The cellular fatty acid composition of 27 strains of gram-negative obligately anaerobic rods was determined by gas-liquid chromatography. Results showed that a variety of acids was detected in these organisms, and comparison of the relative amount of each of the acids was useful in distinguishing several species. A dendrogram based on their fatty acid composition was made, and this analysis appeared to be an aid in the grouping of the bacteria.

Two similar Gram-positive rods were isolated from 10 "6 dilutions of ruminal fluid from a sheep receiving a mixed grass hay/concentrate diet, using a medium containing pancreatic casein hydrolysate as sole source of carbon and energy. The isolates did not ferment sugars, but grew on pyruvate or trypticase, forming caproate as the main fermentation product and valerate to a lesser extent. Acetate and propionate were utilized. One of these strains, I-6 T , was selected for further study. Strain I-6 T was a non-motile coccal rod, 1?260?4 mm, with a Gram-positive cell wall ultrastructure and a G+C content of 56?8 mol%. No spores were visible, and strain I-6 T did not survive heating at 80˚C for 10 min. Its rate of NH 3 production was 375 nmol (mg protein) "1 min "1 , placing it in the 'ammonia-hyperproducing' (or HAP) group of ruminal bacteria. 16S rDNA sequence analysis (1296 bases) indicated that it represents a novel species within the 'low-G+C' Grampositive group, for which the name Eubacterium pyruvativorans sp. nov. is proposed. Among cultivated bacteria, strain I-6 T was most closely related (89 % identity) to other asaccharolytic Eubacterium isolates from the mouth and the rumen. It was 98 % identical to uncultured bacterial sequences amplified by others from ruminal digesta.

The genera of sixty-three bacterial strains isolated from bovine rumen contents were determined according to the identification schemes of Hungate, and Bergey's Manual of Determinative Bacteriology (8th Ed.). Then, the cellular fatty acid and aldehyde composition of all these organisms was analyzed by gas-liquid chromatography, gas chromatography-mass spectrometry, and mass fragmentography. The distinctive features of these organisms were presented. Succinivibrio contained 18: 1 (w-7), 16: 0, 14:0,16: 1, and 3-OH-14: 0 as the most abundant fatty acids. Lachnospira contained 18: 0, 16: 0, and 18: 1 (w-7) as the most abundant acids. Anteiso-l5: 0 and 15: 0 were the most abundant acids in Bacteroides. Iso-16: 0, anteiso-17: 0, 16: 0, and 18: 0 were the most abundant acids in an unidentified gram-negative obligately anaerobic rod. The Butyrivibrio strains were divided into two major groups based on fatty acid and fatty aldehyde composition. One group contained branched-chain fatty acids and fatty aldehydes as major components, and the other had straight-chain ones. The former group contained anteiso-15: 0 fatty acid (FA) and anteiso-15: 0 aldehyde (ALD) as major components. In the latter group, a considerable variation was found in the relative amount of each fatty acid and fatty aldehyde. The major fatty components in Selenomonas were 16:1 FA, 14: 0 FA, 3-OH-14: 0 FA, and 16: 1 ALD. Lactobacillus contained 16: 0 FA, 16: 0 ALD, 16: 1 ALD, and 16: 1 FA as major components, and in Bifidobacterium they were 18: 1 (w-9) FA, 16: 0 FA, 14: 0 FA, and 18: 1 ALD. The major fatty acids of Propionibacterium were iso-15:0,16: 0, and iso-17: 0, and those of unidentified gram-positive chainforming anaerobic coccus were anteiso-15: 0 and anteiso-17: 0. Each of the nine different kinds of bacteria mentioned above, but not Butyrivibrio, formed a single dendrogram cluster according to their fatty acid and fatty aldehyde composition. The Butyrivibrio species were divided into three clusters.

To estimate the contribution of uncultured bacterial groups to fiber degradation, we attempted to retrieve both ecological and functional information on uncultured groups in the rumen. Among previously reported uncultured bacteria, fiber-associated groups U2 and U3, belonging to the low-GC Gram-positive bacterial group, were targeted. PCR primers and fluorescence in situ hybridization (FISH) probe targeting 16S rRNA genes or rRNA were designed and used to monitor the distribution of targets. The population size of group U2 in the rumen was as high as 1.87%, while that of group U3 was only 0.03%. Strong fluorescence signals were observed from group U2 cells attached to plant fibers in the rumen. These findings indicate the ecological significance of group U2 in the rumen. We succeeded in enriching group U2 using rumen-incubated rice straw as the inoculum followed by incubation in an appropriate medium with an agent inhibitory for Gram-negative bacteria. Consequently, we successfully isolated two strains, designated B76 and R-25, belonging to group U2. Both strains were Gram-positive short rods or cocci that were 0.5 to 0.8 m in size. Strain B76 possessed xylanase and ␣-L-arabinofuranosidase activity. In particular, the xylanase activity of strain B76 was higher than that of xylanolytic Butyrivibrio fibrisolvens H17c grown on cellobiose. Strain R-25 showed an ␣-L-arabinofuranosidase activity higher than that of strain B76. These results suggest that strains B76 and R-25 contribute to hemicellulose degradation in the rumen.

Examination of non-methanogenic anaerobic bacteria in sewage digestor fluid was attempted by the anaerobic roll tube method. For the enumeration of anaerobic bacteria in digestor fluid, rumen fluid-glucosecellobiose-agar (RGCA) gave as good results as previously reported in the rumen and other ecosystems like intestines and feces, under the 100% CO2 gas phase. However, under the mixed gas phase (95 % N2 and 5 % C02), higher colony counts were obtained with YLS agar, which contained the supernatant of autoclaved sewage digestor fluid, than with RGCA. Colony counts of anaerobic bacteria decreased with the progress of fermentation of waste and the proportion of facultative anaerobes in the isolated anaerobes also decreased. Large variations in the distribution pattern of bacteria were usually observed between the results obtained from three digestors investigated. However, Streptococcus and Gram-negative curved rods were commonly isolated from the three digestors as predominant groups.The anaerobic roll tube method devised by HUNGATE (1) facilitated isolation and cultivation of strictly anaerobic bacteria, and a significant progress has been made in the studies of microbial ecosystem in the rumen (2). This method was also applied to isolate anaerobic bacteria from intestines (3, 4) and feces (5, 6).However, application of this method to the enumeration and isolation of anaerobic bacteria in other ecosystems, such as the soil and sewage digestors has been very scarce. Our knowledge about anaerobic bacteria in these ecosystems is restricted to only a few kinds of bacteria and increase in the information about other anaerobic bacteria in these ecosystems is being expected.The anaerobic ecosystem of sewage digestor resembles that of rumen. The process of fermentation in digestors involves two stages, non-methanogenic and methanogenic. At the non-methanogenic stage, organic matters poured into digestors are decomposed to lower fatty acids and other low-molecular compounds. 317