Algae Strain Report 9
How are you?
Participating in the conferences in Italy,Japan & London, I am back here with report of some species which are being tested at Albert Einstein Science Institute,both in India & Germany..
Description of the research performed at AESI in the area of microalgal collection and
screening. In addition to performing actual research in this area, AESI personnel were
responsible for coordinating the efforts of the many subcontractors performing similar activities,
and for standardizing certain procedures and analyses. These efforts ultimately resulted in the
development of the AESI Microalgal Culture Collection, which is more fully described in
Brief review of algal taxonomic groups and characteristics:
For the purposes of this report, microalgae are defined as microscopic organisms that can grow
via photosynthesis. Many microalgae grow quite rapidly, and are considerably more productive
than land plants and macroalgae (seaweed). Microalgae reproduction occurs primarily by
vegetative (asexual) cell division, although sexual reproduction can occur in many species under
appropriate growth conditions.
There are several main groups of microalgae, which differ primarily in pigment composition,
biochemical constituents, ultrastructure, and life cycle. Five groups were of primary importance
to the ABP: diatoms (Class Bacillariophyceae), green algae (Class Chlorophyceae), goldenbrown
algae (Class Chrysophyceae), prymnesiophytes (Class Prymnesiophyceae), and the
eustigmatophytes (Class Eustigmatophyceae). The blue-green algae, or cyanobacteria (Class
Cyanophyceae), were also represented in some of the collections. A brief description of these
algal groups follows.
? Diatoms. -Diatoms are among the most common and widely distributed groups
of algae in existence; about 100,000 species are known. This group tends to
dominate the phytoplankton of the oceans, but is commonly found in fresh- and
brackish-water habitats as well. The cells are golden-brown because of the
presence of high levels of fucoxanthin, a photosynthetic accessory pigment.
Several other xanthophylls are present at lower levels, as well as β-carotene,
chlorophyll a and chlorophyll c. The main storage compounds of diatoms are
lipids (TAGs) and a β-1,3-linked carbohydrate known as chrysolaminarin. A
distinguishing feature of diatoms is the presence of a cell wall that contains
substantial quantities of polymerized Si. This has implications for media costs
in a commercial production facility, because silicate is a relatively expensive
chemical. On the other hand, Si deficiency is known to promote storage lipid
accumulation in diatoms, and thus could provide a controllable means to induce
lipid synthesis in a two-stage production process. Another characteristic of
diatoms that distinguishes them from most other algal groups is that they are
diploid (having two copies of each chromosome) during vegetative growth;
most algae are haploid (with one copy of each chromosome) except for brief
periods when the cells are reproducing sexually. The main ramification of this
from a strain development perspective is that it makes producing improved
strains via classical mutagenesis and selection/screening substantially more
difficult. As a consequence, diatom strain development programs must rely
heavily on genetic engineering approaches.
? Green Algae-. Green algae, often referred to as chlorophytes, are also abundant;
component. However, N-deficiency promotes the accumulation of lipids in
certain species. Green algae are the evolutionary progenitors of higher plants,
and, as such, have received more attention than other groups of algae. A
member of this group, Chlamydomonas reinhardtii (and closely related species)
has been studied very extensively, in part because of its ability to control sexual
reproduction, thus allowing detailed genetic analysis. Indeed, Chlamydomonas
was the first alga to be genetically transformed. However, it does not
accumulate lipids, and thus was not considered for use in the ABP. Another
common genus that has been studied fairly extensively is Chlorella.
? Golden-Brown Algae-. This group of algae, commonly referred to as
chrysophytes, is similar to diatoms with respect to pigments and biochemical
composition. Approximately 1,000 species are known, which are found
primarily in freshwater habitats. Lipids and chrysolaminarin are considered to
be the major carbon storage form in this group. Some chysophytes have lightly
silicified cell walls.
? Prymnesiophytes. -This group of algae, also known as the haptophytes, consists
of approximately 500 species. They are primarily marine organisms, and can
account for a substantial proportion of the primary productivity of tropical
oceans. As with the diatoms and chrysophytes, fucoxanthin imparts a brown
color to the cells, and lipids and chrysolaminarin are the major storage products.
This group includes the coccolithophorids, which are distinguished by
calcareous scales surrounding the cell wall.
? Eustigmatophytes-. This group represents an important component of the
?picoplankton?, which are very small cells (2-4 μm in diameter). The genus
Nannochloropsis is one of the few marine species in this class, and is common
in the world?s oceans. Chlorophyll a is the only chlorophyll present in the cells,
although several xanthophylls serve as accessory photosynthetic pigments.
*Cyanobacteria-. This group is prokaryotic, and therefore very different from all
other groups of microalgae. They contain no nucleus, no chloroplasts, and have
a different gene structure. There are approximately 2,000 species of
cyanobacteria, which occur in many habitats. Although this group is
distinguished by having members that can assimilate atmospheric N (thus
eliminating the need to provide fixed N to the cells), no member of this class
produces significant quantities of storage lipid; therefore, this group was not
deemed useful to the ABP.
Six promising strains were analyzed in AESI Type I, Type II, and ASW (Rila) using the
temperature-salinity gradient described previously. These included the diatoms Chaetoceros
muelleri , Navicula , Cyclotella , Amphora 1 & 2, and the chlorophyte Monoraphidium minutum . Navicula and
Cyclotella were actually collected from the Florida keys; the remaining strains were collected in
Colorado and Utah.) All strains exhibited rapid growth over a wide range of conductivities in at
least two media types. Furthermore, all strains exhibited temperature optima of 30?C or higher.
Maximal growth rates of these strains, along with the optimal temperature, conductivity, and
media type determined in these experiments are shown in Table II.A.1. (Higher growth rates
were determined for some of these strains in subsequent experiments.
Experiments were also conducted in an attempt to identify the chemical components of AESI
Type I and Type II media most important for controlling the growth of the various algal strains.
Bicarbonate and divalent cation concentrations were found to be important determinants in
controlling the growth of Boekelovia sp. and Monoraphidium . The
growth rate of Monoraphidium minutum 2 . increased by more than five-fold as the bicarbonate concentration of
Type II/25 medium was increased from 2 to 30 mM, and the growth of BOEKE1 by
approximately 60% over this range. These results make sense, since media enriched in
bicarbonate would have more dissolved carbon available for photosynthesis. An unexpected
finding was that there was a decrease of nearly 50% in the growth rate of BOEKE1 as the
divalent cation concentration increased from 5 mM to 95 mM (in Type I/10 medium containing
altered amounts of calcium and magnesium). The effects of magnesium and calcium
concentration on the growth of Monoraphidium minutum . were less pronounced. These results indicate that
matching the chosen strain for a particular production site to the type of water available for mass
cultivation will be important.
The lipid contents of several strains were determined for cultures in exponential growth phase
and for cultures that were N-limited for 7 days or Si-limited for 2 days. In general, nutrient
deficiency led to an increase in the lipid content of the cells, but this was not always the case.
The highest lipid content occurred with Navicula 1, which increased from 22% in exponential
phase cells to 49% in Si-deficient cells and to 58% in N-deficient cells. For the green alga
Monoraphidium minutum ., the lipid content increased from 22% in exponentially growing cells to 52% for cells
that had been N-starved for 7 days. Chaetoceros
muelleri also exhibited a large increase in lipid content in
response to Si and N deficiency, increasing from 19% to 39% and 38%, respectively. A more
modest increase occurred for nutrient-deficient AMPHO1 cells, whereas the lipid content of
Cyclotella was similar in exponential phase and nutrient-deficient cells, and actually decreased in
Amphora 2 as a result of nutrient deficiency.
These results suggested that high lipid content was indeed achievable in many strains by
manipulating the nutrient levels in the growth media. However, these experiments did not
provide information on actual lipid productivity in the cultures, which is the more important
factor for developing a commercially viable biodiesel production process. This lack of lipid
productivity data also occurred with most of the ABP subcontractors involved in strain screening
and characterization, but was understandable because the process for maximizing lipid yields
from microalgae grown in mass culture never was optimized. Therefore, there was no basis for
designing experiments to estimate lipid productivity potential.
Six algal Strains
(B. braunii, Dunaliella primolecta, Isochrysis sp., Monallanthus salina, Phaeodactylum
tricornutum, and Tetraselmis sueica) were obtained from existing culture collections and
analyzed with respect to lipid, protein, and carbohydrate content under various growth
conditions. For these experiments, all cultures except for B. braunii were grown in natural
seawater that was enriched with N, P, and trace metals. B. braunii was grown in an artificial
seawater medium. Initial experiments to determine productivities of these species were
performed using batch cultures in 9-L serum bottles. Of the strains tested, the highest growth
rates were observed with P. tricornutum (Thomas strain) and M. salina.
Additional experiments were performed in plexiglas vessels that were 5 cm thick, 39 cm deep,
2,000-watt tungsten-halide lamp, which was placed behind a water/CuSO4 thermal filter. In
these experiments, the cultures were typically maintained for 40 to 90 days. In the early stages of
an experiment, the cultures were maintained in a batch mode, and then converted to a continuous
or semi-continuous dilution mode. Various culture parameters (including light intensity, dilution
rate, and N status) were manipulated during the course of these experiments to determine their
effects on the productivities and proximate chemical composition of the strains. The results of
these experiments with each species tested are discussed below. These experiments are difficult
to compare because the experiments were all carried out slightly differently (i.e., different light
intensities, different culturing methods [batch, semi-continuous, and continuous], different means
of obtaining N-deficient cultures, and inconsistent use of a CuSO4 heat filter, which resulted in
differences in light quality and culture temperature). Nonetheless, the general conclusions of this
study are of interest.
This strain has been used for several past studies, and was concomitantly being tested in outdoor
mass culture by another subcontractor (the University of Hawaii; principal investigator Dr.
Edward Laws; discussed in Section III). Therefore, this strain was subjected to more extensive
testing than the other strains in this subcontract. In one experiment reported for this strain, the
effects of light intensity on productivity were determined in batch cultures (i.e., in the Plexiglas
culture apparatus described earlier without culture replacement and dilution). The maximum
productivity observed for this strain (21 to 22 g dry weight?m-2?d-1)3 was observed at a total daily
illumination of 63-95 kcal (representing approximately 40%-60% of full sunlight in southern
California during the summer). This value was slightly higher than the productivity observed
with a total daily illumination of 70% full sunlight (17.1 g dry weight?m-2?d-1). Productivities
under N-limiting, continuous growth mode conditions were between 7 and 11 g dry
weight?m-2?d-1. Likewise, productivities under N-sufficient, continuous growth mode conditions
were reduced relative to batch cultures.
In addition to measuring overall productivities, the levels of protein, carbohydrate, lipid, and ash
were determined for cells grown under the various conditions described earlier. Illumination of
the cultures from 40% to 70% of full sunlight did not have a large impact on the cellular
composition. Growth of P. tricornutum cells under N-deficient conditions resulted in a reduction
of the protein content from 55% (in N-sufficient cells) to 25% of the cellular dry weight.
Carbohydrate content increased from 10.5% to 15.1%, and the mean lipid content increased from
19.8% to 22.2%, although these differences in carbohydrate and lipid contents did not appear to
be statistically significant. At one stage of the experiment, however, a time course of N
deficiency led to a consistent rise in lipid content from 19.9% to 30.8% over the course of 7 days.
The actual rate of lipid production did not increase, however, because the overall productivity of
the cultures was reduced under N-deficient growth.
The maximum productivity observed for this species (12.0 g dry weight?m-2?d-1 ) occurred during
continuous culture at 60% full sunlight under N-sufficient conditions. Doubling the light
intensity lowered the productivity to 6.1 g dry weight?m-2?d-1. The chemical composition of Nsufficient
cells (as an average percentage of total cell dry weight) was 64.2% protein, 12.6%
carbohydrate, and 23.1% lipid. After 7 days of growth under N-deficient conditions, the
composition was 26.8% protein, 59.7% carbohydrate, and 13.7% lipid. Therefore, this alga
accumulates carbohydrates rather than lipids in response to nutrient deficiency, limiting its
usefulness as a lipid production strain.
This alga reportedly contained high levels of lipids when grown under N-deficient conditions.
The highest productivity (13.9 g dry weight?m-2?d-1) was observed under N-sufficient conditions
at a light intensity of 50% full sunlight, although detailed experiments with regards to the effects
of light intensity on productivity were not conducted. There was little difference in the lipid
3Reporting of productivities in g dry weight?m-2 ?d-1 derives from the goal of mass culturing the algae in
shallow open ponds. The objective would be to maximize biomass produced per area of pond. However, it
is often difficult to compare results between experiments when the data are reported in this manner, as
factors such as culture depth and vessel design would significantly affect productivity of the cultures.
content of cells grown under N-sufficient and N-deficient conditions (20.7% and 22.1%,
The highest productivity observed for this strain was 19.1 g dry weight?m-2?d-1, which occurred
in N-sufficient batch cultures grown under a light intensity of 60% full sunlight. N deficiency
resulted in a large increase in carbohydrate content (from a mean value of 10.7% to a mean value
of 47.1%). On the other hand, protein content was reduced substantially (from 67.6% to 28.3%),
and the lipid content decreased from 23.1% to 14.6% in response to N deficiency.
Isochrysis sp.(Tahitian strain T-ISO):
This strain is commonly used as a feed organism in aquaculture production systems. A
productivity of 11.5 g dry weight?m-2?d-1 was typical for batch cultures of this species, which was
approximately 33% higher than the value recorded during semi-continuous growth (dilution of
0.15 L/d). Productivity was lowered during N-deficient growth to 5.5-7.6 g dry weight?m-2?d-1.
This strain accumulated carbohydrate in response to N deficiency (from a mean value of 23.1%
to 56.9%). Lipid content also increased slightly (from 28.5% to 33.4%), whereas protein content
was reduced from 44.9% to 27.3%. The higher lipid content of N-deficient cells did not translate
to higher lipid productivities, however, because of the lower overall productivity of the stressed
Some very limited experiments were conducted with this species, which is known to accumulate
hydrocarbons. A culture grown under a light intensity of 60% full sunlight had a productivity of
only 3.4 g dry weight?m-2?d-1. The lipid content of these cells was 29% of the cellular dry
weight; the N status of the cells was not reported, but it is assumed that the cells were grown
under N-sufficient conditions.
Of the species examined, P. tricornutum and T. sueica had the highest overall productivities.
These species also had the highest lipid productivities, which were 4.34 and 4.47 g lipid?m-2?d-1,
respectively. For both species, the maximal productivities were obtained in batch cultures, as
opposed to semi-continuous or continuous cultures. Although the lipid contents of cells were
often higher in response to N deficiency, the lipid productivities of all species tested were
invariably lower under N deficiency because of an overall reduction in the culture growth rates.
For the species tested under continuous or semi-continuous growth conditions, lipid
productivities were reduced from 14% to 45% of the values measured for N-sufficient cultures.
The results also pointed to the importance of identifying strains that are not photoinhibited at
light intensities that would occur in outdoor ponds. Finally, this work highlighted the fact that
some microalgae accumulate carbohydrates during nutrient-deficient growth; such strains are
clearly not acceptable for use as a feedstock for lipid-based fuel production.
The work is going on....
Will share you more when the reports will come..
Please give your views..