ALGAL STRAIN SELECTION AND CHARACTERISATION: A KEY TO SUCCESSFUL ALGAE FEEDSTOCK PRODUCTION

Transcription

1 ALGAL STRAIN SELECTION AND CHARACTERISATION: A KEY TO SUCCESSFUL ALGAE FEEDSTOCK PRODUCTION DR. SUNIL PABBI Principal Scientist CCUBGA, IARI, PUSA, New Delhi

2 ALGAE A large and widespread group of microorganisms found in wide range of habitats including aquatic, terrestrial and extreme environments Show ability to perform oxygenic photosynthesis Morphologically diverse with unicellular, filamentous and colonial forms Contain different combination of photosynthetic pigments like chlorophyll, carotenoids and phycobiliproteins Depict variability in metabolite content and/or N-assimilatory enzymes Exhibit widespread compatability and adaptability to extremes of temperature, dessication, illumination, radiations, salinity, ph, toxicants and nutrient availability Play a significant role in Environmental management as soil conditioners, biofertilizers, ameliorants of degraded wastelands and polluted water bodies, and scavengers of heavy metals. Bioindustry - as a source of natural pigments, nutritional supplements, pharmaceuticals / drugs and BIOFUELS.

3 FLEXIBILITY OF MICROALGAE FOR BIOFUEL PRODUCTION The process of photosynthesis is central to all light driven biofuel production system in microalgae. Biomass produced can be utilized for the extraction of bio-ethanol through fermentation, biodiesel from lipids, bio-methane production and bio-hydrogen production through hydrogenase system.

4 Cont Rupprecht, 2006

5 Advantages of Microalgae Higher photosynthetic efficiency Faster growth and larger biomass Easy mass culture Easily controllable environmental conditions Round the year production Non polluting and environment friendly No net contribution to atmospheric CO 2 levels

6 Challenges for production of Biofuels from microalgae HARVESTING RECOVERY IMPROVED STRAINS

7 ALGAL CULTURE SELECTION Natural Selection Inherent variability (Genetic & Metabolic) Mutation Selection Recombination Culture stability Growth/nutrient requirement Culture condition

8 Isolation and Purification Collection of water samples Suspend a loopful of the algal growth in 5 ml sterilized distilled water, homogenize and 0.5 to 1 ml is surface plated on agar plates containing the suitable medium for growth. Isolated colonies observed through binocular microscope after incubation are picked up, examined for contamination and transferred to agar slants. Collection of soil samples Representative randomised surface soil samples are collected from 8-10 spots from 0.5 ha area after removing upper 1 cm soil crust. The samples are dried, powered and pooled, and about 100g is preserved in polythene bag for the isolation purpose after thorough mixing. Culture Media Variety of culture media for isolation and maintenance of algae are available. For studying the entire algal component, media with combined nitrogen are used. It is advisable to raise enrichment culture separately in media with and without nitrogen

9 Collection of samples from different Habitats

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11 Algal Crusts in Deserts Source: Chinese Science Bulletin, Vol.50, No.2, January, 2005.

12 Purification Purification can be achieved by streak or spread or pour plate techniques. These are dilution techniques of different types resulting in the physical separation of individual cyanobacterium from the mixture allowing them to form distinct colonies which can be picked up to make pure cultures Methods of purification Streak plate Spread plate Pour plate In these methods, the number of cells are reduced so as to form separate colonies so that isolation of pure culture becomes easy In a streak plate, from the loop containing cyanobacteria, varying numbers of blue green algae adhere to the surface of the medium and towards the end of the streak, the number gets reduced so much that the separate colonies are formed. In spread plate, the cyanobacteria in liquid medium are directly spread over the entire surface of the solid medium resulting in the separation. In pour plate, there is direct dilution of blue green algae while being suspended in the pour agar resulting in separation at the time of plating. The addition of solidifying substance to the liquid medium containg blue green algal cells, traps the individual cells in place. In agar medium,they produce a fixed colony of cells or filaments and grow as separate colonies

13 Streaking Petri Plates with medium Isolated colonies at the end of the streak are expected to be unialgal

14 Pour plate

15 With presently 2213 strains(representing 510 genera and1273 species) the SAG is among the three largest culture collections of algae in the world. Among the several repositories of the world CCUBGA, is a large national repository for fresh water BGA in India (housing more than 550 strains)

16 Among the several repositories of the world CCUBGA, I.A.R.I is a large National Repository for fresh water Cyanobacteria in India (housing more than 550 strains) Isolation, identification & strengthening of cyanobacterial germplasm is one of the major activities of our center. Cyanobacterial isolates are constantly being added, characterized for various attributes. A large number of heterocystous & non-heterocystous strains isolated from different parts of the country are regularly sub cultured and maintained in unialgal form. The centre also functions as service as well as repository unit in the country for fresh water blue green algae. Some of the common forms available in the culture collection are heterocystous and non heterocystous blue green algae.

17 Preservation techniques for microalgae The primary purpose of preservation is to maintain algal population in a viable state for longer periods. During preservation, the growth gets considerably slowed down, and preserved culture can be reactivated by providing suitable growth conditions. Different methods of preservation are Lyophilization Cryopreservation Immobilization

18 Preservation techniques Short term and Long term A. Agar Slants B. Immobilized Alginate Beads C. Cryopreservation D. Lyophilization

19 Characterization MORPHOLOGY PHYSIOLOGY MOLECULAR BIOLOGY

20 Phormidium Microcoleus Oscillatoria Lyngbya

21 Aulosira Scytonema Cylindrospermum Microchaete

22 Chemotaxonomic / biochemical parameters Chemotaxonomic markers have shown to be useful. However, problems of consistency and variations due to factors such as growth conditions have not been systematically investigated (Holten 1981).Biochemical diversity in enzymology and regulatory patterns in aromatic amino acid pathway could provide useful taxonomic markers. Lipid composition (Kenyon et al. 1972; Caudales and Wells, 1992) Polyamines (Hamana et al., 1983) Pigments and Phycobiliprotein Patterns (Schenk and Kuhfitting,1983 ; Hertzberg 1971;Aakermann et al., 1992) Nitrogen fixing potential (Rippka et al., 1979) Protein electrophoresis and isozyme patterns ( Stulp and Stam, 1980; 1982) Immunological studies (Wood and Townsend, 1990)

23 Cyanobacterial isolates have been examined with respect to pigments, nitrogenase activity and metabolites. Variability was observed in these attributes in different cyanobacterial isolates studied. Out of various isolates from J &K, Anabaena comprised six strains, Nostoc comprised two strains, Calothrix comprised three,tolypothrix two & Westiellopsis comprised only one strain. Highest chlorophyll in Anabaena & lowest in Calothrix. Nitrogenase activity highest as well as lowest in Anabaena strains. Soluble proteins & carbohydrates maximum in Tolypothrix strains & minimum in Anabaena strains. Cyanobacterial isolates & their characterization for physiological attributes Strains Chlorophyll ( g/ml) Nitrogenase Activity ( mole C 2 H 4 /mg chl/h) Anabaena An Anabaena An Anabaena An Anabaena An Anabaena An Anabaena An Nostoc Ns Nostoc Ns Calothrix Cl Calothrix Cl Calothrix Cl Tolypothrix To Tolypothrix To Westiellopsis Ws

24 1: AJ Microchaete tenera...[gi: ] DEFINITION Microchaete tenera partial 16S rrna gene. ACCESSION AJ AUTHORS Dhaulakhandi,.B., Pabbi,S., Singh,P.K. and Ahluwalia,K.B. TITLE Ribosomal RNA sequence Analysis of unclassified cyanobacteria REFERENCE 2 (bases 1 to 999) ORIGIN 1 tacggttacc ttgttacgac ttcaccccag tcaccagcac tgccttaggc atcctcctcc 61 tcgaaaggtt ggagtaatga cttcgggcgt tgccagcttc catggtgtga cgggcggtgt 121 gtacaaggcc cgggaacgaa ttcactgcag tatgctgacc tgcaattact agcgattccg 181 acttcacgca ggcgagttgc agcctgcgat ctgaactgag ctacggttta tgagatttgc 241 ttgctatcac tagcttgctg ccctttgtcc gtagcattgt agtacgtgtg tagcccaaga 301 cgtaaggggc atgctgactt gacgtcatcc ccgtgccagc agccgcggta atacggaggg 361 tgcgagcgtt gtccggattt attgggttta aagggtgcgt aggtggccta ataagtcagt 421 ggtgaaatac ggttgctcaa caatcgaggt gccattgata ctgtgaggct tgaaataatt 481 ggaggctgcc ggaatggatg gtgangcggt gaaatgcata gatatcatcc agaacaccga 541 ttgcgaaggc aggtggctac gattggtttg acactgaggc acgaaagcat ggggagcaaa 601 caggattaga taccctggta gtccatgctg taaacgatga ggactcgttg tttggctgca 661 aagctgagcg acttaaggaa accgttaagt cctccacctg gggagtacgc acgcaagtgt 721 gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agtatgtggt ttaattcgat 781 gcaacgcgaa gaaccttacc aagacttgac atgtcgcgaa cctctttgaa aggagagggt 841 gccttaggga gcgcgaacac aggtggtgca tggctgtcgt cagctcgtgt cgtgagatgt 901 tgggttaagt cccgcaacga gcgcaaccct cgtttttagt tgccagcatt aagttgggca 961 ctctagagag actgccggtg acaaaccgga ggaaggtgg //

25 PHYLOGENETIC TREES COMPRISING DIFFERENT MICROALGAE

26 Total lipids in different classes of algae Algal classes Total Lipids Average Range Bacillariophyceae Chlorophyceae Chrysophyceae Cyanophyceae Dinophyceae Haptophyceae Phaeophyceae Prasinophyceae Rhodophyceae Xanthophyceae *per cent dry weight basis Source: Narayan et al.

27 18:1 (n-9) Oleic acid O 2 18:3 (n-6) Υ- linolenic acid O 2 18:2 (n-6) Linoleic acid O 2 18:3 (n-3) Α-Linolenic acid 20:3 (n-6) Dichromo- Υ linolenic acid O 2 20:4 (n-6) Arachidonic acid 22:3 (n-3) Elcosatrienoic acid O 2 22:4 (n-3) Elcosatetraenoic acid 22:4 (n-6) Adrenic acid O 2 22:5 (n-3) Elcosapentaenoic acid O 2 22:5 (n-6) Docosapentaenoic acid 22:5 (n-3) Docosapentaenoic acid Biosynthesis of polyunsaturated fatty acids in microalgae 22:6 (n-3) Docosahexenoic acid

28 Major Abiotic Stresses For Higher Lipids Synthesis Drought Exclusively salt affected Salt affected and water eroded soils Exclusively acidic soils Acidic and water eroded soils Water logging (Permanent surface inundation) Water erosion

29 Species Stress % Lipid (dry weight) Cyclotella cryptica Nitrogen deficiency 18 Dunaliella salina Osmotic stress and Nitrogen deficiency Nitrogen deficiency Non environmental stress Nitzschia sp. Non environmental stress Phaeodactylum tricornutum Non environmental stress Botryococcus braunii Nitrogen deficiency Non environmental stress Chlamydomonas sp. Non environmental stress 23 Chlorella sp. Chlorella vulgaris Non environmental stress Non environmental stress Nitrogen deficiency Non environmental stress Nannochloris sp. Non environmental stress Nannochloropsis sp. Nannochloropsis salina ENHANCED LIPID PRODUCTION BY DIFFERENT MICROALGAE UNDER DIFFERENT STRESS Nitrogen deficiency Non environmental stress Nitrogen deficiency Non environmental stress Spirulina platensis Non environmental stress Tetraselmis suecia Isochrysis sp. Nitrogen deficiency Non environmental stress Nitrogen deficiency Non environmental stress

30 ENHANCEMENT & ALTERATION OF LIPID PROFILE UNDER STRESS CONDITIONS Carbon dioxide Nitrogen stress Phosphorus and Sulphur stress Other chemical factors Physical factors Immobilization

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32 OIL CONTENT OF SOME MICROALGAE Micoralgae Oil content(%dry wt) Botryococcus brauni Chlorella sp Crypthecodinium cohni 20 Cylindrotheca sp Dunaliella primolecta 23 Isochrysis sp Monnallanthus salina >20 Nannochloris sp Nannochloropsis sp Neochloris oleoabundans Nitzschia sp Phaeodactylum tricornutum Schizochytrium sp Tetraselmis sueica (Chisti 2007)

33 IMPROVEMENT FOR HIGHER PRODUCTIVITY

34 ALGAL METABOLITES HAVING POTENTIAL FOR BIOFUEL USES Triglycerides and fatty acids Lipids, long chain hydrocarbons - botryococcene Sugars and starches Ethanol or other alcohols Cellulose or other biomass

35 Engineering Algae towards Biofuel Production Genetic Manipulations Efficient expression of transgenes Inducible promoters Random genomic integration Isolation of knockout mutants Random insertional mutagenesis Targetted gene disruption Over expression

36 STRAIN IMPROVEMENT BY GENETIC ENGINEERING Genetic engineering for Lipid metabolism 1. Lipid biosynthesis Enzymes like ACCase, KAS have been overexpressed with the objective to enhance lipid production But no significant increase Enzymes involved in TAG synthesis may be better candidates for genetic engineering as overexpression resulted in increase in lipid accumulation Unfortunately most studies carried out in Plants but homologues in algae also have the potential to be engineered

37 2. Metabolic blocking of the pathways resulting in accumulation of energy rich compounds For example, two different starch-deficient strains of C. reinhardtii, the sta6 and sta7 mutants (having disruptions in the ADPglucose pyrophosphorylase or isoamylase genes) respectively accumulate increased levels of TAG during nitrogen deprivation (Radakovits, 2010) Starchless mutant of Chlorella pyrenoidosa has also been shown to have elevated polyunsaturated fatty acid content (Ramazanov and Ramazanov, 2006). 3. Knocking out genes involved in lipid catabolism Genes involved in the activation of both TAG and free fatty acids, as well as genes directly involved in β-oxidation of fatty acids, have been inactivated, sometimes resulting in increased cellular lipid content. RNA silencing is preferred for this as silencing strategy for C. reinhardtii is already available 4. Modification of lipid characteristics Algal fatty acids: Mostly 16:1, 16:0, 18:1 ; Fatty acids suitable for biodiesel: 12:0, 14:0 Incorporation of genes coding for 12:0 or 14:0 biased Thioesterases into algal species can significantly improve the suitability of microalga-derived diesel feedstock

38 Genetic Engineering for Carbohydrate metabolism 1. Increasing glucan storage Introduction of designer or recombinant AGPase into AGPase - microalgal backround can improve the glucan production 2. Decreasing starch degradation A prospective startegy but degradation mechanisms in algae are largely unknown. But information from plant system can throw some light

39 3. Engineering secretion and transport systems for soluble sugars Hydrogenases characteized till date are O 2 sensitive, hence bioprospecting for O 2 tolerant Hydrogenase and genetic engineering of the enzyme to improve oxygen tolerance is urgently needed Soluble sugars may be preferred over polysaccharides because soluble sugars are smaller and easier to process, in addition to likely being more amenable for engineered secretion because many transporters have been described. Engineering for Hydrogen production For enhancing Hydrogen production, genetic techniques have been applied with the aim of -- Decreasing light-harvesting antenna size C. reinhardtii mutant (tla1) that exhibited a truncated LHC and showed higher Hydrogen production Inhibiting state transitions stm6 state transition mutant of C. reinhardtii over accumulates starch showing higher rates of cellular respiration and inhibition of cyclic electron transfer around PS I leading to increased Hydrogen production Hydrogenase engineering

40 DESIRABLE TRAITS IN ENGINEERED MICROALGAE High level production of enzymes for lipid biosynthesis Integration of genes into the host chromosome High level of expression Free from catabolic repression Engineering one microalgae with the ability to produce variable lipids Adding desired metabolic capabilities- mutagenesis, addition of multiple genes/operons Knocking out undesirable genes

41 FUTURE PROSPECT To improve photosynthetic efficiency of microalgae To develop high lipid content microalgae To improve light penetration properties of microalgal culture To improve bioreactor efficiency and higher yield

42 Thank You