Relationship between methanogenesis and sulfate reduction in anaerobic digestion of municipal sewage sludge was investigated. The density of methanogenic bacteria in the sludge was estimated to be at the order of 104-106 cells/ml. The density of sulfate reducing bacteria was at the order of 105 colony forming units/ml, while the concentration of sulfate in the sludge was low (<0.2mM). Addition of sulfate to the sludge markedly enhanced sulfate reduction without significantly affecting methanogenesis. In the sludge supplemented with sulfate, both methanogenesis and sulfate reduction were significantly enhanced upon the addition of H2. In the presence of exogenous H2, inhibition of methanogenesis or that of sulfate reduction resulted in enhancement of sulfate reduction or that of methanogenesis, respectively. The addition of acetate markedly enhanced methanogenesis but did not affect sulfate reduction, and the addition of propionate markedly enhanced both methanogenesis and sulfate reduction. Degradation of propionate essentially depended on sulfate reduction, and acetate accumulated in response to the propionate degradation when methanogenesis was inhibited. In conclusion, in the sludge, acetate was used only in methanogenesis, and H2 was used in both methanogenesis and sulfate reduction. Sulfate reduction degraded propionate to acetate and enhanced electron flow to methanogenesis.
Extracellular proteases from three psychrotrophic strains of Pseudomonasfluorescens were purified and characterized. The cultivation temperature decisively influenced the excretion of protease into the medium. Highest enzyme production occurred at 10°C (one strain) or 20°C (two strains). The purified proteases showed a molecular weight between 47kDa and 51kDa, the pI values ranged from 7.8 to 7.4. All three proteases were characterized by several closely focused bands. Sensitivity to metalchelating agents indicates that P. fluorescens proteases are metalloproteases (EC 3.4.24). The pH optimum for azocaseinolytic activity was at pH 6-7, the temperature optimum was at 40-45°C. The apparent energies of activation were 36.9-38.0kJ mol-1. The proteases were thermolabile. By adding calcium ion an increasing thermostability was observed. SDS, urea and several metal ions inhibited proteolytic activities to different extents. Casein was the preferred substrate for the proteases. With casein concentrations up to 0.4-0.75% a Michaelis-Menten-type kinetics was observed, at higher casein concentrations a substrate inhibiton occurred. None of the proteases showed aminopeptidase activity.
Direct fermentation of cellulosic materials to acetic acid and ethanol was studied using a cellulolytic strain of Fusarium oxysporum DSM 841. It produces extracellular enzyme complex consisting of avicelase, CMCase, cellobiase and xylanase activities with hydrolytic potential of 80-87% of different cellulosic substrates. Acetic acid (12g/l) and ethanol (3.6g/l) were obtained with yields of 0.36g/g and 0.1 g/g from potato waste (cellulosic waste from starch industries). F. oxysporum 841 was able to ferment D-xylose as efficiently as it could D-glucose. It exhibited relatively higher product tolerance. Mechanism of inhibition of cellulase effectiveness in the presence of acetic acid/ethanol is also discussed.
A bacterial haloacetate dehalogenase (H-1 type enzyme, EC 184.108.40.206) was proved to be one of the useful probes for detecting a genetically engineered microorganism (GEM) from environmental samples. A mono-fluoroacetate assimilating bacterium (Pseudomonas sp.), characteristically encoding H-1 gene on its plasmid pUO12, was applied to two sorts of intact paddy soil and was detected by colony hybridization probing 32P-labeled H-1 DNA fragment (1.6kb). Target bacteria could be quantitatively counted by autoradiography with the samples of no less than 10-3-fold soil dilution while using nutrient agar plates for colonization. Thus, the detection limit of the target bacteria was regarded as 400 CFU (colony-forming unit) per gram of dry weight of soil introducing the principle of most-probable-number method. When the same experiment was carried out using minimal agar containing monofluoroacetate as a sole source of carbon, target colonies were clearly detected even in the 10-1 soil dilution and thus, the detection limit was improved to as low as 4 CFU per gram of dry weight of soil. These redults suggested that H-1 gene probe was very valuable for tracing a GEM in the environment because of its unique nucleotide sequence and high sensitivity.
Seven strains of Pichia abadieae, P. carsonii, P. etchellsii, P. humboldtii, and Candida ingens were examined for partial base sequences in positions 493-622 (130 bases) and positions 1611-1835 (225 bases) of 26S rRNA and in positions 1451-1618 (168 bases) of 18S rRNA. These three partial base sequencings indicated that P. abadieae and P. humboldtii (and C. ingens) are phylogenetically distant from P. carsonii and P. etchellsii. In contrast, the latter two species, P. carsonii and P. etchellsii had very close relationships to Debaryomyces species. Some discussions were made, especially on transferring the two Pichia species to the genus Debaryomyces.
Seasonal variation in distribution of Vibrio cholerae non-O1 and other indicator organisms in Gomati River water along with pH, temperature and percent transmittance was determined for a period of one year. The increased incidence of V. cholerae non-O1 was observed in sewagecontaminated water during warmer months. A significant correlation was found among the heterotrophs, fecal coliforms and fecal streptococci. Of the test strains isolated during summer and monsoon (March-October), 12% showed hemolysin production, while 52%were found to cause fluid accumulation in suckling mice. The isolates also exhibited highest resistance for ampicillin.
The effects of low-temperature (0°C) incubation on nitrogenase-catalyzed acetylene reduction, alkaline phosphatase activity and cellular ATP pool(s) have been studied in Nostoc linckia and Nostoc muscorum. The cultures incubated at 0°C showed higher enzymatic activities than those incubated at 25°C but the ATP content was lower at 0°C. Our experiments with chloramphenicol and rifampicin suggest that de novo protein synthesis is required for nitrogenase, but not for alkaline phosphatase even at low temperature. The increase in alkaline phosphatase activity at 0°C may possibly be due to enhanced catalytic activity of this enzyme. The ability to protect enzyme activities at 0°C implies that N. linckia and Nostoc muscorum may be tolerant to low-temperature cellular damage.
Nisin is a 34 amino acid peptide antibiotic having a molecular weight of about 3, 340, produced and exported into culture medium by some strains of Lactococcus lactis. It is used as a food preservative for its high potency against certain gram-positive bacteria. The structural genes of nisin have been found and sequenced from several nisin-producing strains, which revealed the existence of 23 residue leader peptide and confirmed posttranslational processing of this substance. Recently a newly found strain of L. lactis, JCM7638 (previous IO-1), was reported to secrete a new peptide antibiotic having a molecular weight of about 2, 700 and a spectrum of action similar to nisin. Several chemical analyses showed that it is quite similar to nisin. In this paper, we have ascertained the resistance of JCM7638 to nisin, showed the existence of the gene which is highly homologous to the structural gene of nisin in a nisin-producing strain, L. lactis NCDO497, and cloned a 1.7kb Sau 3AI fragment encoding this gene. Sequence analysis proved that the deduced product differs from nisin by a single amino acid substitution (His27→Asn27), which strongly suggested that JCM7638 secretes degraded nisin and that His27 is not essential to its antibiotic activity.