A.1.d Collection and Screening Activities

In 1985, the strain enrichment procedure utilizing the rotary screening apparatus described previously was modified to include incubation of samples in SERI Type I and Type II media (25

and 55 mmho•cm-1 _{conductivity) and in artificial seawater, in addition to the original site water.}

The cultures that exhibited substantial algal growth were further treated to isolate the predominant strains as unialgal (clonal) isolates. These strains were then tested for growth using the temperature-salinity matrix described earlier.

Collection activities.

Collection efforts by SERI researchers in 1985 again focused on shallow inland saline habitats. This time collecting trips were also made to New Mexico and Nebraska, in addition to Colorado and Utah. Eighty-six sites were sampled during the year, 53 of which were sampled in the spring. From these 53 sites, 17 promising strains were isolated. An analysis was conducted comparing the results of the new protocol with those that would have resulted from the protocol used in prior years. This analysis indicated that the revised protocol was in fact superior to the older protocol. For example, only six of the 17 strains selected via the new protocol would also have been selected using the old protocol. Only three of the 17 strains grew best in the artificial medium type that most closely resembled the collection site water; in fact, only six strains were even considered to grow well in the collection site water relative to growth in at least one of the artificial medium. This analysis clearly indicated the value of performing the initial screening and enrichment in a variety of relevant media. The results suggest that the shallow saline environments sampled probably contain a large number of species whose metabolism is arrested at any given time. In other words, the water quality of such sites varies greatly, depending on precipitation and evaporation, so probably only a few of the many species present are actively growing at any given time. This also may explain the wide range of salinities and temperatures tolerated by many of these strains.

Growth rates.

Six promising strains were analyzed in SERI Type I, Type II, and ASW (Rila) using the

temperature-salinity gradient described previously. These included the diatoms Chaetoceros

muelleri (CHAET14), Navicula (NAVIC1), Cyclotella (CYCLO2), Amphora (AMPHO1 and

AMPHO2), and the chlorophyte Monoraphidium minutum (MONOR2). (NAVIC1 and

CYCLO2 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 30ºC 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; see results presented in Barclay et al. [1987]). Temperature-salinity growth contours are provided for these strains in the 1986 ASP Annual Report (Barclay et al. 1986).

Table II.A.1. Growth characteristics of various microalgal strains collected in 1985. Strain Maximum Growth Rate (doublings•day-1₎ Optimal Temperature (ºC) Optimal Conductivity (mmho•cm-1)

Optimal Medium Type (dependent on temperature and conductivity used)

AMPHO1 1.7 30 10-25 Type I, ASW

AMPHO2 2.48 30-35 40-70 Type I, Type II

CHAET14 2.87 35 25-70 Type II, ASW

CYCLO2 1.63 30-35 10-70 Type I, ASW

MONOR2 2.84 25-30 25 Type I, II, ASW

NAVIC1 2.77 30 10-40 Type I, Type II

Experiments were also conducted in an attempt to identify the chemical components of SERI 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. (BOEKE1) and Monoraphidium (MONOR2). The

growth rate of MONOR2 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 MONOR2 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 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.

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 ASP 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.

Publications:

Barclay, B.; Nagle, N.; Terry, K. (1986) “Screening microalgae for biomass production potential:

protocol modification and evaluation.” FY 1986 Aquatic Species Program Annual Report, Solar

Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 22-40.

Barclay, W.R.; Terry, K.L.; Nagle, N.J.; Weissman, J.C.; Goebel, R.P. (1987) “Potential of new

strains of marine and inland saline-adapted microalgae for aquaculture.” J. World. Aquaculture

Soc. 18:218-228.