ALGAL STRAIN SELECTION AND CHARACTERISATION: A
KEY TO SUCCESSFUL ALGAE FEEDSTOCK PRODUCTION
DR. SUNIL PABBI
Principal Scientist
CCUBGA, IARI, PUSA, New Delhi 110012
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
carotenoids and phycobiliproteins
of
photosynthetic
pigments
like
chlorophyll,
 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.
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.
Cont…
Rupprecht , 2006
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 CO2 levels
Challenges for production of Biofuels from microalgae
HARVESTING
RECOVERY
IMPROVED
STRAINS
ALGAL CULTURE SELECTION
 Natural Selection
 Inherent variability (Genetic & Metabolic)
 Mutation Selection
 Recombination
 Culture stability
 Growth/nutrient requirement
 Culture condition
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
Collection of samples from different Habitats
Algal Crusts in Deserts
Source: Chinese Science Bulletin, Vol.50, No.2, January, 2005.
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
Streaking
Petri Plates with medium
Isolated colonies at the end of the streak are expected to be unialgal
Pour plate
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)
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.
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
Preservation techniques
•Short term and Long term
A. Agar Slants
B. Immobilized Alginate
Beads
C. Cryopreservation
D. Lyophilization
Characterization
• MORPHOLOGY
• PHYSIOLOGY
• MOLECULAR BIOLOGY
Phormidium
Microcoleus
Oscillatoria
Lyngbya
Scytonema
Aulosira
Cylindrospermum
Microchaete
Chemotaxonomic
parameters
/
biochemical
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)

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
Anabaena
Anabaena
Anabaena
Anabaena
Anabaena
Anabaena
Nostoc
Nostoc
Calothrix
Calothrix
Calothrix
Tolypothrix
Tolypothrix
Westiellopsis
An-1
An-2
An-3
An-4
An-5
An-6
Ns-1
Ns-2
Cl-1
Cl-2
Cl-3
To-1
To-2
Ws-1
Chlorophyll
(g/ml)
Nitrogenase Activity
(mole C2H4/mg chl/h)
4.893
0.683
0.474
0.241
3.698
3.921
0.785
2.057
2.652
0.086
3.964
2.628
2.131
1.432
7.19
61.2
41.77
73.03
23.9
24.69
56.05
17.12
21.57
46.31
23.87
30.14
53.68
32.26
1: AJ438184.
Microchaete tenera...[gi:19577330]
DEFINITION
ACCESSION
AUTHORS
Microchaete tenera partial 16S rRNA gene.
AJ438184
Dhaulakhandi,.B., Pabbi,S., Singh,P.K.
and Ahluwalia,K.B.
Ribosomal RNA sequence Analysis of
unclassified cyanobacteria
2 (bases 1 to 999)
TITLE
REFERENCE
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
//
•
PHYLOGENETIC TREES
COMPRISING DIFFERENT
MICROALGAE
Total lipids in different classes of algae
Algal classes
Total Lipids
Average
Range
Bacillariophyceae
16
1-39
Chlorophyceae
19
1-53
Chrysophyceae
29
12-39
Cyanophyceae
8
2-13
Dinophyceae
21
5-36
Haptophyceae
29
5-48
Phaeophyceae
4
1-9
Prasinophyceae
11
3-18
Rhodophyceae
9
1-14
Xanthophyceae
12
6-16
*per cent dry weight basis
Source: Narayan et al.
18:1 (n-9)
Oleic acid
O2
O2
18:2 (n-6)
Linoleic acid
O2
18:3 (n-6)
Υ- linolenic acid
18:3 (n-3)
Α-Linolenic acid
20:3 (n-6)
Dichromo- Υ linolenic acid
22:3 (n-3)
Elcosatrienoic acid
O2
20:4 (n-6)
Arachidonic acid
O2
22:4 (n-3)
Elcosatetraenoic acid
O2
22:4 (n-6)
Adrenic acid
22:5 (n-3)
Elcosapentaenoic acid
O2
22:5 (n-6)
Docosapentaenoic acid
Biosynthesis of polyunsaturated fatty acids in microalgae
22:5 (n-3)
Docosapentaenoic acid
22:6 (n-3)
Docosahexenoic acid
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
ENHANCED LIPID PRODUCTION BY DIFFERENT
MICROALGAE UNDER DIFFERENT STRESS
Species
Stress
% Lipid (dry weight)
Cyclotella cryptica
Nitrogen deficiency
18
Dunaliella salina
Osmotic stress and Nitrogen deficiency
Nitrogen deficiency
Non environmental stress
18.5
14.4
6
Nitzschia sp.
Non environmental stress
45-47
Phaeodactylum tricornutum
Non environmental stress
20-30
Botryococcus braunii
Nitrogen deficiency
Non environmental stress
54.2
25-75
Chlamydomonas sp.
Non environmental stress
23
Chlorella sp.
Non environmental stress
Non environmental stress
20.7
28-32
Chlorella vulgaris
Nitrogen deficiency
Non environmental stress
18
14-22
Nannochloris sp.
Non environmental stress
20-35
Nannochloropsis sp.
Nitrogen deficiency
Non environmental stress
33.3-37.8
31-68
Nannochloropsis salina
Nitrogen deficiency
Non environmental stress
54
28.6
Spirulina platensis
Non environmental stress
16.6
Tetraselmis suecia
Nitrogen deficiency
Non environmental stress
20-30
15-23
Isochrysis sp.
Nitrogen deficiency
Non environmental stress
26-45
25-33
ENHANCEMENT & ALTERATION OF LIPID PROFILE
UNDER STRESS CONDITIONS
 Carbon
dioxide
 Nitrogen stress
Phosphorus and Sulphur stress
 Other chemical factors
 Physical factors
Immobilization
OIL CONTENT OF SOME MICROALGAE
Micoralgae
Oil content(%dry wt)
Botryococcus brauni
25-75
Chlorella sp.
28-32
Crypthecodinium cohni
20
Cylindrotheca sp.
16-37
Dunaliella primolecta
23
Isochrysis sp.
25-33
Monnallanthus salina
>20
Nannochloris sp.
20-35
Nannochloropsis sp.
31-68
Neochloris oleoabundans
35-54
Nitzschia sp.
45-47
Phaeodactylum tricornutum
20-30
Schizochytrium sp.
50-70
Tetraselmis sueica
15-23
(Chisti 2007)
IMPROVEMENT FOR HIGHER
PRODUCTIVITY
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
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
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
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
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
3. Engineering secretion and transport systems for soluble sugars
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
Hydrogenases characteized till date are O2 sensitive, hence bioprospecting for O2
tolerant Hydrogenase and genetic engineering of the enzyme to improve oxygen
tolerance is urgently needed
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
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
Thank You