BIOTECHNOLOGY AND BIOENGINEERING

VOL. X I X (1977)

An Improved Method f o r Optical Density Measurement of the Semimicroscopic Blue Green Alga Spirulina maxima
Our research interest is to study the continuous cultivation of Spirulina maxima in a controlled annular photochemical reactor as well as the dynamic behavior and the optimal control of this particular culture system in order to increase the productivity of algal protein. For the control purpose, the determination of biomass concentration in the culture medium by dry weight (DW) as used elsewhere1e2was found inconvenient because it took a t least two hours to obtain the result. Furthermore, the cell count method is not suitable for this alga because the filaments do not have the same numbers of spirals at any physiological state (the distribution of numbers of spirals ranged from less than one to more than ten). Thus the optical density (OD) reading of the culture sample at 560 nm as used by Zarrouk3 seemed the most suitable method for the rapid determination of the S. maxima biomass concentration. A unit for the continuous measurement of the OD of recycled culture medium has been tested; however, it gave no reproducible results. Errors were due to the fouling of cells in the tubes and in the measuring chamber, the semimicroscopic size of S. maxima cells (the filaments are usually 5-10 pm broad and 200-300 pm long), and the nonuniformity of the cell lengths (number of spirals). Recently, an automatic device for measuring and controlling the OD has been developed and successfully used for three species of green algae.4 In this study, the sampling tube for OD measurement was coated with a silicone agent in order to reduce the cells fouling to a minimum. Furthermore, the three algal species tested by these authors4 were of microscopic sizes (maximum lengths of -20 pm) and were unicellular (Dunaliella viridis and Tetraselmis suecica) or coenobial (Scenedesmus acutus). Therefore, the objective of our work was to develop an improved method for the spectrophotometric determination of the cell mass concentration of the semimicroscopic filamentous S . maxima algal culture. The original culture of S. maxima was received from the Institut Frangais du P6trole in 1970 and was periodically transferred and maintained a t room temperature in Roux bottles containing 600 ml of a synthetic medium described by Zarr~uk.~ The bottles were illuminated with Westinghouse Cool White fluorescent lights (18.8 klux) and bubbled with a mixture of air-COz (3 v/v% of CO,) to maintain the culture at p H -8.5. The batch cultivation experiments were carried out in Roux bottles, under the same conditions, and using the same culture medium. The DW's were determined by filtering 10 or 20 ml of culture medium through a 0.45 pm Gelman membrane filter, then rinsing with distilled water, and drying in an oven at 105OC to a constant weight. Homogenates were prepared from 20 ml of culture medium using the Model W185 Sonifier Cell Disruptor (Heat Systems-Ultrasonics, Inc., Plainview, N.Y.) equipped with a standard horn (a special alloy titanium 1.27 cm disruptor horn). Homogenization a t maximum power (-150 W) for 7 min was found to be satisfactory, resulting in a green, clear solution. Diluted and undiluted homogenates stored in the refrigerator (4C) were stable for a t least one day. The OD's of the cell suspension and the
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@ 1977 by John Wiley & Sons, Inc.

1220 BIOTECHNOLOGY AND BIOENGINEERING VOL. X I X (1977) homogenate samples were measured a t 560 nm, in a 20 mm test tube, using the Bausch & Lomb Spectronic 70 Colorimeter spectrophotometer.

OD OF CELL SUSPENSIONS VERSUS DW Spirulina maxima was grown in triplicate Koux bottles which were inoculated with 1 v/v% of inoculum (0.0.500-0.0515 g of I>W/liter). Twenty ml of culture sample were taken daily for measurements of the OD of cell suspensions, DW, and pH. The experiment was continued for 14 days and 20 ml of fresh culture medium were added to the Roux bottles immediately after each sampling in order to maintain the same volume of the culture medium during the cultivation. The plot of the OD of the cell suspensions versus DW for S. maxima is shown in Figure 1. For all samples within the first three days and two samples a t the fourth day of cultivation which contain relatively small concentration of biomass (-0.5 g of DW/liter or less), readings fell within the accurate range of the OD scale (0.1 to 1.6) and they could be read directly from the spectrophotometer without dilution. However, for all samples during the later cultivation periods which contained higher concentration of biomass (-0.5 g of I>W/liter or more), dilution

Fig. 1. Correlation between OD of cell suspensions and DW of S. mazima grown in a synthetic medium. D :Diluted samples ( 0 : bottle 1, A: bottle 2, bottle 3) with slope = 3.845 OD unit-liter/g of DW and correlation coefficient 0.920, U : Undiluted samples ( 0 : bottle 1, A : bottle 2, 0 : bottle 3) with slope = 3.052 OD unit-liter/g of DW and correlation coefficient = 0.993.

.:

DRY WEIGHT

,G/L

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of the samples with distilled water was necessary prior to OD readings. The straight lines D and U in Figure 1 represent the correlations between the OD of the cell suspensions and the DW of S. maxima for diluted and undiluted samples, respectively. Examination of these data illustrates that the correlation between the OD of cell suspensions and the DW of S. maxima is obviously not practicable because the straight lines representing diluted and undiluted samples have dissimilar slopes. Moreover, the data for diluted samples show a scattering around line D which renders this calibration curve inaccurate and undesirable. It was observed that the cells in the samples of culture medium containing high concentrations of S. maxima readily form small flocs which partially floated toward the surface or the bottom of the container. Dilution of these samples with distilled water did not alter these observations. This would explain the scattered nature of the data obtained for diluted samples. The deviation of the straight line D from the straight line U was due to the dilution of the samples with distilled water. This was confirmed by the results shown in Figure 2 . In this figure, many dilutions were performed on a two-week old sample (all cells a t the same physiological state) and both their OD and DW were measured. I t is shown that the slope of the straight line in Figure 2 is the same as that of the straight line D in Figure 1. Thus, the correlation between the OD of the cell suspensions and the DW of S. maxima was not satisfactory for practical determination of the biomass concentration in the culture medium over a wide spectrum of concentration.

1.5 I

0.4 DRY WEIGHT, G/L

Fig. 2 . Correlation between OD of cell suspensions and DW of S. m z i m a a t one physiological state. Slope = 3.845 OD unit-liter/g of DW; correlation coefficient = 0.997.

1222 BIOTECHNOLOGY AND BIOENGINEERING VOL. X I X (1977)

OD OF CELL HOMOGENATES VERSUS DW

I n order to avoid flocculation of S. maxima cells during OD readings, we a& tempted to use cell hornogenates instead of the cell suspensions. An appropriate wavelength for OD readings was chosen and verified by testing the linearity of these OD readings against various dilutions of the homogenates with distilled water. A homogenate was prepared from a culture medium containing 4.076 f 0.032 g of DW/liter of S. maxima and subjected to various dilutions. The plot of the OD of this hornogenate a t 560 nrn versus DW is shown in Figure 3. From this figure, a perfectly linear correlation between OD and DW was observed. It suggested that a concentrated hornogenate could be diluted a t least 32 times for OD readings without deviation from its linearity. Later in this work, it was found that homogenates prepared from a11 culture samples of S. maxima required no more than two dilutions for OD readings. Furthermore, no significant fluctuations were observed for individual OD readings of the undiluted or diluted hornogenates. Finally, a calibration curve of homogenate OD readings versus DW a t various concentrations and different physiological states of S. maxima cells was prepared. For this purpose, the cultures were grown for 11 days in four Roux bottles initially inoculated with 1, 5, 10, and Z y of inoculum, respectively. The Oo results in Figure 4 show a very satisfactory correlation between the OD and the DW of S. maxima as compared with the results shown in Figure 1. The difference in physiological states of S. maxima cells and the dilution of the homogenates with distilled water did not significantly affect the slope of the calibration curve.

DILUTION 2

k
u )

1.0

z
W

0
0

1
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DRY WEIGHT, G/L

Fig. 3. Correlation between OD of diluted homogenate and DW of S. maxima at one physiological state. Slope = 0.663 OD unit-liter/g of IIW; correlation coefficient = 1.000..

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0.0 0 . 5

1.0

1.5

2.0 2.5

3.0 3.5

DRY WEIGHT, G/L

Fig. 4. Correlation between OD of homogenates and DW of S. maxima grown in a synthetic medium. All samples above 1.5 OD were diluted twice with distilled water. Numbers indicate the cultivation age of samples. Bottles were inoculated with 1% ( 0 ) ,5%(0), 10% ( A ) , and 20% (A)of inoculum. Slope = 0.692 OD unit-liter/g of DW; correlation coefficient 0.979. The slope of this calibration curve (Fig. 4) was found to be very close to that obtained from dilutions of a single homogenate (Fig. 3). This is a distinct advantage in the rapid preparation or checking of the calibration curve according to our method. Another experiment was performed in order to verify whether or not there is any effect on the slope of the calibration curve when a culture system was grown under a different set of conditions. With this objective in mind, a calibration curve was prepared from the growth in both batch and continuous operations of our builbin 8 liter annular photochemical reactor under an entirely different set of conditions (illumination source, light intensity, gas mixture ratio, flow rate, and temperature). The resultant slope of the calibration curve (not shown) obtained from this reactor from a set of 22 samples taken a t different physiological states was 1.58 g of DW/liter per OD unit while the one obtained from Roux bottles (Fig. 4) represented 1.4.5.

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As a rule of thumb, under conditions of experiment,ation observed for the growth of S . maxima in a synthetic medium, one OD unit of cell homogenate represents 1.50 g of DW/liter with an absolute error of 5% or less.

References
1. N. Kosaric, H. T. Nguyen, and M. A. Bergougnou, Biotechnol. Bioeng., 16, 881 (1974). 2. H. T. Nguyen, N. Kosaric, and M . A. Bergougnou, Can. Znst. Food Sci. Technol. J., 7, 114 (1974). 3. C. Zarrouk, Contribution b 16tude dune CyanophycCe. Influence de divers facteurs physiques et chimiques sur la croissance et la photosynthbe de Spirulina maxima (Setch. et Gardner) Geitler. Thesis of doctorat of Applied Science, Universit6 de Paris, 1966. 4. P. Sorgeloos, E. Van Outryve, G. Persoone, and A. Gattoir-Reynaerts, A p p l . Environ. Microbiol., 31, 327 (1976). A. LEDUY N. THERIEN

Department of Chemical Engineering University of Sherbrooke Sherbrooke, Quebec, Canada J1K 2 R l Accepted for Publication February 25, 1977