Biogas plasticization coupled anaerobic digestion: continuous flow anaerobic pump test results.

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 degrees Celsius). 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 CH(4) per kilogram COD fed and specific methane yield of 0.78 m(3) CH(4) per cubic meter per day. This was more than three times the CFSTR specific methane yield (0.22 m(3) CH(4) per cubic meter per day) and more than double the CFSTR methane production (0.15 m(3) CH(4) 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.

Butyrate- and propionate-degrading syntrophs from permanently cold marine sediments in Skan Bay, Alaska, and description of Algorimarina butyrica gen. nov., sp. nov.

Two anaerobic, psychrotolerant, syntrophic strains were enriched from permanently cold, shallow anoxic marine sediments in Skan Bay, Alaska. One strain, AK-B(T), oxidized butyrate syntrophically and was isolated in defined coculture with a H(2)-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-B(T) 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-B(T) 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-B(T) was only distantly related to cultivated sulfate-reducing bacteria, and that this strain represented a new genus. We propose Algorimarina butyrica, with strain AK-B(T) (=OCM 842(T)), as the type strain. This report is the first description of psychrotolerant as well as marine butyrate--and propionate-oxidizing syntrophic organisms.

Cultivation of methanogens from shallow marine sediments at Hydrate Ridge, Oregon.

Little is known about the methanogenic degradation of acetate, the fate of molecular hydrogen and formate or the ability of methanogens to grow and produce methane in cold, anoxic marine sediments. The microbes that produce methane were examined in permanently cold, anoxic marine sediments at Hydrate Ridge (44 degrees 35' N, 125 degrees 10' W, depth 800 m). Sediment samples (15 to 35 cm deep) were collected from areas of active methane ebullition or areas where methane hydrates occurred. The samples were diluted into enrichment medium with formate, acetate or trimethylamine as catabolic substrate. After 2 years of incubation at 4 degrees C to 15 degrees C, enrichment cultures produced methane. PCR amplification and sequencing of the rRNA genes from the highest dilutions with growth suggested that each enrichment culture contained a single strain of methanogen. The level of sequence similarity (91 to 98%) to previously characterized prokaryotes suggested that these methanogens belonged to novel genera or species within the orders Methanomicrobiales and Methanosarcinales. Analysis of the 16S rRNA gene libraries from DNA extracted directly from the sediment samples revealed phylotypes that were either distantly related to cultivated methanogens or possible anaerobic methane oxidizers related to the ANME-1 and ANME-2 groups of the Archaea. However, no methanogenic sequences were detected, suggesting that methanogens represented only a small proportion of the archaeal community.

Methanococcus aeolicus sp. nov., a mesophilic, methanogenic archaeon from shallow and deep marine sediments.

Three strains of CO(2)-reducing methanogens were isolated from marine sediments. Strain PL-15/H(P) was isolated from marine sediments of the Lipari Islands, near Sicily and the other two strains, Nankai-2 and Nankai-3(T), were isolated from deep marine sediments of the Nankai Trough, about 50 km from the coast of Japan. Analysis of the cellular proteins and 16S rRNA gene sequences indicated that these three strains represented a single novel species that formed a deep branch of the mesophilic methanococci. Phylogenetic analysis indicated that the three strains were most closely related to Methanothermococcus okinawensis (95 % 16S rRNA gene sequence similarity). However, strains PL-15/H(P), Nankai-2 and Nankai-3(T) grew at temperatures that were more similar to those of recognized species within the genus Methanococcus. Strain Nankai-3(T) grew fastest at 46 degrees C. Results of physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strains PL-15/H(P), Nankai-2 and Nankai-3(T) from closely related species. The name Methanococcus aeolicus sp. nov. is proposed, with strain Nankai-3(T) (=OCM 812(T)=DSM 17508(T)) as the type strain.

Isolation and characterization of methylotrophic methanogens from anoxic marine sediments in Skan Bay, Alaska: description of Methanococcoides alaskense sp. nov., and emended description of Methanosarcina baltica.

Three novel strains of methylotrophic methanogens were isolated from Skan Bay, Alaska, by using anaerobic cultivation techniques. The water was 65 m deep at the sampling site. Strains AK-4 (=OCM 774), AK-5T (=OCM 775T=DSM 17273T) and AK-9 (=OCM 793) were isolated from the sulfate-reducing zone of the sediments. Each of the strains was a non-motile coccus and occurred singly. Cells grew with trimethylamine as a catabolic substrate and strain AK-4 could also catabolize methanol. Yeast extract and trypticase peptones were not required for growth, but their addition to the culture medium slightly stimulated growth. Each of the strains grew at temperatures of 5-28 degrees C; they were slight halophiles and grew fastest in the neutral pH range. Analysis of the 16S rRNA gene sequences indicated that strain AK-4 was most closely related to Methanosarcina baltica. DNA-DNA hybridization studies showed 88 % relatedness, suggesting that strain AK-4 represents a novel strain within this species. Strains AK-5T and AK-9 had identical 16S rRNA gene sequences that were most closely related to the sequence of Methanococcoides burtonii (99.8 % sequence similarity). DNA-DNA hybridization studies showed that strains AK-5T and AK-9 are members of the same species (88 % relatedness value), but strain AK-5T had a DNA-DNA relatedness value of only 55 % to Methanococcoides burtonii. This indicates that strains AK-5T and AK-9 should be considered as members of a novel species in the genus Methanococcoides. We propose the name Methanococcoides alaskense sp. nov., with strain AK-5T (=OCM 775T=DSM 17273T) as the type strain.

Isolation of a methanogen from deep marine sediments that contain methane hydrates, and description of Methanoculleus submarinus sp. nov.

We isolated a methanogen from deep in the sediments of the Nankai Trough off the eastern coast of Japan. At the sampling site, the water was 950 m deep and the sediment core was collected at 247 m below the sediment surface. The isolated methanogen was named Nankai-1. Cells of Nankai-1 were nonmotile and highly irregular coccoids (average diameter, 0.8 to 2 micro m) and grew with hydrogen or formate as a catabolic substrate. Cells required acetate as a carbon source. Yeast extract and peptones were not required but increased the growth rate. The cells were mesophilic, growing most rapidly at 45 degrees C (no growth at /=55 degrees C). Cells grew with a maximum specific growth rate of 2.43 day(-1) at 45 degrees C. Cells grew at pH values between 5.0 and 8.7 but did not grow at pH 4.7 or 9.0. Strain Nankai-1 grew in a wide range of salinities, from 0.1 to 1.5 M Na(+). The described phenotypic characteristics of this novel isolate were consistent with the in situ environment of the Nankai Trough. This is the first report of a methanogenic isolate from methane hydrate-bearing sediments. Phylogenetic analysis of its 16S rRNA gene sequence indicated that it is most closely related to Methanoculleus marisnigri (99.1% sequence similarity), but DNA hybridization experiments indicated a DNA sequence similarity of only 49%. Strain Nankai-1 was also found to be phenotypically similar to M. marisnigri, but two major phenotypic differences were found: strain Nankai-1 does not require peptones, and it grows fastest at a much higher temperature. We propose a new species, Methanoculleus submarinus, with strain Nankai-1 as the type strain.

Methanogenium marinum sp. nov., a H2-using methanogen from Skan Bay, Alaska, and kinetics of H2 utilization.

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.