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Abstract The Subsurface Microbial Culture Collection (SMCC) was established by the U.S. Dept. of Energy (DOE) and contains nearly 10,000 strains of microorganisms (mostly bacteria) isolated from terrestrial subsurface environments. Selected groups of bacterial isolates from three sample sites situated above geochemically and hydrologically different subsurface environments have been characterized by phylogenetic analysis of 16S ribosomal RNA (rRNA) gene nucleotide sequences. Among these isolates were members of six major phylogenetic groups of bacteria: the high-G+C and low-G+C Gram-positive bacteria; the α-, β-, and γ-subdivisions of the Proteobacteria; and the Flexibacter/Cytophaga/Bacteroides group. A small number of the SMCC strains may be members of new bacterial genera, but most of them could be placed with reasonable confidence into more than 35 previously described genera. The majority of the Gram-positive isolates were species of Arthrobacter, Bacillus, or Streptococcus, whereas Acinetobacter, Comamonas, Pseudomonas, Sphingomonas, and Variovorax were among the most frequently encountered Gram-negative genera. A high proportion of the strains were placed in fewer than 10 genera, implying that there is substantial duplication within the SMCC at the genus level. When groups of isolates assigned to Acinetobacter, Arthrobacter, or Sphingomonas were analyzed in more detail, however, it was found that each group consisted of subgroups of strains that probably differed at the species level. Restriction endonuclease analysis (applied to the strains from one sample site) indicated that additional diversity was present at the strain level. Most of the SMCC isolates assigned to some genera (e.g., Acinetobacter) were very closely related to previously described species in those genera, but most of the isolates assigned to other genera (e.g., Arthrobacter and Sphingomonas) appeared (or were shown) to be new species, thereby indicating that a reasonable amount of novelty is present within the SMCC at the species level.

Two anaerobic, psychrotolerant, syntrophic strains were enriched from permanently cold, shallow anoxic marine sediments in Skan Bay, Alaska. One strain, AK‐BT, oxidized butyrate syntrophically and was isolated in defined coculture with a H2‐using methanogen or in a dixenic coculture that also contained an acetate‐scavenging methanogen. The other enrichment culture syntrophically oxidized propionate. The growth of these syntrophic cultures was very slow: approximately 1 year for cocultures of strain AK‐BT to form colonies and >1 year for the propionate‐oxidizing enrichment to form colonies. Neither culture grew axenically when supplied with the catabolic substrates crotonate, pyruvate, malate, or sulfate plus butyrate or propionate. Strain AK‐BT catabolized iso‐butyrate in syntrophic coculture but did not catabolize valerate or caproate. Phylogenetic analyses of the 16S rRNA gene sequence suggested that strain AK‐BT was only distantly related to cultivated sulfate‐reducing bacteria, and that this strain represented a new genus. We propose Algorimarina butyrica, with strain AK‐BT (=OCM 842T), as the type strain. This report is the first description of psychrotolerant as well as marine butyrate‐ and propionate‐oxidizing syntrophic organisms.

In this investigation, the Anaerobic Pump (®TAP) and a conventional continuous flow stirred tank reactor (CFSTR) were tested side by side to compare performance. TAP integrates anaerobic digestion (AD) with biogas plasticization-disruption cycle to improve mass conversion to methane. Both prototypes were fed a “real world” 50:50 mixture of waste-activated sludge (WAS) and primary sludge and operated at room temperature (20°C). The quantitative results from three steady states show TAP peaked at 97% conversion of the particulate COD in a system hydraulic residence time (HRT) of only 6 days. It achieved a methane production of 0.32 STP cubic meter CH₄ per kilogram COD fed and specific methane yield of 0.78 m³ CH₄ per cubic meter per day. This was more than three times the CFSTR specific methane yield (0.22 m³ CH₄ per cubic meter per day) and more than double the CFSTR methane production (0.15 m³ CH₄ per kilogram COD fed). A comparative kinetics analysis showed the TAP peak substrate COD removal rate (R o) was 2.24 kg COD per cubic meter per day, more than three times the CFSTR substrate removal rate of 0.67 kg COD per cubic meter per day. The three important factors contributing to the superior TAP performance were (1) effective solids capture (96%) with (2) mass recycle and (3) stage II plasticization-disruption during active AD. The Anaerobic Pump (®TAP) is a high rate, high efficiency-low temperature microbial energy engine that could be used to improve renewable energy yields from classic AD waste substrates like refuse-derived fuels, treatment plant sludges, food wastes, livestock residues, green wastes and crop residuals.

Hot, anoxic fluids emerging from deep-sea hydrothermal vents mix suddenly with cold oxygenated sea water, providing ideal microbial niches for organisms that need limited amounts of oxygen. We have now identified and grown the first microaerophilic, thermophilic eubacterium from a deep-sea hydrothermal chimney. In view of the likely abundance of this type of microenvironment in hydrothermal structures, these newly discovered thermophilic microbes could constitute a large part of the microbial populations in seafloor hydrothermal systems. In 1995, using the submersible DSV Nautile, we deployed an in situ growth chamber on the top of a hydrothermal vent at the Mid-Atlantic Ridge (Snake Pit, 23 degree 22' N, 44 degree 57' W). After deployment for 5 days (with an in situ chamber temperature between 70 and 25 degree C), we extracted DNA from samples collected from the chamber. We amplified the genes encoding small-subunit ribosomal RNA (16S rRNA genes) by using the polymerase chain reaction, then cloned and screened them by restriction-fragment length-polymorphism analysis.

Hot, anoxic fluids emerging from deep-sea hydrothermal vents mix suddenly with cold oxygenated sea water, providing ideal microbial niches for organisms that need limited amounts of oxygen. We have now identified and grown the first microaerophilic, thermophilic eubacterium from a deep-sea hydrothermal chimney. In view of the likely abundance of this type of microenvironment in hydrothermal structures, these newly discovered thermophilic microbes could constitute a large part of the microbial populations in seafloor hydrothermal systems.

Abstract Two anaerobic, psychrotolerant, syntrophic strains were enriched from permanently cold, shallow anoxic marine sediments in Skan Bay, Alaska. One strain, AK-BT, oxidized butyrate syntrophically and was isolated in defined coculture with a H2-using methanogen or in a dixenic coculture that also contained an acetate-scavenging methanogen. The other enrichment culture syntrophically oxidized propionate. The growth of these syntrophic cultures was very slow: approximately 1 year for cocultures of strain AK-BT to form colonies and >1 year for the propionate-oxidizing enrichment to form colonies. Neither culture grew axenically when supplied with the catabolic substrates crotonate, pyruvate, malate, or sulfate plus butyrate or propionate. Strain AK-BT catabolized iso-butyrate in syntrophic coculture but did not catabolize valerate or caproate. Phylogenetic analyses of the 16S rRNA gene sequence suggested that strain AK-BT was only distantly related to cultivated sulfate-reducing bacteria, and that this strain represented a new genus. We propose Algorimarina butyrica, with strain AK-BT (=OCM 842T), as the type strain. This report is the first description of psychrotolerant as well as marine butyrate- and propionate-oxidizing syntrophic organisms.

The Subsurface Microbial Culture Collection (SMCC) was established by the U.S. Dept. of Energy (DOE) and contains nearly 10,000 strains of microorganisms (mostly bacteria) isolated from terrestrial subsurface environments. Selected groups of bacterial isolates from three sample sites situated above geochemically and hydrologically different subsurface environments have been characterized by phylogenetic analysis of 16S ribosomal RNA (rRNA) gene nucleotide sequences. Among these isolates were members of six major phylogenetic groups of bacteria: the high-G+C and low-G+C Gram-positive bacteria; the α-, β-, and γ-subdivisions of the Proteobacteria; and the Flexibacter/Cytophaga/Bacteroides group. A small number of the SMCC strains may be members of new bacterial genera, but most of them could be placed with reasonable confidence into more than 35 previously described genera. The majority of the Gram-positive isolates were species of Arthrobacter, Bacillus, or Streptococcus, whereas Acinetobacter, Comamonas, Pseudomonas, Sphingomonas, and Variovorax were among the most frequently encountered Gram-negative genera. A high proportion of the strains were placed in fewer than 10 genera, implying that there is substantial duplication within the SMCC at the genus level. When groups of isolates assigned to Acinetobacter, Arthrobacter, or Sphingomonas were analyzed in more detail, however, it was found that each group consisted of subgroups of strains that probably differed at the species level. Restriction endonuclease analysis (applied to the strains from one sample site) indicated that additional diversity was present at the strain level. Most of the SMCC isolates assigned to some genera (e.g., Acinetobacter) were very closely related to previously described species in those genera, but most of the isolates assigned to other genera (e.g., Arthrobacter and Sphingomonas) appeared (or were shown) to be new species, thereby indicating that a reasonable amount of novelty is present within the SMCC at the species level.

A methanogen, strain AK-1, was isolated from permanently cold marine sediments, 38- to 45-cm below the sediment surface at Skan Bay, Alaska. The cells were highly irregular, nonmotile coccoids (diameter, 1 to 1.2 microm), occurring singly. Cells grew by reducing CO2 with H2 or formate as electron donor. Growth on formate was much slower than that on H2. Acetate, methanol, ethanol, 1- or 2-propanol, 1- or 2-butanol and trimethylamine were not catabolized. The cells required acetate, thiamine, riboflavin, a high concentration of vitamin B12, and peptones for growth; yeast extract stimulated growth but was not required. The cells grew fastest at 25 degrees C (range 5 degrees C to 25 degrees C), at a pH of 6.0-6.6 (growth range, pH 5.5-7.5), and at a salinity of 0.25-1.25 M Na+. Cells of this and other H2-using methanogens from saline environments metabolized H2 to a very low threshold pressure (less than 1 Pa) that was dependent on the methane partial pressure. We propose that the threshold pressure may be limited by the energetics of catabolism. The sequence of the 16S rDNA gene of strain AK-1 was most similar (98%) to the sequences of Methanogenium cariaci JR-1 and Methanogeniumfrigidum Ace-2. DNA-DNA hybridization between strain AK-1 and these two strains showed only 34.9% similarity to strain JR-1 and 56.5% similarity to strain Ace-2. These analyses indicated strain AK-1 should be classified as a new species within the genus Methanogenium. Phenotypic differences between strain AK-1 and these strains (including growth temperature, salinity range, pH range, and nutrient requirements) support this. Therefore, a new species, Methanogenium marinum, is proposed with strain AK-1 as type strain.

A methanogen, strain AK-1, was isolated from permanently cold marine sediments, 38- to 45-cm below the sediment surface at Skan Bay, Alaska. The cells were highly irregular, nonmotile coccoids (diameter, 1 to 1.2 mu m), occurring singly. Cells grew by reducing CO sub(2) with H sub(2) or formate as electron donor. Growth on formate was much slower than that on H sub(2). Acetate, methanol, ethanol, 1- or 2-propanol, 1- or 2-butanol and trimethylamine were not catabolized. The cells required acetate, thiamine, riboflavin, a high concentration of vitamin B sub(12), and peptones for growth; yeast extract stimulated growth but was not required. The cells grew fastest at 25 degree C (range 5 degree C to 25 degree C), at a pH of 6.0 - 6.6 (growth range, pH 5.5 - 7.5), and at a salinity of 0.25 - 1.25 M Na super(+). Cells of this and other H sub(2)-using methanogens from saline environments metabolized H sub(2) to a very low threshold pressure (less than 1 Pa) that was dependent on the methane partial pressure. We propose that the threshold pressure may be limited by the energetics of catabolism. The sequence of the 16S rDNA gene of strain AK-1 was most similar (98%) to the sequences of Methanogenium cariaci JR-1 and Methanogenium frigidum Ace-2. DNA-DNA hybridization between strain AK-1 and these two strains showed only 34.9% similarity to strain JR-1 and 56.5% similarity to strain Ace-2. These analyses indicated strain AK-1 should be classified as a new species within the genus Methanogenium. Phenotypic differences between strain AK-1 and these strains (including growth temperature, salinity range, pH range, and nutrient requirements) support this. Therefore, a new species, Methanogenium marinum, is proposed with strain AK-1 as type strain.

Hot, anoxic fluids emerging from deep-sea hydrothermal vents mix suddenly with cold oxygenated sea water, providing ideal microbial niches for organisms that need limited amounts of oxygen. We have now identified and grown the first microaerophilic, thermophilic eubacterium from a deep-sea hydrothermal chimney. In view of the likely abundance of this type of microenvironment in hydrothermal structures, these newly discovered thermophilic microbes could constitute a large part of the microbial populations in seafloor hydrothermal systems.