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Algae Research from Dept of Energy ASP Aquatic Species Program

http://www1.eere.energy.gov/biomass/pdfs/biodiesel_from_algae.pdf

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 ASP: 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 (Class Bacillariophyceae)

Diatoms are among the most common and widely distributed groups of algae. The cells are golden-brown because of the presence of high levels of fucoxanthin, a photosynthetic accessory pigment.The main storage compounds of diatoms are lipids (TAGs) and a β-1,3-linked carbohydrate known as chrysolaminarin.

Several other xanthophylls are present at lower levels, as well as β-carotene, chlorophyll a and chlorophyll c. 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. Examples studied:

• Chaetoceros muelleri (CHAET14)
• Navicula (NAVIC1)
• Cyclotella (CYCLO2)
• Amphora (AMPHO1 and AMPHO2)

NAVIC1 and CYCLO2 were actually collected from the Florida keys; the remaining strains were collected in Colorado and Utah. 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.

Golden-Brown Algae (Class Chrysophyceae)

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.

Green Algae (Class Chlorophyceae)

Green algae, often referred to as chlorophytes, This group has chlorophyll a and chlorophyll b. These algae use starch as their primary storage component.

approximately 8,000 species are estimated to be in existence. 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. Example studied:

• chlorophyte Monoraphidium minutum (MONOR2)
• Chlamydomonas reinhardtii

has been studied very extensively, in part because of its ability to control sexual reproduction, thus allowing detailed genetic analysis. Chlamydomonas was the first alga to be genetically transformed. However, it does not accumulate lipids, and thus was not considered for use in the ASP. Another common genus that has been studied fairly extensively is Chlorella

Class Prymnesiophyceae

This group of algae, also known as the haptophytes, They are primarily marine organisms, and can account for a substantial proportion of the primary productivity of tropical oceans.

consistsof approximately 500 species. 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 (Class Cyanophyceae)

Bacteria, They contain no nucleus, no chloroplasts, and have a different gene structure.

This group is prokaryotic, and therefore very different from all other groups of microalgae. 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 ASP.

growth media

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 NAVIC1, 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 MONOR2, the lipid content increased from 22% in exponentially growing cells to 52% for cells that had been N-starved for 7 days. CHAET14 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 CYCLO2 was similar in exponential phase and nutrient-deficient cells, and actually decreased in AMPHO2 as a result of nutrient deficiency. Table II.A.3. Strains from the Arizona State University collection having the highest Nile Red fluorescence.

lipid production (red nile assay)

Strain Genus Class Triolein equivalents (mg•L-1) Exponential growth Triolein equivalents (mg•L-1) N-deficient growth

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NITZS54 Nitzschia Bacillariophyceae 8 1003 NITZS53 Nitzschia Bacillariophyceae 17 934 NITZS55 Nitzschia Bacillariophyceae 37 908 ASU3004 Amphora Bacillariophyceae 9 593 NAVIC36 Nitzschia Bacillariophyceae 61 579 AMPHO45 Amphora Bacillariophyceae 39 308 FRAGI2 Fragilaria Bacillariophyceae 6 304 AMPHO27 Amphora Bacillariophyceae 38 235 NITZS52 Nitzschia Bacillariophyceae 24 234

Overall Conclusions

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.