DEPARTMENT OF BIOTECHNOLOGY, Govt of India Initiatives taken by Department of Biotechnology in the Biofuel and Bioenergy area

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1 DEPARTMENT OF BIOTECHNOLOGY, Govt of India Initiatives taken by Department of Biotechnology in the Biofuel and Bioenergy area Department of Biotechnology has supported an Energy Bioscience Programme with a broad vision to make available, economically viable alternative fuel from biomass to meet the energy deficit and national targets of biofuel blending. The programme concentrates on utilizing biomass as a source of bioenergy and feedstock for biofuel. The broad objectives of the Programme are R&D for technology development and production of: 2nd Generation biofuels- ethanol from cellulosic biomass including agricultural & forestry waste; (ii) Algae as a feedstock for 2 nd generation biofuel; (iii) Biodiesel from varied feed stocks like Jatropha; (iv) 3rd generation biofuels such as Biohydrogen, Biobutanol etc. The various Programmes that have been undertaken by the Department to achieve these objectives are as follows: I. Biodiesel Quality planting material (ii) Improved feed stock (iii) Process optimization II. Bioethanol Re-engineered feed stock (ii) Re-engineered microorganisms (iii) Process optimization III. Algal Biofuel Collection and characterization (ii) Establishment of repositories (iii) Development of production system IV. Energy Bioscience Centre V. Capacity building I. BIODIESEL 1) DBT Micro-mission on Jatropha Improvement for Biodiesel To meet the requirement of the National Mission on Biodiesel, in the year 2004 the Department of Biotechnology initiated a micro-mission programme on production and demonstration of superior quality planting material of Jatropha. For the first time the country s germplasm has been screened, collected and characterized. Superior material based on oil content (30-35%) and yield (3-5 tons/ha)

2 ha been collected from across the country involving 14 centres covering 12 states. High yielding genotypes were identified for mass multiplication and demonstration. Department also supported a programme for a centralized oil testing and characterization of the accessions collected. Nearly 1500 accessions have been collected and chemically analysed for oil quantity and quality. These were characterized using molecular tools and deposited at the National Bureau of Plant Genetic Resources, New Delhi for conservation. The selections made on the basis of high oil content, healthy and robust growth were accessioned in the National Register with the NBPGR, New Delhi. This is the largest collection of accessioned material which has been characterized chemically and at molecular level. Collections made by institutes 1236 Collections accessioned 1118 Accessions used for trials 253 Institutions involved Area under plantation Number of plants in trials ha Multi location trials to evaluate site specific germplasm In 2009, Multilocation trial has been started at 14 site with 100 best selected accessions. 100 accessions screened from different locations based on their field performance at experimental stations have been bulked up and planted at 14 different sites. Performance, Agronomic and Silvicultural trials have been initiated. Nearly 400 selected accessions were bulked up to raise 17 lakh rooted clonal cuttings for sharing with beneficiaries from State Governments. These clonal saplings are being used to develop Jatropha Germplasm Resource and Conservation centers in different parts of the country. This quality planting material is being distributed to different States/locations to set up Superior Mother Nurseries and this stock can be made available for further multiplication and distribution for large scale plantation. 2) R&D for Jatropha Improvement Research on Improvement of Jatropha germplasm has been supported focusing on metabolic engineering of the oil biosynthesis pathway for improved quality and content, identifying candidate gene markers, prospecting for genes; developing potential hybrids for higher yield and oil content etc. The major achievements in this area are: Five elite selections of high yielding Jatropha curcas plants have been multiplied through tissue culture at LabLand, Mysore. Nearly 1500 plants have been produced and evaluated for genetic fidelity. The RAPD analysis revealed that there are no genetic variations among tissue culture regenerants and their respective mother plants. (ii) Studies have been supported to generate EST s for identification of genes for oil biosynthesis pathway. So far 6000 EST s have been generated and nearly 3000 EST s have been sequenced and the genes of interest are being identified.

3 A total of 130 microsatellite markers have been developed through enrichment of genomic libraries and sequencing. (iii) Candidate genes markers for selection of high oil content genotypes of Jatropha have been identified. (iv) Public Private Partnership Programmes have been supported with Industry. Two genes (GPAT & LPAT) identified for oil enhancement have been cloned into plant expression vectors of Jatropha with the dual gene constructs. Transformation system has been developed and selected Jatropha genotypes transferred with desired genes. Transformed plants have been confirmed by PCR. II. Bioethanol from Lignocellulosic biomass-process Development and Production For meeting the increasing demands of bioethanol a commercially viable production technology using Lignocellulosic biomass as a feed stock (Agriculture and forestry waste), is being developed with support from DBT. Some of the achievements of the programme are as follows: A Labscale process has been developed for ethanol production using lignocellulosic feed stock as a raw material (Prosopis juliflora (a weed), Lantana camara (a shrub), rice straw, corn cob). (ii) Pretreatment process has been developed and is being up-scaled at 5 kg level. Saccharification and fermentation process has been established and is now being scaled up to 10 Ltr capacity. (iii) Strategy to carry out fermentation of both pentose and hexose has been investigated with the help of Co-Culture experiments where fermenting yeast was inoculated simultaneously rather than strategy where hexose fermenting yeast was inoculated first and pentose fermenting yeast was inoculated after 48 h. (iv) Strategy for recycling the enzyme using ultrafiltration has been developed. (v) Two Thermotolerant yeast strains have been developed. (vi) Recombinant yeast strain for converting starch to ethanol tested at pilot scale. (vii)recombinant bacteria has been developed for enhanced cellulose production. III. DBT-ICT Centre for Energy Biosciences To give the area of Energy Bioscience a special impetus, it was felt essential to have a dedicated Centre which addresses all issues of Biomass based Biofuels production. Being a multidisciplinary field it was essential to bring the biochemists, molecular biologists, protein chemists and downstream processing groups together under a common platform. Thus emerged the country s First Energy Bioscience Centre at ICT, Mumbai. The DBT-ICT Centre for Energy Biosciences (DBT-ICTCEB) is the first Energy Biosciences Centre in India where integrated basic and translational science capabilities, meet and amalgamate with each other to add a new dimension to Bioprocess Technology and Biological Engineering.

4 The primary focus is on developing cutting edge technologies, and processes and products from renewable resources. The overall objective is: Improved bio-ethanol production from any given biomass, (ii) Developing a bio-refinery approach for selected biomass (iii) Developing other 2 nd and 3rd generation biofuel technologies such as algal biofuel, bio-hydrogen, biobutanol and bio-methane. Pilot Scale Technology for Cellulosic Ethanol The Centre has developed an industry-ready technology for producing lignocellulosic ethanol. The technology employs engineering a combination of pretreatment methods and novel use of depolymerases for cost-effective breakdown of celluloses and hemicelluloses to respective sugars and their conversion to alcohol. The yield of bioethanol per ton of dry biomass is ~300L and lignin is produced as dry solid up to kg/ton biomass depending on biomass variety. The technology developed is being validated at pilot scale with an industrial partner Indian Glycols Ltd. An MoU with India Glycols Ltd. has been signed for setting up a 10T/day pilot plant at their premises in Uttarakhand. The technology developed has shown promise to bring down both capital and operating cost of production of bioethanol to half of that of the traditional alkali or acid based technology. Besides, extensive work has been carried out and the initial shape of a new technology that gives near pure fractions of cellulose, hemicelluloses and lignin has been developed. Efforts in synthetic biology have been undertaken, with help from collaborating partners, to design enzymes like lipases and cellulases that will be more robust and safe against product/substrate inhibitors. IV. Algal Biofuels : While a number of feedstocks are currently being experimented for biofuel production, Algae is one of the most promising sources, for the following reasons: The yield is in orders of magnitude, higher than those for traditional oilseeds (ii) Algae does not compete with food crops for land availability (iii) Algae can grow in places away from forests, thus minimizing the damages caused to the eco- and food chain systems (iv) Our advantages are : Enormous Diversity; Vast Coastline; Sufficient Solar Energy; Scientific Strength A National Algal Biofuel Network has been launched in with participation of 12 national laboratories/institutions/universities and industry focusing on:

5 Collection and characterisation of algal strains from different ecological niches and deposition of the same in 3 repositories. (ii) Development of different production systems (iii) Improved Algal Strains for more oil/lipid content. Leads Available 3 National Repositories have been set up for depositing Algal Collections:Marine: Bhartidasan University Brackish water: Orissa Fresh Water: IBSD, Imphal (ii) Nearly 400 unialgal microalgal isolates of freshwater cyanobacteria and other algae have been collected and characterized by different centers in North-East. More than 400 marine cyanobacterial samples have been collected and out of this 50 samples have been screened for lipid content and others are under process of screening and characterization at Bhartidasan University, Tamil Nadu. About 150 algal species have been collected from brakish waters, of which 14 species have been submitted to the repository at IMMT. Preliminary screening of lipid content for six algal species have been carried out and further screening of species is continuing. (iii) A common SOP s (Standard Operational Practices) for algal screening, characterization as well as for maintenance and documentation of algal strains has been prepared on a global pattern with the help of researchers among the Network as well as other well known scientists and researchers in India involved in Algal related research. The SOP will help in proper documentation of strains deposited at various repositories (iv) The parameters such as salinity, ph, light intensity, light wavelength on the growth and lipid profile of marine algae, eg. Dunaliella or Chaetocerosor Chlorella etc. have been studied and standardized. (v) A photobioreactor of 5-10 L capacity, which can also act as seed development photobioreactor for the pilot plant has been designed and fabricated. V. New Initiatives There is a need to look for future generation biofuels beyond ethanol using bio-based biorefinery & synthetic biology approach. The future focus will be on 3 rd Generation and 4th Generation biofuels biobutanol, bio-acetone, bio-hydrogen, biomethyl furan (BIODMF), Biodimethyl ether (BIODME), Green diesel etc. Studies have been initiated to develop biohydrogen and biobutanol as a potential biofuel with a focus on production, process optimization, purification, scale up and evaluation. 1. Biohydrogen: Recently Department has started a project on Dark- Fermentative Bio-hydrogen production from renewable sources with following objectives :

6 Selection of suitable cost effective substrates for dark fermentative hydrogen production. Standardization of process parameter( including scale up and optimization studies) to be carried out in 1 Ltr & scaled to 10 Ltr bioreactor and then upscaled to 30 Ltr & subsequently to 1000 Ltr bioreactor capacity. Selection of suitable substrate for biohydrogen production, enhancement of BH2 production by optimization of growth and fermentation conditions and take scale up studies in various sizes of bioreactors for BH2 production. 2. Biobutanol Since biobutanol has been termed as future fuel by researchers as well as different agencies of the world therefore, the Department has also initiated project on biobutanol which deals with production, process optimization, scale up and evaluation.. Cost effective technology for Butanol fermentation to provide butanol at fuel cost is being envisaged. Some major DBT R&D initiatives on Biofuels are listed project wise at Annexure II VI. Capacity building for generating skilled human resource in Bioenergy For any research program to be implemented with high precision, competence of the human resource available is a key to the success of the programme. The Department has initiated two schemes for the Energy Bioscience Area: National Energy Bioscience Chair to have excellent team leaders in the Bioenergy area who can help in building innovative teams to address the major challenges in this sector. The main objective of the scheme is to utilize the expertise of senior scientists of international repute for providing leadership to the bioenergy Programme being implemented at different places across the country. This will help in the process of capacity building for strengthening the R&D base in the energy bioscience area in the country. It is expected to have at least 5 chairs at any one given time. The duration of the Chair is for a period of five years. During the year, two candidates have been shortlisted for the Chair. (ii) Energy Bioscience Overseas Fellowships for scientists of Indian origin who are working outside the country in the field of Energy Biosciences The main objective of the scheme is to support scientists of Indian origin who wish to return to the home country and pursue research of high caliber in the area of Energy Biosciences.