide tolerance. Sulfide and thiosulfate are oxidized to sulfate without an intermediate accumulation of elemental sulfur. The new species seems to be one of the most versatile types of photosynthetic bacteria isolated thus far.

Cleavage of dimethylsulfoniopropionate and reduction of acrylate by Desulfovibrio acrylicus sp. nov.

Abstract From anoxic intertidal sediment, a dimethylsulfoniopropionate (DMSP)-cleaving anaerobe (strain W218) was isolated that reduced the acrylate formed to propionate. The bacterium was vibrio- to rod-shaped and motile by means of multiple polar flagella. It reduced sulfate, thiosulfate, and acrylate, and used lactate, fumarate, succinate, malate, pyruvate, ethanol, propanol, glycerol, glycine, serine, alanine, cysteine, hydrogen, and formate as electron donors. Sulfate and acrylate were reduced simultaneously; growth with sulfate was faster than with acrylate. Extracts of cells grown in the presence of DMSP contained high DMSP lyase activities (9.8 U/mg protein). The DNA mol% G+C was 45.1. On the basis of its characteristics and the 16S rRNA gene sequence, strain W218 was assigned to a new Desulfovibrio species for which the name Desulfovibrio acrylicus is proposed. A variety of other sulfate-reducing bacteria (eight of them originating from a marine or saline environment and five from other environments) did not reduce acrylate.

Occurrence of polyglucose as a storage polymer in Desulfovibrio species and Desulfobulbus propionicus

The occurrence of organic storage compounds in six strains of sulfate-reducing bacteria was investigated.In Desulfovibrio HL21 and Desulfovibrio vulgaris NCIB 8303 accumulation of polyglucose was brought about by limiting the Fe2+ or NH4+concentration in the growth medium. Desulfobulbus propionicus 1pr3 and especially Desulfovibrio gigas NCIB 9332 already synthesized large amounts of polyglucose in normal media, whereas the synthesis of polyglucose in two strains of Desulfovibrio desulfuricans was far less pronounced.Suspensions of Desulfovibrio HL21 cells in media without an energy source and sulfate degraded the polyglucose to acetate, hydrogen and small amounts of ethanol and succinate; Desulfobulbus propionicus formed acetate, propionate and some hydrogen under these conditions. In the presence of sulfate both strains produced acetate and corresponding amounts of sulfide.None of the strains synthesized PHB as a storage polymer.

Pathway of propionate formation from ethanol in Pelobacter propionicus

Whole cells of Pelobacter propionicus fermented (1-13C) ethanol and CO2 to nearly equal amounts of (2-13C) and (3-13C) propionate and to (1-13C) acetate indicating a randomizing pathway of propionate formation. Enzymes involved in the fermentation were assayed in cell-free extracts and cetyltrimethylammonium bromide-permeabilized cells grown with ethanol as sole substrate. Alcohol dehydrogenase, aldehyde dehydrogenase (benzylviologen-reducing), phosphate acetyl transferase, acetate kinase, pyruvate synthase, methylmalonyl CoA: pyruvate transcarboxylase, propionyl CoA: succinate CoA transferase, and the enzymes of the succinate-methylmalonyl CoA pathway all were detected at activities sufficient to be involved in ethanol fermentation. Very low amounts of a b-type cytochrome were detected in ethanol-grown cells (46 nmol δ g protein−1). Low cell yields obtained with ethanol as substrate indicate that P. propionicus does not conserve energy by electron transport-linked fumarate reduction. Despite the presence of a hydrogenase and a shift in the fermentation of lactate towards the formation of more propionate in the presence of hydrogen, P. propionicus was unable, to catalyze, the reduction of acetate and CO2 to propionate, unlike Desulfobulbus propionicus.

Immunocytochemical localization of APS reductase and bisulfite reductase in three Desulfovibrio species

The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were embedded and ultrathin sections were incubated with antibodies and subsequently labeled with protein A-gold. The bisulfite reductase in all three strains and APS reductase in d. gigas and D. vulgaris were found in the cytoplasm. The labeling of d. thermophilus with APS reductase antibodies resulted in a distribution of gold particles over the cytoplasmic membrane region. The localization of the two enzymes is discussed with respect to the mechanism and energetics of dissimilatory sulfate reduction.

METHANOL, A POTENTIAL FEEDSTOCK FOR BIOTECHNOLOGICAL PROCESSES

Abstract A wide variety of bacteria and yeasts is able to grow in inexpensive synthetic media with methanol as the sole or major source of carbon and energy. This is due to the presence of a few unique enzymes which enable these organisms to generate metabolic energy and synthesize cell constituents from this one-carbon substrate. In the chemical industry there is currently much interest in the production of fuels and chemicals from methanol. As a feedstock for industrial fermentations methanol is also attractive because of its low cost, ease of handling and abundant availability. In many countries methanol-utilizing microbes are being studied and their potential utility in biotechnological processes is explored. These studies are aimed at making use of their characteristic properties, exploiting known organisms and new strains for improving existing processes and developing novel products.

ETHANOL DISSIMILATION IN DESULFOVIBRIO

During growth of ethanol plus sulfate Desulfovibrio gigas and three other Desulfovibrio strains tested contained high NAD-dependent alcohol dehydrogenase activities and dye-linked aldehyde dehydrogenase activities. In lactate-grown cells these activities were lower or absent. In D. gigas an NADH dehydrogenase activity was found which was higher during growth on ethanol than during growth on lactate. The NADH dehydrogenase activity appeared to consist of at least three different soluble enzymes. The aldehyde dehydrogenase activity in D. gigas was highest with benzylviologen as an acceptor and was strongly stimulated by potassium ions. Coenzyme A or phosphate dependency could not be shown, indicating that acetyl-CoA or acetyl phosphate are not intermediates in the conversion of acetaldehyde to acetate.In the absence of sulfate D. gigas was able to convert ethanol to acetate by means of interspecies hydrogen transfer to a methanogen. This conversion, however, did not lead to growth of the Desulfovibrio.