Hydrogen-dependent autotrophic growth in phototrophic and chemolithotrophic cultures of thermophilic bacteria, Chloroflexus aggregans and Chloroflexus aurantiacus, isolated from Nakabusa hot springs

ABSTRACTThe genus Chloroflexus is a deeply branching group of thermophilic filamentous anoxygenic phototrophic bacteria. The bacteria in this genus have been shown to grow well heterotrophically under anaerobic photosynthetic and aerobic respiratory conditions. We examined autotrophic growth in new isolates of Chloroflexus strains from hot springs in Nakabusa, Japan. The isolates belonging to Chloroflexus aggregans (98.7% identity of 16S rRNA gene sequence to the respective type strain) and Chloroflexus aurantiacus (99.9% identity to the respective type strain) grew photoautotrophically under a 24% H2 atmosphere. We also observed chemolithotrophic growth of these isolates under 80% H2 and 5% O2 conditions in the dark. This is the first report showing that Chloroflexus grew under both photoautotrophic and chemolithotrophic conditions in addition to photoheterotrophic and aerobic chemoheterotrophic conditions.

Gas Fermentation Enhancement for Chemolithotrophic Growth of Cupriavidus necator on Carbon Dioxide

Cupriavidus necator, a facultative hydrogen-oxidizing bacterium, was grown on carbon dioxide, hydrogen, and oxygen for value-added products. High cell density and productivity were the goal of gas fermentation, but limited by gas substrates because of their low solubility in the aqueous medium solution. Enhancement of gas fermentation was investigated by (i) adding n-hexadecane as a gas vector to increase the volumetric mass transfer coefficient (kLa) and gas solubility, (ii) growing C. necator under a raised gas pressure, and (iii) using cell mass hydrolysates as the nutrients of chemolithotrophic growth. In contrast to previous studies, little positive but negative effects of the gas vector were observed on gas mass transfer and cell growth. The gas fermentation could be significantly enhanced under a raised pressure, resulting in a higher growth rate (0.12 h−1), cell density (18 g L−1), and gas uptake rate (200 mmole L−1 h−1) than a fermentation under atmospheric pressure. The gain, however, was not proportional to the pressure increase as predicted by Henry’s law. The hydrolysates of cell mass were found a good source of nutrients and the organic nitrogen was equivalent to or better than ammonium nitrogen for chemolithotrophic growth of C. necator on carbon dioxide.

Carboxydothermus islandicus sp. nov., a thermophilic, hydrogenogenic, carboxydotrophic bacterium isolated from a hot spring

An anaerobic, thermophilic bacterium, strain SET IS-9T, was isolated from an Icelandic hot spring. Cells of strain SET IS-9T are short, slightly curved, motile rods. The strain grows chemolithotrophically on CO, producing equimolar quantities of H2 and CO2. It also grows fermentatively on lactate or pyruvate in the presence of yeast extract (0.2 g l−1). Products of pyruvate fermentation are acetate, CO2 and H2. Growth occurs at 50–70 °C, with an optimum at 65 °C, and at pH 5.0–8.0, with an optimum at pH 5.5–6.0. The generation time during chemolithotrophic growth on CO under optimal conditions is 2.0 h. 16S rRNA gene sequence analysis suggested that the organism belongs to the genus Carboxydothermus. On the basis of phenotypic features and phylogenetic analysis, Carboxydothermus islandicus sp. nov. is proposed, with the type strain SET IS-9T ( = DSM 21830T = VKM B-2561T). An emended description of the genus Carboxydothermus is also given.

Hydrogenophilus islandicus sp. nov., a thermophilic hydrogen-oxidizing bacterium isolated from an Icelandic hot spring

A novel chemolithotrophic bacterium, strain 16CT, was isolated from a hot spring in Graendalur, south-west Iceland. Cells of this organism were Gram-negative, rod-shaped and motile. The isolate was aerobic and capable of chemolithotrophic growth on hydrogen and carbon dioxide, heterotrophic growth on butyrate and several other organic compounds, and mixotrophic growth on butyrate, hydrogen and carbon dioxide. Heterotrophic growth was generally enhanced in the presence of yeast extract. Autotrophic growth on hydrogen was observed at pH values between 6.0 and 10.0 and temperatures between 35 and 60 °C; optimum growth conditions were pH 7.0 and 55 °C. The DNA G+C content was 63.9 mol%. 16S rRNA gene sequence analysis showed that strain 16CT was a member of a distinct species belonging to the class Betaproteobacteria and was most closely related to Hydrogenophilus thermoluteolus NBRC 14978T and Hydrogenophilus hirschii DSM 11420T. The major cellular fatty acids were straight-chain C16 : 0 (44.98 %) and C18 : 1 ω7c (17.93 %), as well as cyclic C17 : 0 (13.90 %) and C19 : 0 ω8c (4.67 %) fatty acids. Based on its physiological and molecular properties, it is concluded that strain 16CT represents a novel species within the genus Hydrogenophilus, for which the name Hydrogenophilus islandicus is proposed; the type strain is 16CT (=DSM 21442T=JCM 16106T).

Thiomonas islandica sp. nov., a moderately thermophilic, hydrogen- and sulfur-oxidizing betaproteobacterium isolated from a hot spring

A novel, hydrogen- and sulfur-oxidizing bacterium, designated strain 6CT, was isolated from a hot spring in Graendalur, south-western Iceland. Cells of this organism were Gram-reaction-negative, rod-shaped and motile. The strain grew aerobically and was capable of chemolithotrophic growth on thiosulfate and hydrogen, heterotrophic growth on pyruvate, oxalate, acetate and on glutamate in the presence of yeast extract and mixotrophic growth on several organic compounds, thiosulfate and/or hydrogen. During growth on thiosulfate, the final product was sulfate, resulting in a drop in pH from 6.8 to 2.7. Heterotrophic growth on pyruvate was observed at pH 4–7 (optimum pH 4) and 35–50 °C (optimum 45 °C). The DNA G+C content was 65.2 mol%. As determined by 16S rRNA gene sequence analysis, strain 6CT represents a distinct species belonging to the class Betaproteobacteria and is most closely related to Thiomonas intermedia DSM 18155T and Thiomonas perometabolis DSM 18570T. DNA–DNA hybridization between strain 6CT and Thiomonas intermedia DSM 18155T and Thiomonas perometabolis DSM 18570T gave relatedness values below 32 %. These results, together with physiological characteristics, showed that strain 6CT represents a novel species within the genus Thiomonas, for which the name Thiomonas islandica sp. nov. is proposed. The type strain is 6CT (=DSM 21436T =JCM 16107T).

Thiosulfate-Dependent Chemolithoautotrophic Growth of Bradyrhizobium japonicum

ABSTRACT Thiosulfate-oxidizing sox gene homologues were found at four loci (I, II, III, and IV) on the genome of Bradyrhizobium japonicum USDA110, a symbiotic nitrogen-fixing bacterium in soil. In fact, B. japonicum USDA110 can oxidize thiosulfate and grow under a chemolithotrophic condition. The deletion mutation of the soxY 1 gene at the sox locus I, homologous to the sulfur-oxidizing (Sox) system in Alphaproteobacteria, left B. japonicum unable to oxidize thiosulfate and grow under chemolithotrophic conditions, whereas the deletion mutation of the soxY 2 gene at sox locus II, homologous to the Sox system in green sulfur bacteria, produced phenotypes similar to those of wild-type USDA110. Thiosulfate-dependent O2 respiration was observed only in USDA110 and the soxY 2 mutant and not in the soxY 1 mutant. In the cells, 1 mol of thiosulfate was stoichiometrically converted to approximately 2 mol of sulfate and consumed approximately 2 mol of O2. B. japonicum USDA110 showed 14CO2 fixation under chemolithotrophic growth conditions. The CO2 fixation of resting cells was significantly dependent on thiosulfate addition. These results show that USDA110 is able to grow chemolithoautotrophically using thiosulfate as an electron donor, oxygen as an electron acceptor, and carbon dioxide as a carbon source, which likely depends on sox locus I including the soxY 1 gene on USDA110 genome. Thiosulfate oxidation capability is frequently found in members of the Bradyrhizobiaceae, which phylogenetic analysis showed to be associated with the presence of sox locus I homologues, including the soxY 1 gene of B. japonicum USDA110.

Elemental Sulfur Oxidation in Acidiphilium spp.

The alpha-proteobacterial genus Acidiphilium consists of several acidophilic species, generally known as a part of the mesophilc microbial flora of leaching biotopes. All of them can grow chemoorganotrophically on carbon sources like sugars and many express additional photosynthetic pigments. Thus far, only Ap. acidophilum is known to be capable of chemolithotrophic growth on elemental sulfur oxidation. The oxidation potential of inorganic sulfur species by the other strictly heterotrophic species has not yet been thoroughly investigated. Here, we demonstrate the unequivocal evidence of inorganic sulfur compound oxidation by strains of Ap. cryptum and other Acidiphilium species. Evolutionary and biochemical aspects of this new feature among the heterotrophic Acidiphilium spp. are discussed. This finding will possibly help to solve the long-standing question about the biochemical nature of elemental sulfur oxidation in mesophilic leaching bacteria.