Isolation of psychrotolerant methangogens

Progress Report
on
STUDY OF COLD TOLERANT
BIOGAS PRODUCING BACTERIA
Submitted To
Ministry of Environment, Science &Technology (MoEST)
Submitted By
Nepal Academy of Science & Technology (NAST)
Chaitra, 2065
PROJECT TITLE: Study of Cold Tolerant Biogas Producing Bacteria
IMPLEMENTING AGENCY: Nepal Academy of Science & Technology (NAST),
Khumaltar, Lalitpur, Nepal
COLLABORATING AGENCY: Research Laboratory for Biotechnology and Biochemistry
(RLABB), Maitidevi, Kathmandu, Nepal
FINANCING AGENCY: Ministry of Environment, Science & Technology (MoEST),
Government of Nepal, Sighdurbar, Kathmandu
PROJECT GOAL: Development of microbial inoculants for biogas production and technology
transfer to biogas users in cold regions
PROJECT TEAM:
Prof. Dr. V. P. Agrawal (Director, RLABB)
Dr. Kayo D. Yami
Ms. Tista Prasai (Scientific Officer, NAST)
Ms. Trishna Manandhar (Research Associate, NAST)
Mr. Upendra Thapa Shrestha (Research Scientist, RLABB)
Mr. Gopal BK (Lab Assistant, NAST)
Mr. Dilip Maharjan (Lab Assistant, RLABB)

INTRODUCTION
Biogas is a term used to represent a mixture of different gases (varied composition) produced as a result of action of anaerobic microorganisms on domestic and agricultural wastes. Bio-gas usually contains about 60 to 70 percent methane, 30 to 40 percent carbon dioxide, and other gases, including ammonia, hydrogen sulfide, mercaptans and other noxious gases.
Anaerobic digestion is a complex process wherein, a variety of bacteria are involved. It involves three steps: hydrolysis, acetogenic and methanogenic bacteria. (i) hydrolysis, which converts organic polymers into monomers (with the help of hydrolytic bacteria); (ii) acid formation, which involves conversion of monomers into simple compounds such as CO2, NH3 and H2, using a group of acid forming bacteria (acetogenic bacteria) and (iii) methane formation involving conversion of simple compounds into methane CH4 and CO2 utilizing anaerobic bacteria (methanogenic archaebacteria).
Methanogenic bacteria belong to the species of genera Methanobacterium, Methanomicrobium, Methanogenium, Methanosarcina, Methanospirillum and Methanococcus. Along with on going research, newer genera of methanogenic archaea namely : Methanosphaera, amarthanothermus, Methanoplanus, Methanocorpusculum, Methanocelleus, Methanobolus, Methanohalobium, Methanococcoides, Methanohalophilus, Methanothrix and Methanopyrus have been described.

JUSTIFICATION OF THE STUDY

Knowledge of the fundamental processes involved in methane fermentation is necessary for planning, building and operating biogas plants. Anaerobic fermentation involves the activities of different bacterial communities. The process of biogas production depends on various parameters. For example, changes in ambient temperatures especially lowering of temperature can have a negative effect on bacterial activity. Furthermore, Biogas production in cold hilly areas is generally very slow due to inadequate temperature of biogas plant for the active conversion of biodegradable substrates into methane gas. Psychrophilic methanogenic bacteria pose potentials for biogas production in cold climates. The project focuses on isolation and development of inoculums of cold tolerant biogas producing bacteria so that biogas users of high altitude/ cold regions as well as biogas users of non-high altitudes in cold season can enjoy this rural technology.

OBJECTIVE
Development of cold tolerant Microbial Inoculant for expediting biodegradation of substrates used in biogas plants of cold regions

METHODOLGY

-Selection of cold tolerant Methanogenic bacteria capable of producing biogas growing them at 150C, 100c and 50C

-Biogas Slurry Sample collection

-Isolation and Identification of methonaogenic microorganisms (basal Medium (LPBM))
Anaerobic culture of Methanogenic bacteria at 150C, 100c and 50C


PROGRESS ACTIVITIES (Poush, 2065-Chaitra 2065)

Ø Field Survey on biogas users of Chapagaon (Total of 50 questionnaires)
Ø Biogas slurry Sample collection from Chapagaon, Lalitpur
Ø Preparation of Anaerobic media for methanogenic bacteria (Modified Hungate's media/ basal Medium /LPBM) used for selective enrichment and growth of methanogenic bacteria )
Ø Isolation and Identification of methanogenic bacteria


Based on the presumptive identification of anaerobic bacteria (Bergey's Manual of Systematic Bacteriology, Vol. 3) isolated from the LPBM Media were suspected to be Methanobacterium spp., Methanobrevibacter spp., Methanogenium spp., Methanosarcina spp. and Methanomicrobium spp.

RESULTS obtained at RLABB (ISOLATION AND IDENTIFICATION OF COLD TOLERANT METHANOGENS FROM YAK DUNG OF DARCHULA)

REFERENCES

1. Boone, D. R. and Mah, R. A (1986). Methanogenic Archaeobacteria. In Bergey's Manual of Systematic Bacteriology, Vol. 3; section 5: pp. 2173-2216
2. Bryant, M. P. (1972) Commentary on the Hungate technique for culture of anaerobic bacteria. Am. J. Clin. Nutr. 25:1324-1328.
3. Daniels, L., and Zeikus, J. G (1975). Improved culture flask for obligate anaerobes. Appl. Microbiol. 29:710-711.
4. Roberts, D.J (2002) Methods for Assessing Anaerobic Biodegradation Potential: In Manual of Environmental Microbiology 2nd Edition ( Eds. Hurst, C.J, Crawfold, R. L, McInernet, M. J, Kndsen, G. R, Stetzenbach, L. D) American Society of Microbiology Press, Washington D. C, PP 1008-1017
5. http://www.bsp.org.np/
6. Zeikus, J. G., and Bowen, V. G. (1975) Comparative ultrastructure of methanogenic bacteria. Can. J. Microbiol. 21:121-129.


Glimpses on Biogas Project Activities 2065/66

Biogas plants in Chapagaon VDC

Stove operated by Biogas

Microbiological Analysis of Biogas slurry

RANDOM AMPLIFIED POLYMORPHIC DNA-POLYMERASE CHAIN REACTION ANALYSIS OF BACILLUS THURINGIENSIS ISOLATES FROM THE KHUMBU REGION OF EVEREST BASE CAMP

(Published in Journal of Institute of Science and Technology, Vol. 15, 2007-08, pp. 90-102.)

Random Amplified Polymorphic DNA-polymerase chain reaction analysis of Bacillus thuringiensis isolates from the Khumbu region of Everest Base Camp

G. S. Sahukhall, U. T. Shrestha1, B. Lekhak2, A. Singh2, V. P. Agrawal1

1. Research Laboratory for Biotechnology and Biochemistry (RLABB);
Maitidevi Plaza, Maitidevi, Kathmandu, Nepal
2. Central Department of Microbiology, Tribhuvan University;
Kirtipur, Kathmandu, Nepal.

ABSTRACT
Genetic similarity and diversity among the Bacillus thuringiensis isolates were examined using Random Amplified Polymorphic DNA-polymerase chain reaction (RAPD-PCR). Relationship between the species was determined by comparing their unique RAPD fingerprint information. A total of 86 endotoxin producing B. thuringiensis strains isolated from soil samples of Khumbu region were tested against a series of 100 decamer RAPD primers (codes 201-300). Nine primers: 208, 254, 256, 268, 275, 276, 284, 292 and 299 with GC%: 50-80, were found to amplify genomic DNA fragments with reproducible polymorphisms. Primer 284 with GC% 70 that produced more polymorphic bands was used to obtain RAPD profiles. The reaction mixture was optimized in 25ul system with PCR buffer containing 10mM Tris HCl, 50 mM KCl, 3 mM MgCl2 with pH 8.3.; 200μm dNTPs each, 1U Taq polymerase, 40 pmol decamer primers, 20 ng template DNA and 1% DMSO. The thermal programme was set as: initial denaturation temperature at 94oC for 5 mins followed by 35 cycles with denaturation at 94oC for 1 min, annealing at 36oC for 1 min and extension at 72oC for 2 mins with final extension temperature at 72oC for 10 mins. Higher polymorphic fragments were produced in the range between 700-900 base pairs. The range of 400-700 and 1200-1600 bp were also highly polymorphic. The Discriminatory capacity (D) of the RAPD-PCR was 0.9901 and hence the cold tolerant B. thuringiensis isolates from high altitude regions were very rich in genomic polymorphism.

Antimicrobial Efficacies of Nepalese Toothpastes against Dental Pathogens and Prevalence of Dental caries

(Abstract published in the Fifth National Conference on Science and Technology, Nov.10-12, 2008, Kathmandu pp. 353-354)

Antimicrobial Efficacies of Nepalese Toothpastes against Dental Pathogens and Prevalence of Dental caries

A. Acharya1, B. Subedi1, B.Paudyal1, M. Jnawali1, P. Shakya1, U. KC 1,
U. T. Shrestha2, K.B. Tiwari1, 2, A. Singh1 and V. P. Agrawal2
1Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu
2Research Laboratory for Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu
E-mail: md.jnawali@gmail.com

ABSTRACT
Dental caries is a multifactorial oral disease developed by the localized dissolution of the tooth hard tissues, caused by acids produced by bacteria in the biofilms (dental plaque) which eventually leads to cavities. Frequently encountered bacteria in caries are Streptococci (S. mutans, S. salivarious, S. sanguis, S. sobrinus and S. mitis) and others including some species of Actinomyces, Lactobacillus, Candida albicans and some anaerobes together with Bacteroids, Fusobacterium, and Prevotella etc. Maintainace of oral health is highly interrelated with use of toothpaste. Toothpaste need to contain various antimicrobial agents in order to reduce, control and prevent different kind of dental caries. Different brands have their own composition and concentration of ingredients for their efficacy. The consumers should aware about the facts associated with their health. Present study was carried out to determine the antimicrobial status of locally used toothpastes against common dental pathogens and to know dental caries condition of school children of Kathmandu valley. The dental pathogens were isolated and identified from various dental samples. Antibacterial activities of 25 different toothpastes available locally in markets were assessed against the isolates by standard agar well diffusion method. Altogether 638 students (325 of age group 12-13 years and 313 of age group 5-6 years) from 30 different schools of Kathmandu valley were included in the study and their dental status was examined and recorded by using WHO caries recording form with DMFT and DMFS as standard tools. Both monomicrobial and polymicrobial infections were observed in all cases. The dental pathogens isolates and identified were Streptococcus mutans, S. salivarious, S. sanguis, S. sobrinus, S. mitis, Lactobacillus species and Candida albicans. Out of 25 assayed toothpastes only 7 were found to be highly effective against all the tested pathogens. DMFS and caries percentage of age group 5-6 years and 12-13 years were found to be 3.79, 69% and 1.60, 53% respectively. But no significance difference at p less than 0.001 was observed among male and female of both age group. Strong correlation was found in dental caries status and type of schools, diet and toothpaste using habit. The result showed that the toothpastes containing Triclosan followed by fluoride as a major chemical ingredient possess significant antibacterial activities. In both age group caries percentage was found to be above the recommended level of WHO where as DMFS and DMST varies within the level.