Draft genome sequence of Methanoculleus sp. MH98A, a novel methanogen isolated from sub-seafloor methane hydrate deposits in Krishna Godavari basin.

Members of the genus Methanoculleus are among the most prevalent methanogens in biomethanation processes especially in marine and brackish environments. A methanogen, identified as a novel species of the genus Methanoculleus, was isolated from deep sub-seafloor sediment obtained from the Krishna Godavari Basin off the eastern coast of India. This methanogen is thought to be the supplier of the methane in the submarine methane hydrate deposits. Further study of this microorganism could possibly help to revolutionize the energy industry. The draft genome of Methanoculleus sp. MH98A is presented.

Megasphaera indica sp. nov., an obligate anaerobic bacteria isolated from human faeces.

Two coccoid, non-motile, obligately anaerobic, Gram-stain-negative bacteria, occurring singly or in pairs, or as short chains, with a mean size of 1.4-2.5 µm were isolated from the faeces of two healthy human volunteers, aged 26 and 56 years, and were designated NMBHI-10(T) and BLPYG-7, respectively. Both the strains were affiliated to the sub-branch Sporomusa of the class Clostridia as revealed by 16S rRNA gene sequence analysis. The isolates NMBHI-10(T) and BLPYG-7 showed 99.1 and 99.2% 16S rRNA gene sequence similarity, respectively, with Megasphaera elsdenii JCM 1772(T). DNA-DNA hybridization and phenotypic analysis showed that both the strains were distinct from their closest relative, M. elsdenii JCM 1772(T) (42 and 53% DNA-DNA relatedness with NMBHI-10(T) and BLPYG-7, respectively), but belong to the same species (DNA-DNA relatedness of 80.9 % between the isolates). According to DNA-DNA hybridization results, the coccoid strains belong to the same genospecies, and neither is related to any of the recognized species of the genus Megasphaera. Strains NMBHI-10(T) and BLPYG-7 grew in PYG broth at temperatures of between 15 and 40 °C (optimum 37 °C), but not at 45 °C. The strains utilized a range of carbohydrates as sources of carbon and energy including glucose, lactose, cellobiose, rhamnose, galactose and sucrose. Glucose fermentation resulted in the formation of volatile fatty acids, mainly caproic acid and organic acids such as succinic acid. Phylogenetic analysis, specific phenotypic characteristics and/or DNA G+C content also differentiated the strains from each other and from their closest relatives. The DNA G+C contents of strains NMBHI-10(T) and BLPYG-7 are 57.7 and 54.9 mol%, respectively. The major fatty acids were 12 : 0 FAME and 17 : 0 CYC FAME. On the basis of these data, we conclude that strains NMBHI-10(T) and BLPYG-7 should be classified as representing a novel species of the genus Megasphaera, for which the name Megsphaera indica sp. nov. is proposed. The type strain is NMBHI-10(T) ( = DSM 25563(T) = MCC 2481(T)).

Upflow anaerobic filter for the degradation of adsorbable organic halides (AOX) from bleach composite wastewater of pulp and paper industry.

The removal of AOX from bleach plant effluent of pulp and paper industry was studied using upflow anaerobic filter. In this paper biodegradation of AOX at different concentrations and effect of electron donors like acetate and glucose thereon in an upflow anaerobic filter at 20 d HRT is described. Results showed significant improvement in AOX degradation when electron donors such as acetate and glucose were supplemented to the influent. AOX degradation was 88% at 28 mg AOX L(-1) and 28% at 42 mg AOX L(-1). The percent degradation efficiency was enhanced to 90.7, 90.2, and 93.0 at 28 mg AOX L(-1) when the influent was supplemented with glucose, acetate and both glucose and acetate, respectively. Similarly, the efficiency was 57, 56.6 and 79.6 at 42 mg AOX L(-1) when the influent was supplemented with glucose, acetate and both glucose and acetate, respectively. The GC-MS analysis data indicated that supplementation of the influent with electron donor increased the biodegradability of number of chlorinated organic compounds.

Anaerobic degradation of adsorbable organic halides (AOX) from pulp and paper industry wastewater.

Adsorbable organic halides (AOX) are generated in the pulp and paper industry during the bleaching process. These compounds are formed as a result of reaction between residual lignin from wood fibres and chlorine/chlorine compounds used for bleaching. Many of these compounds are recalcitrant and have long half-life periods. Some of them show a tendency to bioaccumulate while some are proven carcinogens and mutagens. Hence, it is necessary to remove or degrade these compounds from wastewater. Physical, chemical and electrochemical methods reported to remove AOX compounds are not economically viable. Different types of aerobic, anaerobic and combined biological treatment processes have been developed for treatment of pulp and paper industry wastewater. Maximum dechlorination is found to occur under anaerobic conditions. However, as these processes are designed specifically for reducing COD and BOD of wastewater, they do not ensure complete removal of AOX. This paper reviews the anaerobic biological treatments developed for pulp and paper industry wastewater and also reviews the specific micro-organisms reported to degrade AOX compounds under anaerobic conditions, their nutritional and biochemical requirements. It is imperative to consider these specific micro-organisms while designing an anaerobic treatment for efficient removal of AOX.

Two-stage anaerobic digestion process for complete inactivation of enteric bacterial pathogens in human night soil.

Anaerobic digestion offers a good alternative for human waste treatment. However, the fate of enteric bacterial pathogens present in human night soil (HNS) remains a major concern for hygienic safety of the process. A two-stage anaerobic digestion process, consisting of separate acidogenic and methanogenic digesters, was designed and its efficacy in the inactivation of Salmonella typhi was compared to a single-stage digestion process. In a single-stage digestion, complete pathogen inactivation was achieved only in the digesters with high levels of volatile fatty acids (VFA approximately equal to 18,000 mg/l) and acidic pH (approximately equal to 6.0). These digesters, however, showed drastic reduction in methane yield. In the two-stage digestion process, S. typhi was completely inactivated in the acidogenic digester and the methanogenic digester was free from the pathogen even after receiving a daily dose of the pathogen. The process also achieved complete inactivation of other enteric pathogens, viz., Shigella dysenteriae and Vibrio cholerae. The two-stage process was efficient in biogas generation from HNS. Thus, the two-stage process ensures complete hygienic safety in anaerobic digestion of human night soil.

Application of Methanobrevibacter acididurans in anaerobic digestion.

To operate anaerobic digesters successfully under acidic conditions, hydrogen utilizing methanogens which can grow efficiently at low pH and tolerate high volatile fatty acids (VFA) are desirable. An acid tolerant hydrogenotrophic methanogen viz. Methanobrevibacter acididurans isolated from slurry of an anaerobic digester running on alcohol distillery wastewater has been described earlier by this lab. This organism could grow optimally at pH 6.0. In the experiments reported herein, M. acididurans showed better methanogenesis under acidic conditions with high VFA, particularly acetate, than Methanobacterium bryantii, a common hydrogenotrophic inhabitant of anaerobic digesters. Addition of M. acididurans culture to digesting slurry of acidogenic as well as methanogenic digesters running on distillery wastewater showed increase in methane production and decrease in accumulation of volatile fatty acids. The results proved the feasibility of application of M. acididurans in anaerobic digesters.

Methanobrevibacter acididurans sp. nov., a novel methanogen from a sour anaerobic digester.

A novel acid-tolerant, hydrogenotrophic methanogen, isolate ATMT, was obtained from an enrichment performed at pH 5.0 using slurry from an acidogenic digester running on alcohol distillery waste. The original pH of the slurry was 5.7 and the volatile fatty acid concentration was 9000 p.p.m. Cells of isolate ATMT were Gram-positive, non-motile and 0.3-0.5 microm in size. They did not form spores. The isolate could grow in the pH range 5.0-7.5, with maximum growth at pH 6.0. The optimum temperature for growth was 35 degrees C. Formate, acetate, methanol, trimethylamine, 2-propanol and 2-butanol were not utilized as growth substrates. Rumen fluid and acetate were required for growth on H2/CO2. Coenzyme M and 2-methylbutyric acid were not required in the presence of rumen fluid. 16S rDNA sequence analysis confirmed the signature sequence of the genus Methanobrevibacter. Morphological and biochemical characteristics of the isolate, together with the 16S rDNA sequence analysis, clearly revealed that the isolate could not be accommodated within any of the existing species of the genus Methanobrevibacter. Therefore, it is proposed that a novel species of the genus Methanobrevibacter should be created for this isolate, Methanobrevibacter acididurans sp. nov., and the type strain is

Selenomonas lipolytica sp. nov., an obligately anaerobic bacterium possessing lipolytic activity.

A novel, oligately anaerobic bacterium capable of hydrolysing lipids was isolated from a tropical anaerobic lagoon receiving waste water from an edible oil mill. The isolate had many characteristics similar to those of members of the genus Selenomonas. The isolate showed lipolytic activity on tributyrin, triolein and groundnut oil in qualitative plate clearance assays, which has not been reported for the type strain of the genus Selenomonas. It did not require n-valerate supplementation for growth on glucose. Acetate and propionate were the only volatile fatty acids produced from glucose fermentation with propionate as the major end product. The isolate could grow optimally at pH 6.8 and at a temperature of 40 degrees C. It could tolerate NaCl concentrations of up to 40 g l-1. The G&C content of the DNA was 40 mol% as determined by thermal denaturation analysis. Comparison of partial 165 rRNA gene sequences revealed that the isolate was most closely related to genus Selenomonas with 91% sequence similarity (250 bp compared) to Selenomonas ruminantium strain GA 192. On the basis of the results obtained in the present investigation, it is suggested that a new species of Selenomonas should be created for this novel isolate and the name Selenomonas lipolytica is proposed for this new species. The type strain is strain CF1BT (= MCMB 505T).

Inactivation of Salmonella typhi by high levels of volatile fatty acids during anaerobic digestion.

Survival of Salmonella typhi was investigated in an anaerobic digester for cattle dung with volatile fatty acid (VFA) levels of 5000 mg l(-1) and pH 6.0. The organism was added to the digester only once in the first experiment and daily in the other. Survival was monitored on alternate days. In the single dose experiment, the counts of Salm. typhi declined rapidly and the pathogen was completely eliminated within 12 d in the experimental digester (VFA ca 5000 mg l(-1) and pH 6.0), whereas 26 d were required in the control digester (VFA ca 100 mg l(-1) and pH 6.8). T90 values for the experimental and control digesters were 2.44 d and 4.80 d, respectively. In the daily dose experiment, a four log reduction in the pathogen count was observed in the experimental digester, but only a two log reduction in the control digester at the end of the experimental period. The mean T90 values for the experimental and the control digester were 4.22 d and 18.63 d, respectively. In both the experiments, statistical analysis of the data showed significant differences in the survival pattern of Salm. typhi in the two digesters.

Short communication: Butyrate-degrading syntrophic culture from an anaerobic digester treating cattle waste.

A co-culture of bacteria responsible for the conversion of butyrate to methane and CO2 was isolated from a cattle-waste treatment plant. The non-methanogenic partner of the co-culture was Syntrophomonas wolfei and the methanogenic partner was Methanobacterium formicicum. Although butyrate degradation occurred at pH<6.0 and below 45°C, methanogenesis was observed at pH>6.5 and above 40°C.

Inhibition of methanogens by n- and iso-volatile fatty acids.

n-Butyrate, n-valerate and n-caproate were more inhibitory towards Methanobacterium byrantii, Methanobacterium formicicum and Methanosarcina barkeri than the corresponding iso-acids. Butyrate caused maximum inhibition irrespective of isomer. Methanobacterium bryantii was more sensitive to inhibition than Methanobacterium formicicum.

Morphology of the ovaries of Sphaerodema (Diplonychus) rusticum (Heteroptera, Belostomatidae).

The female reproductive system of Sphaerodema rusticum consists of a pair of ovaries, two lateral oviducts, a median common oviduct, and a median spermatheca. Accessory glands are absent. Each ovary has five free ovarioles branching from the oviduct. Each ovariole consists of a terminal filament, germarium, vitellarium, brown mass, and an exceptionally long pedicel. The terminal filament consists of a central core, interstitial cells, and an outer sheath. In the germarium, which consists of trophic and prefollicular regions, the trophic region or nurse cell chamber is divided into four histologically differentiated zones, distinguished as zones I-IV. Nutritive cords, originating from the posterior end of the trophic core in zone IV extend centrally and join the developing oocytes in the prefollicular chamber and the vitellarium. The compact prefollicular tissue at the base of the trophic core gives rise to prefollicular cells which, after encircling the young oocytes, become modified into follicular epithelial cells, the interfollicular plug, and epithelial plug. The young oocytes descend into the vitellarium and gradually develop into mature oocytes. A compound corpus luteum is observed simultaneously in all the ovarioles of both ovaries after ovulation. Below the epithelial plug there is an accumulation of material, the "brown mass," which develops cyclically in correlation with the ovulation cycle. Each pedicel stores five mature chorionated eggs ready for oviposition. The epithelium of the anterior region of the pedicel secretes a PAS-positive material. General morphology and histology of the subdivisions of the ovarioles are described.

A note on survival of salmonellas during anaerobic digestion of cattle dung.

Anaerobic digestion of night soil with cattle dung slurry in biogas plants is advocated in Indian villages as a means of disposal of human excreta in the absence of conventional sanitary systems. Although intestinal pathogens are likely to be eliminated during anaerobic digestion, there is no conclusive evidence that this is so. Large numbers of saprophytic organisms in the fermenting mass make it impossible to detect the residual pathogens. Use of an antibiotic-resistant strain of Salmonella typhimurium as a test organism to study its survival during anaerobic digestion showed that the organism is totally eliminated in nine days.