Laboratory Studies

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.

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bmcfteeul turds 24 cm wedumsuotates .area -t94 0s tcmns teTbeC,cUl¡etfeg hesr egiliu\icafTratttícS

wore 0he esrved with P . t2líC00riWfttmf(TTgsIieals]MlfsiTl)lancpMyh4Clnawas 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 P. tricornutum (Thomas iStrain): .

carried out slightly differently (i.e., different light intensities, different culturing in giffeiiTac efvestigghr Dtia!liCYv^lfTcL^W3SttCrec^(essFeiTaltífeioTeeíITle,ifee]fisSc reththi|§Tr§ria wascsubocso t0fmsfudyfienf Tiefi§g 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 • d ) 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 • d ). Productivities under N-limiting,

continuous growth mode conditions were between 7 and 11 g dry weight • m • d . Likewise, productivities under

NrfUiaCfffi'OnPttOU <WegSWltbinjodOVifrtlOhpW(e'CCfllCtilVfdiffSatit]b^ iecels^ ep rotein, 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 De Minc^ ^HcC to a consistent rise in lipid content from 19.9% to 30.8% over the

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Doubling the light intensity lowered the productivity to 6.1 g dry weight- m • d .

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%

Mrbí^1hyaCrate' and 13.7% lipid. Therefore, this alga accumulates carbohydrates rather than lipids in response to nutrient deficiency, limiting its usefulness as a lipid This alga reportedly contained nigh levels of lipids when grown under iN-aeficient production strain. _2 _!

conditions. The highest productivity (13.9 g dry weight- m • d ) was observed under N-sufficient conditions at a light intensity of 50% full sunlight, although

CRltaMg|spgFoaufttVitwSiR r'egryraSeiightthfh-ff^fc(!t§ eaVeSgbOiibergiial oMSO ^CUM^

WfrfIgat ifrPtSbcal1<otf tip eff hetlw ashU ttlu eCtfVerwOCfd n eh© lipd mize 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%, respectively).

T. sueica:

The highest productivity observed for this strain was 19.1 g dry weight- m • d , 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

CiOCtSrl|SiWap (Fedutiti<m StrbSAaptiSOiy from 67.6% to 28.3%), and the lipid content decreased from 23.1% to 14.6% in response to N deficiency.

This strain is commonly used as a feed organism in aquaculture production systems.

A productivity of 11.5 g dry weight- m • d was typical for batch cultures of this

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WSHihi^nIimlO1ds gIPli®i1stiiilUliicnulHulated 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, Boweurnii because of the lower overall productivity of the stressed cultures.

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 • d . The lipid content of these cells was 29% of the cellular dry weight; the N status of the cells was not Overl? <C pncluSlp m§sumed 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 • d , 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 PUbniUatl0na-(deficient growth; such strains are clearly not acceptable for use as a feedstock for lipid-based fuel production.

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 cultures. I Introduction and Phaeodactylum tricornutum experiments." Biomass 5hfima§oW' H Seibert, D1R 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 ri3 ferences listed in the following section.)

II.A.2.c. Selection of High-Yielding Microalgae from Desert Saline Environments

VA SERI/NREL/DOE REPORTS AND PUBLICATIONS 263

VB ADDITIONAL REFERENCES 293

V.C. PATENTS 294

The work carried out under this subcontract represented one of the first efforts to collect microalgae from inland saline habitats and to screen those strains for rapid growth rates and lipid content. Collecting trips were made to eastern California and western Nevada, and initial culturing efforts were conducted at the Sierra Nevada Aquatic Research Laboratory near Mammoth Lakes, California, and the Desert Studies Center (Zzyzx Springs), which is near Baker, California. Various saline waters and soils were sampled during these collecting trips. The collection sites included Pyramid Lake, Black Lake, Owens Lake, Walker Lake, Saline Valley, Zzyzx Springs, Armagosa standardized conditions in various

"Zzyzx medium" before algal isolation (see Thomas et al. [1986] for media compositions).

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