Hello Friends,

      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

Chapter II.A.3.

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.

Growth rates.

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.

Lipid content.

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.

P. tricornutum):

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.

D. primolecta:

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.

M. salina: