Subcontractor: University of California, San Diego Principal Investigator: William H. Thomas
Period of Performance: 1980 - 1983 Subcontract Number: XK-0-9111-1
Work carried out under this subcontract represented one of the first attempts by an ASP subcontractor to characterize the productivity and lipid yields of various microalgae. 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,
and 24 cm wide (surface area ~940 cm2). The cultures were illuminated from the side with a
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 (Thomas strain):
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 N-
sufficient 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:
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
content of cells grown under N-sufficient and N-deficient conditions (20.7% and 22.1%, respectively).
T. sueica:
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 cultures.
B. braunii:
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.
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.
Publications:
Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Neori, A. (1981) “Effects of light intensity and nitrogen deficiency on yield, proximate composition, and photosynthetic efficiency of Phaeodactylum.” Proceedings of the Subcontractors’ Review Meeting—Aquatic Species Program, Solar Energy Research Institute, Golden, Colorado, SERI/CP-624-1228, pp. 33-58. Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Neori, A.; Eldridge, P. (1984a) “Yields, photosynthetic efficiency, and proximate composition of dense marine microalgal cultures. I.
Introduction and Phaeodactylum tricornutum experiments.” Biomass 5:181-209.
Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Neori, A.; Eldridge, P. (1984b) “Yields, photosynthetic efficiency, and proximate composition of dense marine microalgal cultures. II. Dunaliella primolecta and Tetraselmis suecica experiments.” Biomass 5:211-225.
(Also see references listed in the following section.)
II.A.2.c. Selection of High-Yielding Microalgae from Desert Saline Environments