Ashok Pandey, Ph.D.
Scientist F & Head, Biotechnology Division, National Institute for Interdisciplinary Science and Technology-CSIR), Thiruvananthapuram, India
Alternative renewable resources: Issues and perspectives for India – the case of transport fuels
With the increase in the urbanization way of life and also more and more dependence on materialistic life, there is substantial growing demand for the energy. The science and technological policy of the India has looked several avenues to fulfill this demand through alternative resources such as solar energy, wind energy, tidal energy, bioenergy, etc. The demand for the transport sector is largely met through the import (~70%). Biofuels, in particular bioethanol from lignocellulosic biomass offer attractive possibilities in this regard.
The sugar platform which generates ethanol is considered to be the most valuable solution to the transport fuel demand. Bioethanol can be generated from grains as well as from lignocellulosic plant material by their saccharification to sugars and subsequent fermentation of the sugars to produce ethanol. Bio-ethanol as a transportation fuel is attractive since it is more energy efficient than gasoline and produces less emissions. The benefits of developing biomass to ethanol technology(s) include: increased national energy security, reduction in GHG emissions, use of renewable resources, economic benefits and creation of employment and the foundation of a carbohydrate based chemical industry. However, the utilization of lignocellulosic biomass for fuel generation has not been given the sort of attention it ought to receive. It is known that the technology for ethanol production from biomass has to evolve greatly for an economical commercial scale utilization of the renewable biomass resources. Biomass requires extensive processing involving multiple steps for hydrolysis and fermentation of the raw material for producing ethanol. Feed stock availability, pretreatment, saccharification, fermentation and ethanol recovery are all factors which influence the production of ethanol and which needs R&D efforts for overall improvement of the production economics.
Bioconversion of lignocellulosic biomass (LB) can contribute significantly to the production of organic chemicals also. LB is also considered to be the only foreseeable source of energy. LB is mainly composed of (dry wt basis): cellulose, 40-60; hemicellulose, 20-40; and lignin, 10-25%. Most efficient method of biomass hydrolysis is through enzymatic saccharification5 using cellulases and hemicellulases. Fungal cellulases (FCs) have proved to be a better candidate than other microbial cellulases, with their secreted free cellulase complexes comprising all three components of cellulase [endoglucanases, exoglucanases and cellobiases (glucosidases).
The Centre for Biofuels at NIIST, Trivandrum, India aims ultimately to develop technologies and processes which will address the nation’s need for making fuel ethanol from the renewable resource: biomass. It is proposed to direct R&D activities at the major requirements of a biomass-ethanol technology, which include production of cellulases, hydrolysis of biomass, and ethanol fermentation. Viable technologies for each of these processes will contribute to the overall process development for fuel alcohol production from cheap and renewable biomass resources.
The lecture would present perspectives on bioethanol from lignocellulosic feedstocks.
References
- Biofuels- Alternative Feedstocks and Conversion Processes, Editors- Ashok Pandey, C Larroche, SC Ricke, CG Dussap & E Gnansounou, Academic Press, Elsevier Inc; San Diego, USA, p629 (2011) ISBN: 978-0-12-385099-7
- Handbook of Plant-Based Biofuels, Editor- Ashok Pandey, CRC Press, Francis & Taylors, Boca Raton, USA, p 297 (2008) ISBN 978-q-5602-2175-3
- Biofuels II, Special issue of Journal of Scientific & Industrial Research, Guest Editors- E Gnansounou, C Larroche and Ashok Pandey, 67(11), 837-1040 (2008) ISSN: 0022-4456
- Biofuels, Special issue of Journal of Scientific & Industrial Research, Guest Editors- C Larroche and Ashok Pandey, 64(11), 797-988 (2005) ISSN: 0022-4456
Abhijeet Kate, Arpana G Panicker, Diana Writer, Giridharan P, Keshav K V Ramamoorthy, Saji George, Shailendra K Sonawane
Protoplast fusion and transformation: A tool for activation of latent gene clusters
In the quest to discover new bioactive leads for unmet medical needs, actinomycetes present a treasure trove of undiscovered molecules. The ability of actinomycetes to produce antibiotics and other bioactive secondary metabolites has been underestimated due to sparse studies of cryptic gene clusters. These gene clusters can be tapped to explore scaffolds hidden in them. The up-regulation of the dormant genes is one of the most important areas of interest in the bioactive compounds discovery from microbial resources. Genome shuffling is a powerful tool for the activation of such gene clusters. Lei Yu, et al.1, reported enhancement of the lactic acid production in Lactobacillus rhamnosus through genome shuffling brought about by protoplast fusion. D. A. Hopwood et al.2 suggested that an interspecific recombination between strains producing different secondary metabolites, generate producers of ‘hybrid’ antibiotics. They also mentioned that an intraspecific fusion of actinomycetes protoplast bring about random and high frequency recombination. Protoplasts can also be used as recipients for isolated DNA, again in the presence of polyethylene glycol (PEG). In our study we had undertaken random genome shuffling by protoplast fusion of two, rather poorly expressed actinomycetes strains A (Figure 1) & B (Figure 2), mediated by PEG; and also by naked DNA transformation of Strain A protoplast with the DNA of Strain B. We generated eight protoplast fusants and seven transformants from parents considering their morphological difference from the two parent strains. These 15 recombinants were checked for their same colony morphologies for five generations to ensure phenotypic stability. Antibiotic resistance pattern was established by using antibiotic octodisc to generate a marker profile of the recombinants and the parent strains. Eight fusants (AP-18, AP-25, AP-2, AP-11, AP-14, AP-19, AP-11 and AP-27) and four transformants (TAP-30, TAP-31, TAP-32 and TAP-33) (Table 1) have shown a different antibiotic sensitivity pattern as compared to the parent strains. We envisage that these recombinants harbor shuffled gene clusters. To support array of conditions to express such shuffled/cryptic genes the recombinants were fermented in 11 different nutrient stress variants. The extracts generated were subjected to metabolite profiling by HPLC-ELSD, bioactivity screening for cytotoxicity and anti-infective capabilities. Two fusants AP-11 (Figure 3) and AP-25; one transformant TAP-32 (in growth media MBA-5 and MBA-7) displayed antifungal activity unlike parent strains (Table 2) Fusant AP-11 (Table 5) exhibited significant cell growth inhibition of five different cancer cell lines. The parents Strain A and Strain B did not exhibit any cell growth inhibition of these cell lines (Table 5). The metabolite profiling of fusant AP-11 and transformant TAP-32 was done by HPLC-ELSD. AP-11 showed the presence of five additional peaks (Figure 5 & Figure 6); TAP-32 extract from medium MBA-5 (Figure 7 & Figure 8) showed the presence of four additional peaks and TAP-32 extract from MBA-7 (Figure 9 & Figure 10) showed 14 additional peaks as compared to parent strains in similar medium and media controls. The study indicated that protoplast fusion and transformation have not only caused morphological changes but also shuffled genes responsible for synthesis of bioactive molecules. Further characterization of these new peaks is warranted.
Nader Pourmand, Ph.D.
Director, UCSC Genome Technology Center,University of California, Santa Cruz
Biosensor and Single Cell Manipulation using Nanopipettes
Approaching sub-cellular biological problems from an engineering perspective begs for the incorporation of electronic readouts. With their high sensitivity and low invasiveness, nanotechnology-based tools hold great promise for biochemical sensing and single-cell manipulation. During my talk I will discuss the incorporation of electrical measurements into nanopipette technology and present results showing the rapid and reversible response of these subcellular sensors to different analytes such as antigens, ions and carbohydrates. In addition, I will present the development of a single-cell manipulation platform that uses a nanopipette in a scanning ion-conductive microscopy technique. We use this newly developed technology to position the nanopipette with nanoscale precision, and to inject and/or aspirate a minute amount of material to and from individual cells or organelle without comprising cell viability. Furthermore, if time permits, I will show our strategy for a new, single-cell DNA/ RNA sequencing technology that will potentially use nanopipette technology to analyze the minute amount of aspirated cellular material.
Sunilkumar Sukumaran, Ayyappan Nair, Madhuri Subbiah, Gunja Gupta, Lakshmi Rajakrishna, Pradeep Savanoor Raghavendra, Subbulakshmi Karthikeyan, Salini Krishnan Unni and Ganesh Sambasivam
Genotoxicity is defined as DNA damage that leads to gene mutations which can become tumorigenic. Genotoxicity testing is important to ensure drug safety and is mandatory prior to Phase I/II clinical trials of new drugs. The results from genetic toxicology studies help to identify hazardous drugs and environmental genotoxins. Currently, among others there are four tests recommended by regulatory authorities (Ames test-bacterial, chromosome aberrations; in vitro gene mutation-eukaryotic cells and in vivo test). These assays are laborious, time consuming, require large quantities of test compounds and limited by throughput challenges. The site and mechanism of genotoxicity are not revealed by these assays and data obtained from bacterial tests might not translate the same in mammals. To address these we have developed a novel, versatile, human cell based, high throughput, reporter based genotoxicity screen (Anthem’s Genotox screen). This screen is performed on genetically engineered human cell lines that express 3 reporter genes under transcriptional control of ‘early DNA damage sensors’ (p53, p21 and GADD153). These genes are involved in DNA repair, cell cycle arrest and/or apoptosis. p21 and GADD are also known to be induced in a p53 independent manner. p53 blocks G1/S transition of cell cycle while the p53 independent DNA damage block G2/M transition. Identification of the mechanism of genotoxicity helps in rational drug designing. Additionally, the platform can be used to screen other potential genotoxins from cosmetics, food and environment. Initial validation studies of the Genotox screen was performed with over 60 compounds chosen from a variety of chemical classes. The genotoxic potential of metabolites was tested using rat liver S9 fractions. The results demonstrated a sensitivity of 86.7–92.3% and a specificity of 70–78.6% when compared with currently available in vitro genotoxicity assays. This Genotox screen would prove to be an invaluable human cell based tool to weed out potential genotoxins in various industries.