Captain Green Reducing the global warming gases and increasing fertility of paddy fields The Team: E. Varma, S.Tejpal, S. Jha, P. Rana, P. Sharma, N. Aggarwal, V. Manohar, P. Sharma, M. Sharda Professors: Dr. Antresh Kumar Dr. Saurabh Bansal Dr. R.S Chauhan
J U I T India
Global Green House Gases Em ission Others, 1% CH4, 9% N 2O, 20% CO2 N2O CO2, 70% CH4 Others IPCC Fourth Assessment Report, 2008
Energy Supply- Main contributor Effects (concentration – 1 to 8 %) Drowsiness, Increased blood pressure, Dizziness, shortness of breath, panic attacks, headaches. Adopted from U.S. Environmental Protection Agency
CO2 emissions from industrial processes CO2 emissions from land use change Source: www.oup.co.uk/vsi
Agricultural Soil - Main Contributor of N 2 O Man-made Nitrous oxide Natural Nitrous oxide sources sources ~ 10 million tonnes each year ~ 8 million tonnes each year. Effects (>100ppm) Asphyxiation, Kidney damage, headaches Adopted from GHG online
Rice Cultivation- Main contributor of CH4 emission Rice cultivation 14% 29% Biomass burning Landfills 18% Sewage treatment 8% Animal waste 11% 6% Termites 3% 4% 3% 4% CH4 hydrates and ocean Wetlands Coal & miming/ natural gas Enteric fermentation Effects Dizziness, headaches, nausea, drowsiness (Dangerous at 5-15%concentration in air) and unconsciousness Adopted from National Aeronautics & Space Administration
2010 estimates FAO estimates 2010 FAO Estimates 2010
International Grains Council estimates, 2012 FAO Estimates 2010
Rice production in India increased from 44.69 million tons (2001) to 95.32 million tons (2011). Sharad Pawar, Agriculture Minister, 2011
• Rice is a high energy calorie food. • Major part of rice – carbohydrate (starch) - 72-75%. • Protein (glutelin) content of rice is 7% • Nutritive value of rice protein (biological value = 80) • Rice contains most of the minerals ( 4% phosphorus). • Rice also contains some enzymes. • India has become one of the top five countries with rice exports (2011). • Employment of a large number of people. • Products of the rice plant are used for a number of different purposes, such as fuel, thatching, industrial starch, and artwork.
80% 70% 60% H % GHG emission Contribution to global warming 50% 40% Contribution by paddy fields 30% 20% 10% 0% Methane Nitrous oxide Carbon dioxide U.S. Environmental Protection Agency Adopted from GHG online &
Bacillus Sp. Paddy and the microbes Methanogens Thermo Cyanobacteria sp. Tolerancy Methylococcus capsulatus Facultative Anaerobe . p s m u i b o z i h R Denitrifying gene present MMO encoding gene. Methanotrophic bacteria – R.S. Hanson and T.E. Hanson, 1996,60(2)
Captain Green: To Do List – Convert CH 4 into CO 2 at a faster rate. Convert N 2 O coming from the nitrogen fertilizers into NO 3 . Constitutively show it ’ s skills to be really called a captain and show no competition with the natural flora.
O U R NifA MxaF C O N S T R U NosZ SacB C T
MxaF Present in Methanotrophs • Located upstream of genes of • Methanol Dehydrogenase Methanol Inducible Promoter • Create a diversion of flux • Leading to faster degradation of • methane. CH3OH MxaF
N o s Z Present in Denitrifying • Bacteria Responsible for • conversion of nitrous oxide into free nitrogen. N 2 O N 2
Transcriptional NifA • Activator for Nif genes Nif genes are a cluster • of genes responsible for conversion of free nitrogen into nitrate i.e. nitrogen fixation.
• Used as a Negative selectable S a c B marker • Codes for levansucrase • Levansucrase is a transfructosylase catalyzing sucrose hydrolysis and levan synthesis • The gram negative microbe cannot survive in the presence of sucrose
MxaF nosZ nifA (Promoter) N 2 O N 2 NO3 CH 4 inducer NH 2 OH CH 3 OH Methylococcus Capsulatus OUR APPROACH
T 1. Design of primers & Optimization of PCR H reactions E SpeI MxaF AceI NosZ EX AceI NdeI P E AscI R SacB NdeI NifA I SpeI M AscI E
THE EXPERIMENT Nos Z NifA Y Y Z X Ligation X Y Z Ligation of Gene Complete Fragment MxaF NosZ NifA SacB Genomic DNA
T H The Detection Systems E Gas Chromatography for detection of methane and EX nitrous oxide. P Flame Ionization Detector - methane Thermal Conductivity Detector - nitrous oxide. E R I M E
References • Genomic Insights into Methanotrophy: The Complete Genome Sequence of Methylococcus capsulatus (Bath) - Naomi Ward et.al • Over-production and Characterization of the nifA Gene Product of Klebsiella pneumoniae - the Transcriptional Activator of nif Gene Expression By R. T U L I t AND M. J . MERRICK • Global Methane Emissions From Wetlands, Rice Paddies, and Lakes* - EOS, vol 90 no. 5, 3 feb 2009 • Methanotrophic bacteria - R S Hanson and T E Hanson- Microbiol. Mol. Biol. Rev. 1996, 60(2):439. Methane and Nitrous Oxide Emissions from a Rice Field in Relation to Soil Redox and • Microbiological Processes - A. X. Hou, G. X. Chen, Z. P. Wang, O. Van Cleemput, and W. H. Patrick, Jr.*
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