Two sheep, with and without ciliate protozoa, were fed on a wood-pulp cellulose, corn starch, soya-bean protein diet and the microbiological and chemical characteristics of the rumen ingesta of both sheep were studied. The purified diet led to a simplified rumen flora enabling some deductions to be made about the interactions of the principal bacterial species and their interactions with the protozoa in relationship to the biochemical analysis of the rumen. Ammonia concentrations were similarly low in each animal. Total volatile fatty acid concentrations were higher in the faunated sheep though the proportion of propionic acid was highest in the unfaunated sheep. Cellulose digestion in the faunated rumen was about twice that in the unfaunated one. Total bacterial concentrations in the unfaunated rumen were over twice those in the faunated rumen, but the numbers of cellulolytic bacteria were higher in the latter while the numbers of amylolytic bacteria were higher in the unfaunated rumen. The principal species of bacteria differed in the two rumens. INTRODUCTIONThe natural rumen flora contains both bacteria and ciliate protozoa, but the role of the protozoa in rumen function and the relationships between the protozoa and bacteria are not completely understood. Animals can be, by partial isolation, reared without rumen ciliate protozoa, or the organisms can be removed from the rumen population by chemical additives or by change in feed to one producing rather acidic rumen conditions (Eadie, 1962;Abou Akkada et al. 1968;Eadie et al. 1970). Investigations of metabolic activities of the protozoa have shown that they are essentially similar to those of the bacteria so far as the main functions of the rumen are concerned, that is they use carbohydrates as energy sources and form acidic fermentation products and cell material which are utilized by the host animal. Because of this, the fact that on most diets the protozoa perform only a minor part of the total rumen metabolism, and in many cases rumen bacterial concentrations have been found to be higher in unfaunated than faunated animals, unfaunated animals are not

ABSTRACT. Experimental studies on the bioremediation of groundwater contaminated with low concentration trichloroethylene (TCE) and cis1,2-dichloroethylene (DCE) were performed with two sets of bioreactors. Reactors No. 1 and No. 2 were operated without and with methane supplement, respectively. No inoculum was used. The concentrations of TCE and DCE in the effluent and the off gas from reactor No. 2 were much lower than those from reactor No. 1. When air and an H 2 O 2 solution were supplied to reactor No. 2, concentrations of TCE and DCE in the effluent and the off gas were lower than the lowest detectable limit. The population of methaneutilizing bacteria in reactor No. 2 was 1,000 times higher than that in groundwater or in the effluent from reactor No. 1. These methaneutilizing bacteria were apparently attributable to the treatment of TCE.-KEY WORDS: bioreactor, methane-utilizing bacteria, trichloroethylene.J. Vet. Med. Sci. 61 (7): [861][862][863] 1999 Two bioreactors were packed with 1,000 ml (an effective volume of 500 ml) cylindrical (4 mm OD , 3 mm ID , 5 mm L ) carriers made of polypropylene [18]. No inoculum was used. These reactors were operated in a single-pass mode with a hydraulic retention time of 8 hr. The influent groundwater flow rate was 1 ml/min, the air flow rate of reactor No. 1 5 ml/min, the air flow rate of reactor No. 2 4 ml/min and the methane flow rate of reactor No. 2 1 ml/ min. The concentration of H2O2 supplied was 0.3% and a 0.3% H2O2 solution flow rate was 5 ml/hr. An experiment which used H2O2 as the oxygen source of supply of the bioreactor was reported by Gerhard et al. [8]. The concentration of H2O2 was so set as to make 13 ppm oxygen in the reactors based on the field experiment made by Semprini et al. [17]. The number of heterotrophic bacteria was calculated by counting colonies, which appeared on standard agar plates (Eiken Co., Tokyo, Japan) incubated at 25°C for 7 days. The methanotrophic bacteria were counted by the most probable number (MPN) method after 8 weeks of incubation in nitrate mineral salts (NMS) medium [26] under an atmosphere of methane-air (1:4) in a desiccator at 30°C. Samples for determination of TCE and DCE concentrations in liquids were prepared by the head-space method [1] and gas samples were analyzed with a gas chromatograph (Type GC-311, HNU Co., U.S.A.). Dissolved oxygen (DO) was analyzed with a DO meter. Groundwater contaminated with about 0.6 ppm of TCE and 0.2 ppm of DCE was used as influent. There were 1 × 10 3 MPN/ml of methane-utilizing bacteria in this groundwater.At first, oxygen was supplied to reactors by aeration. Methane-utilizing bacterial flocculation, a substantial growth of salmon-pink-colored biomass, was seen in the whole reactor No. 2 in two weeks after the beginning the experiment, and then, decrease in TCE became apparent and DO in effluent dropped from 7.5 ppm to 1.0 ppm in reactor No. 2. Finally, more than 90% of TCE and more than 70% of DCE were removed from reactor No. 2 in 3 weeks. However, in reactor No.

ABSTRACT. A mixture of bacteria, having a methane-utilizing ability, was separated from a bioreactor supplied with air and methane gas. The bioreactor was operated to treat trichloroethylene (TCE)-contaminated groundwater. The mixture was composed of an obligate methane-utilizing bacterium and a heterotroph, identified as Methylomonas methanica and Pseudomonas sp., respectively. The mixed culture of these two strains removed TCE. In addition, it appeared that a cooperative metabolic interaction of these strains enabled Meth.methanica to maintain the TCE degradation ability. The bacteria found in enrichment culture were short thick rods and thin rods. These bacteria were isolated as obligate methane-utilizing and heterotrophic bacteria, respectively. The methane-utilizing bacterium formed pink, small, smooth colonies on NMS agar and the heterotrophic bacterium formed yellow, smooth colonies on the standard agar plates. On NMS agar plates, the purified methane-utilizing bacteria formed grow much more slowly than the pre-purified one. Isolates from NMS or standard agar failed to grow on other media.The methane-utilizing isolate was a gram-negative and motile rod with a width of 0.7 µm and a length of about 1.3-1.8 µm. In a static NMS liquid culture, the methaneutilizing isolate gave rise to a pink surface pellicle. A representative electron micrograph of these methaneutilizing bacteria is shown in Fig.