problems of design and ecological considerations in mass culture of algae

RIOTECHNOLOGY AND BIOENGINEERING VOL. VI, PAGES 173-190 (1964)

Problems of Design and Ecological Considerations in Mass Culture of Algae

A. AM. NAYER, U. ZURI, Y. SHAIN, and H. GINZBURG, Botany Department, Hebrew University, Jerusalem, Israel

Summary

A 2000 1. 1 m. deep mass culture of algae was operated with continuous stirring, as an open system. The system behaved as an ecological unit selecting the most favored species. The ecological conditions could be modified by stirring speed and pattern in the tank. Methods for improving yields and utilization of COZ are described. Assessment of algal species for suitability in mass cultures is discussed. Yields obtained were 13 g . dry matter/sq. m. illuminated arealday .

INTRODUCTION

The possibility of using algae as a food source was mooted long ago, and the possibilities have often been discussed (Burlew, Carpenter,2 Meffert,I3 Tamiya, l9 Kanazawa et a1.,8 Kachroo,' and Oswald15). The problem has, therefore, received much attention, but little real progress has been made in realizing the apparent potential of the use of algae. Recently some of the problems which must be solved in order to make the use of algae economical have been discuseed (Ma- yerlO). Attention had tended to veer to other aspects and in particu- lar to the possible use of algae as gas exchangers in space systems (Gafford and Richard~on,~ Gaucher et a1.,6 see also Airforce Sympo- sium 1962). The literature in this latter field is so extensive, and the approaches to the problem so variable, that no attempts will be made to review them.

The work to be reported here was undertaken to investigate the basis of the functioning of a deep mass culture of Chlorella described previously (Mayer et al."). This mass culture unit was operated for two years and yields, factors affecting yield, gas exchange and other

173

174 MAYER ET AL.

parameters were studied. ing.

Thz results will be reported in the follow-

METHODS The large culture unit was the one previously described (Mayer

et al."). The only modification was that a vary-speed control unit, for continuously and smoothly varying stirring speed, was introduced between the stirring motor and the lay shaft, Figure 1. The culture solution, prepared in tap water was also as previously described, modified by using 0.5 g. EDTA/l., 0.31 g. KOH/l., and by adding H3B03, 100 mg./l. and ammonium molybdate 0.1 mg./l., both previously omitted. The final pI-1 was brought, a t the outset to 6.5 and

Fig. 1. View of culture unit, showing stirring mechanism.

was periodically adjusted, as necessary. Gas was supplied from cylinders, and metered by a time clock operating a solenoid value. It was introduced into the tank through a porous porcelain filter candle. Growth was determined as described previousfy and harvest carried out by a Sharples S o . 16.A centrifuge. The product was oven

BIOTKCHNO1,OGY . \NI) BIOENGINEEKING. VOL. V I , ISSUE 1

MASS CULTURE OF ALGAE 175

dried. Dissolved COz was determined by the method of ConwayY using 0.05N HCl and 0.05N Ba(OH)z. Dissolved oxygen was determined according to the Winkler method (Kolthoff and Belchera&) modified according to Placak and Ruchhaft.17

Light intensity was measured using a Weston Light Meter, Nodel 756, to which special water-tight photoelectric cells were attached.

Bacterial counts were obtained by plating out the culture medium, suitably diluted after removal of the algae by centrifugation a t 1000 g., on 2% Bacto nutrient agar (Difco Laboratories). Incubation was at 28°C. for 4 days. Colonies were counted at the end of this period.

Respiration of the algae was determined by conventional Warburg techniques at 25°C.

Photosynthesis was measured in a Warburg apparatus equipped for photosynthetic studies. Algae were centrifuged down and resus- pended in bicarbonate buffer (0.065&! KaHC03 + 0.035M KHCO,) ~

Light intensity reached a maximum of about 2000 ft.c. Protein was determined according to Lowry et al.,9 but the algal

suspension was first treated with 0.3 ml. N SaOH at 100°C. for 10 min. and 3.0 ml. 2% XazC03 were then added.

Chlorophyll was estimated according to Xackinney,12 the pigment being extracted into methanol by boiling for 15 min. in a water bath.

RESULTS

The modification of the tank permitted stirring at speeds between 30 and 120 c.p.m.

Gas Content of Medium

Solubility of gases in the solution was first determined. In tap water, without algae, both oxygen and carbon dioxide content of the solution remained steady throughout the day. When COZ was introduced into the tank, the concentration rose steeply, but after 7 hr. had dropped again to its original level in air, when the stirring speed was 60 r.p.m. When large amounts of' CO, were introduced into the culture solution and algae were present, the gas content changes as shown in Table I. The introduction of 300 1. COZ into thc culture in one lot raises the COz content to levels which must be re- garded as toxic to the algae (Sorokin,'s Xoreover, the algae are unable to use COs at this rate and hence large losses to the atnios-

176 MAYER ET AL.

TABLE I Solubility of gases in tank when algae (C. vulgaris) are also present. Stirring

speed 60 r.p.m.; 300 1. GO2 were introduced between 8:00-9:00

Time, hr.

8 : O O 9:oo 1o:oo 11:oo 12:00 13:OO 14:OO 15:OO

Algal ~oncn. /mm.~

mg. COJ. mg. C02/1. mg. COJL mg. CO2/l. mg. 02/1.

1900 2080 1950 200

0 277.5 188.7 188.7 179.8

33.3 222.0 178.0 144.3 111.0 111 .o

22.1 199.8

111.0 111.0 99.0 22. I

-

32.0 205.0 166.5 155.4 138.0 88.8

10.8 11.2 11.8 14.4 14.8 12.0

13.6

TABLE I1 Change in GO2 and (1% Content of Soiution in Tanka

Change in GO:! content of solution, mg./l.a

Time, hr. Oct. 26-Nov. 3, 1961 NOV. 9-Nov. 17, 1961

8:3& 9:00 9: 00-1 1 : 30

11:3&12:00 12 : W 1 4 : 30 14:30-15:00

+ 28 - 19 +23 -21 +21.6

+ 24 - 20 + 18 - 19 - 20

Change in O2 concentration, mg./l.b

Oct. 26Nov. 17 9:0&12:00 +5.1

12:OO-15:OO +2.7

Data obtained from analysis of solution a t different time. 70 l./hr. C02 supplied 8:3&9:00, 11:3Q-12:00, and 14:3&15:00 (Figures show increase or decrease in GO2 content of solution during periods indicated).

Maxim. concn. at 15:00,26 mg./l. Range: 14.4-26.2 mg./l. Minim. concn. at 9:00, 14.4 mg./l. Range: 14.4-17.8 mg./l.

phere occur. Attempts to introduce GOz at different rates were therefore made. Even if CO, was introduced a t a rate of 35 l./hr., continuously for 6 hr., its level still remained too high. It was therefore decided to supply pure GO, in a different way. GO, was metered into the tank for 70 min. at a rate of 70 l./hr. 3 times a day, between

BIOTECHNOLOGY AND BIOENGINEERING, VOL. VI, ISSUE 2


8 : 30-9 : 00, 11 : 30-12 :OO, and 2 : 30-3 :OO. In later experiments during the summer of 1963 when sunlight periods were very long, four doses were given. Typical figures for changes in dissolved gas are shown in Table 11. This method kept the C02 content in a range suitable for algal photosynthesis. Oxygen content showed little de- viation from that of the solution when it was equilibrated with air without algae. Apparently oxygen is liberated quantitativeIy into the air, while C02 supplied is absorbed almost quantitatively.

Effect of Stirring Speed

The effect of stirring speed on algal yield was determined in the period October-November 1961, Table 111. It will be seen that yield increases with stirring speed. CO, utilization also increases, as calculated from dry production and C02 supplied (1.8 g. COz should give 1 g. algal dry matter). As stirring speed increased, oxygen was expelled from the solution, i.e., its oxygen content decreased somewhat.

Table 111 Dependence of Yield from Culture Unit

on Stirring Speed (October-November 1961)

Stirring speed, r.p.m. 38 70 90 120 Daily yield 35 38 50 50 Efficiency of CO2 utilization % Algal material produced

COz supplied 34.2 35.8 43.2 46.0

Occasionally serious difficulties were encountered, due to the precipitation of salts in the tank. This precipitation appeared at what seemed to be normal operating conditions and at normal pH, around 6.5. Extensive experiments were made to try and define the conditions under which this occurred. Culture solutions were made up with different sources of Nz, urea, NOS-, NH4+, and stood in the light indoors, in sunlight, and in the dark. The concentration of the salts was also varied. It was quite clear that precipitation was light induced, and occurred most strongly in the light and was almost absent in the dark. The source of N, was of no importance. During precipitation there was no change in pH. The precipitates contained N2, P, Mg, and probably also microelements. Although we were unable to find the cause or mechanism of this light-induced precipitation, it

178 MAYER ET AL.

was found by trial and error that reduction of the salt concentration to a half of that originally used largely prevented precipitation and this procedure was therefore used in later experiments.

Effect of Temperature Temperature played an important part in the yield obtained from

the culture unit. A “mean” temperature was calculated from the temperatures of the unit a t 9.00, 12.00, and 15.00 and this was related to the yield on the different days. These data are shown in Figure 2.

a 0

I I 1 1 I I I I I I 1 18 19 20 21 22 2 3 2 4 2 5 2 6 27 2 8 2 9

TEMPERATURE OC

Fig. 2. Effect of temperature on daily yield of algae, a t different stirring speeds.

This figure indicates the importance of the temperature in determining yield. In this, the effect of “mean” temperatures on the daily yield is shown in the period from April 15, 1962 to June 10, 1962. This period can be broken down into two sets, both from the point of view of stirring speed and from the point of view of temperature dependence. In the period April 15- May 10, 1962, the predominant organism was a fairly large Chlorella, presumably C. vulgaris as originally inoculated. In the second period, May 11-June 10 this was gradually replaced by a smaller Chlorella,

This is further indicated by Figure 3.

BIOTKCIINOLOGY .\ND B1Ol3NGINJ3EKING, VOL. V I , LSSUE 2

MASS CUT1TUR.E OF ALGAE 179

60 rpm(11/5-10/6/1962 1 0 120rpm(15/4-10/5/1962)

0 0

60 -

\. 19 20 21 2 2 23 2L 25 26 27 28

TEMPERATURE OC

Fig. 3. Effect of temperature on daily yield of algae showing selective effect of temperature.

presumably C. pyrenoidosa, which showed a temperature optimum some three degrees higher thaii C. vulgaris. Clearly, if the cultures are harvested daily, a very strong tendency prevails toward selection for the most rapidly dividing and growing organism. An open culture, no matter what organism is originally inoculated, will lead to the development, by selection, of a monoculture or culture with a very limited number of species, which is best adapted to the culture conditions prevailing. An idea of the actual yield obtainable from the 2000 1. unit over a period of almost 2 years is given in Table IV.

Clearly, stirring speed, temperature, and possibly light intensity all play an important part in determining yield.

Effect of Stirring on Exposure to Light

In order to determine what part stirring played in exposing the cells to the light, an empirical approach was used. Ping-pong balls were filled with water, so that they had a specific gravity equal to that of the algal suspension. They were suspended in the tank and this was stirred a t various speeds. The period of time the balls were at the top and south illuminated surface was determined with the aid of a stop watch. It can be seen from this table that stirring speed and arrangement of paddles affects the number of appearances of the algae at the light-exposed surfaces and their length of sojourn there. Only a depth of about 10 cm. from the surface was considered in the experiments. The number of exposures

The results are shown in Table V.

TA

BL

E I

V.

Yie

lds

of D

ry A

lgae

/Tan

k at

Diff

eren

t Pe

riods

and

Dif

fere

nt S

tirri

ng S

peed

Dai

ly

Stir

ring

spe

ed

Perio

d sp

ecie

s gn

i. pa

ddle

pos

ition

m

ediu

m

Rem

arks

C

ultu

re

Alg

al

yiel

d,

r.p.

m.,

and

i

m

0

1961

O

ct. 3

-0ct

. 23

8 O

ct. 2

6-N

oV.

17

Nov

. 19

-Dec

. 3

0

+I

Z

Dec

. 19

, 196

1-Fe

b. 2

8 5

Feb.

21-

Mar

ch 1

1 s

Apr

il 15

-May

108

0 * +

May

11-

May

16

z C E o

May

17-

June

10

2 Ju

ne 1

7-Ju

ne 2

2 3

June

24-

July

3

Aug

. 9-A

ug.

16"

Aug

. 26-

Sept

. 4

1962

E E 2 Se

pt. 1

3-Se

pt.

19

r 2 O

ct. 4

-Oct

. 10

Aug

. 17-

Aug

. 24

51 3 O

ct. 1

7-O

ct. 30'

m N

ov.

15-N

ov.

30

C. v

ulga

ris

C. v

ulga

ris

C . v

ulga

ris

C. v

ulga

ris

C. v

ulga

ris

C. v

ulga

ris

C. v

ulga

ris

C. p

yren

oido

sa

C. p

yren

oido

sa

C. p

yren

oido

sa

C. p

yren

oido

sa

C. p

yren

oido

sa

C. p

yren

oido

sa

C. p

yren

oido

sa

C. p

yren

oido

sa

C. p

yren

oido

sa

C. p

yren

oido

sa

and

C. v

ulga

ris

50

50

18

21

34

35.8

29.3

38.0

55

43

63

51

55

53

46

33

54

60/p

aral

lel

9Olp

aral

lel

120/

para

llel

12O

/par

alle

l 12

0/pa

ralle

l 12

0/pa

ralle

l

60/p

aral

lel

60/p

aral

lel

120/

para

llel

6O/p

aral

lel

120/

para

llel

No

stir

ring

9 O

lpar

alle

l 9l

)/par

alle

l

6O/p

aral

lel

90/p

aral

lel

9O/s

pira

l

Stan

dard

St

anda

rd

Stan

dard

Stan

dard

St

anda

rd

Stan

dard

Stan

dard

Stan

dard

Stan

dard

St

anda

rd

Stan

dard

St

anda

rd

Stan

dard

St

anda

rd

Stan

dard

Stan

dard

Stan

dard

Tem

p.:

12-2

0'C

. du

ring

da

y

Tem

p.:

15O

C.

Tem

p.:

20°C

. T

emp.

: 19

-25°

C.

10

3

Tem

p.:

23-2

9°C

. 2

days

23°

C.

days

bel

ow 2

2°C

. 2- 2

3 da

ys 2

9OC.

H

T

emp.

: 21

-31%

. 2-

N

M

12 d

ays

abov

e 25

°C.

?

Av.

dai

ly te

mp.

: 25

OC.

A

v. d

aily

tem

p.:

27°C

. A

v. d

aily

tem

p.:

28°C

. A

v. d

aily

tem

p.:

29°C

. A

v. d

aily

tem

p.:

28°C

. A

v. d

aily

tem

p.:

28O

C.

Av.

dai

ly te

mp.

: 28

OC.

Av.

dai

ly t

emp.

: 25

'C.

Av.

dai

ly t

emp.

: 23

OC.

cone

s ad

ded

cone

s ad

ded

salt

ppt

.

1963

D

ec. 2

4, 1

962-

Jan.

28%

Feb.

19-

Mar

ch 5

8

Mar

ch 1

0-M

arch

31

Apr

il 10

-Apr

il 25

"

Apr

il 26

-May

14

May

24-

June

8'

June

9-Ju

ne

18

Jun

elg

Jun

e 28

July

19-

JUly

298

C. v

ulga

ris

C. p

yren

oido

sa

C. p

yren

oido

sa

C. p

yren

oido

sa

and

C.

vulg

aris

and

C.

vulg

aris

C

. pyr

enoi

dosa

an

d C.

el

lipso

idea

C.

pyr

enoi

dosa

an

d C

. el

lipso

idea

C.

pyr

enoi

dosa

an

d C.

el

lipso

idea

C

hlam

ydom

onas

an

d C

. py

reno

idos

a

C. p

yren

oido

sa

27

34

27

38

29

54

54

49

50

W/s

pira

l

90ls

pira

l

90ls

pira

l

80/p

aral

lel

80/p

aral

lel

80/p

aral

lel

8O/p

aral

lel

80/s

pira

l

80/p

aral

lel

a Fr

esh

nutr

ient

med

ium

pla

ced

in t

ank.

Con

cn.

l/Z

stan

dard

C

oncn

. st

anda

rd,

urea

as

N2 s

ourc

e

stan

dard

, ur

ea a

s N

2 sou

rce

stan

dard

Con

cn.

Con

cn.

Con

cn.

stan

dard

Con

cn.

'/?

stan

dard

Con

cn.

stan

dard

Con

cn.

stan

dard

Stan

dard

Av.

dai

ly t

emp.

: 25

°C.

Av.

dai

ly t

emp.

: 26

OC.

sa

lt p

pt.

Av.

dai

ly t

emp.

: 26

°C.

Av.

dai

ly t

emp.

: 20

OC.

co

nes a

dded

Av.

dai

ly t

emp.

: 21

OC.

Av.

dai

ly t

emp.

: 25

°C.

80 1

. CO

Z per

day

Av.

dai

ly t

emp.

: 26

°C.

140

1. C

OZ p

er d

ay

Av.

dai

ly t

emp.

: 28

°C.

Av.

dai

ly t

emp.

: 28

OC.

182 MAYER ET AL.

at the illuminated surfaces increases between 60 and 90 r.p.m., but did not differ much between 90 and 120 r.p.m. In other words, up to 90 r.p.m. the number of alternations between light and dark increased. For the parallel arrangement of the paddles this was accompanied by a slight decrease in the total time of exposure to the light, while it increases for the spiral arrangement. In addition, in both cases the relative exposures a t the top and side are changed by stirring speed, having a peak at 90 r.p.m. for the spiral arrangement and progres- sively increasing for the parallel arrangement, Table V.

TABLE V The Period of Time and Number of Times That a Ping-Pong Ball Was Present at the Illuminated Surfaces of the Large Tank, with Different Stirring Speeds

and Paddle Arrangements

Paddle Arrangement

Spiral Angle between

Length 65 cm. Distance between paddles 50 cm.

Normal, parallel. Distance half-paddles 60". between paddles, 50 cm.

Paddle length, 65 cm.

Stirring speed, r.p.m. 60 90 I20 60 90 120 Number of exposures/

min. a t illuminated surface 11 15 17 12 17 17

Exposure time in ser. /minute 22 16.5 1 1 . 0 28.8 35.7 34.0

Ratio of exposure time, 0.22 0.48 0.56 0.45 0.78 0.55

toD surface South side

Effect of Light

If these results are considered in conjunction with Table VII it appears that most satisfactory yields are obtained a t stirring speeds of 90 r.p.m. and that the spiral arrangement is particularly desirable when light is limiting. Light intensity at certain periods of the year might limit growth even at the high light intensities prevalent in Jerusalem. An attempt was made to introduce more light into the tank using cones, constructed from translucent perspex (Diffusing Opal 030, I. C. I. Ltd). Three such cones were introduced as shown

BIOTECHNOCOGY AND BIOENGINEERING. VOL. VI. ISSlJE 2


INTRODUCED INTO TANK

AND LIGHT INTENSITIES

MEASURED IN AND ABOVE

TANK ON 31.7.63

SCALE 0 20 40 6Ll cm

- 1 1

3 L -2 I

I

I - 22000 FC.

.2 - 10.000 FC.

3 - 700 FC.

b - 460 FC.

5 - 110 F.C.

TOP V I E W W I T H CONES

- I

6- 1 5 I I I

TOP VIEW

VIEW OF CONE

1:ig. 4 Const,riirtion of cones introduced into tank and light intensities measured in and above tank on July 3 1 , 196.7.

184 MAYER ET AL.

in Figure 4. Light intensities were measured in July 1963, and are shown in Figure 4. In April 1963 these cones somewhat improved yield, compared to May 1963. However, in September-October

130

120

110

100

90

C .- 5 80 -I _I t X a p 70 9 X 0

60

2 a

50

40

30

//’- _ - - p------

/ 0

CH. FVRENOIDOSA

CH. VULGARIS

CHLAMY DOMONAS

CH. ELLIPSOIDEA

0 200 400 600 800 1000 I200 I400 Id00 Id00 2dOO LIGHT INTENSITY F: C.

Fig. 5. Specific rates of photosynthesis of various algae at different light intensities.

BIOTECHNOLOGY AND BIOENGINEERING, VOL. V I , ISSUE 2


1962, when light is abundant, they had no effect on yields (Table IV). Clearly, during this latter period the cones do not make a significant contribution to light energy entering the tank.

Among these were Chlorella ellipsoidea and Chlamydomonas snowiae in addition to C. vulgaris and C. pyrenoidosa already mentioned.

Although the C. ellipsoidea appeared very promising in small scale experiments, it was supplanted by C. pyrenoidosa in the large tank. The same was true to a lesser extent also for Chlamydomonas, but this was able to maintain a steady state of equilibrium with C. pyrenoidosa.

It seemed probable that this behavior was correlated to the photosynthetic efficiency of these various organisms. Photosynthesis was therefore investigated, the results being corrected for respiration. The relative rates of photosynthesis of four algal species are shown in Table VI. The results are given on the basis of rate/mg. chlorophyll. On a cell number basis, results cannot be compared because of enor- mous divergences in cell size. Since it was suspected that differences would be marked even at lower light intensities, photosynthesis was compared a t different light intensities (Fig. 5 ) .

A number of algal species were tested in mass culture.

TABLE VI Relative Photosynthetic Rates of Various Algae a t 2000 ft. c., at 25°C.

~~

Relative photosynthetic rate

pl. Oz/min./mg. Algal species pl. O2/min./1O8 algae chlorophyll

Chlorella vulgaris 8.80 f 3.0 51.27 f 14.00 Chlorella ppenoidosa 9.49 f 3.5 100.71 i 30.0

Chlamydomonas 17.04 f 0.16 132.2 f 3.0 Phueodactylum 2.05 f 0.17 32.8 i 12.0

Chlorella ellipsoidea 0.97 f 0.05 99.0 f 20.0

It can be seen that while C. vulgaris and C. ellipsoidea are beginning to be light saturated at around 600 ft. c., Chlamydomonas and C. pyrenoidosa had photosynthetic rates which increased linearly up to 2000 f t . c. without indication of saturation. Thus the data indicate that at high intensities Chlamydomonas and C. pyrenoidosa have a clear advantage over the other species from a point of view of growth potential, assuming that photosynthesis is a factor for growth. In

I86 MAYER E T AL.

fact, these two species occurred in the tank particularly during the summer months, when light intensity is high. In the winter C. vulgaris seemed to be the most suitable organism, existing almost in mono-algal cultures. C. ellipsoidea was not tested during the winter months, but according to prediction and provided it stands low temperatures, it should be even more suitable than 6. vulgaris in winter.

It is interesting to note that competition between algal species could also be demonstrated in small scale experiments. Mixed cultures were sown in Roux bottles in the greenhouse. They were allowed to grow up and then harvested daily by replacing half the medium by fresh medium. Here again the same trends found in the mass culture were essentially confirmed.

Bacterial Contamination

The question of contamination of a large culture of algae by bacteria is one which has occupied many workers, particularly if the algal product is to be used directly. Bacterial counts were estimated and an attempt was made to correlate them with algal counts. For considerable periods of time it was found that bacterial counts maintained a steady state at a count of around 2 X 106/ml., i.e., about the same order of magnitude as the algal count. The bacteria rapidly reached a steady state, and showed no tendency to increase (Table VII). The bacterial count was not reduced appreciably by centrifugation.

TABLE VII Average Bacterial Counts in the Large Tank During Different Periods

Bacterial count in Period 1 rr. culture medium Remarks

Jan. 21, 1962-Feh. 19, 1903 2 6 x 106 No harvesting Feb. 19-March 5, 1963 4 15 X lo6 March 1O-March 31, 1963 2 38 x 1 0 6 April 1-April 10, 1963 1 i 8 X 1 0 6 No harvesting April 1O-April 25, 1963 3 4 x 106 April 26-May 14, 1963 4 22 x 106 May 15-May 23, 1963 1 8 x 106 iXn harvesting May 24June 8, 1963 2 34 x 106 June 9-June 18, 1963 2 43 x 106 June 19-June 28, 1963 1 84 x 106 June 29-July 18, 1963 2 5 x 106 July 19-July 29, 1963 1 3 x 106

RIOTECHNOLOGP AND RIOENCINEERING. VOL. VI, ISSUE 2


Although the bacteria were not identified, the majority of the colonies were small and pigmented. Apparently the vast majority were Micrococci with the occurrence of occasional Streptococci. Patho- genity of the aIgae was not examined.

DISCUSSION

Perhaps the most important feature of this work is that i t has been possible to operate continuously with the same nutrient medium, for a period of a few months at a time, a culture unit containing some 2000 1. algal suspension. This culture unit is extremely simple to operate and could be made fully automatic if desired. It gives rise to very few technical snags. The salient features of this unit are that both the top and a south facing wall are transparent and that the depth of liquid is 1 meter, the whole being vigorously stirred.

Because of lack of hydrodynamical data, the stirring problem was approached empirically, in order to determine various effects of stirring. The effect of stirring on the gas balance of the tank was studied and was found to be small. Stirring did, however, influence the yields obtained. The optimal yields obtained from the tank were ;),)-60 g. dry matter per day when the tank was harvested 6 days out of 7 and the results calculated for 7 days (Table IV). This corre- sponds to a yield of about 13 g. per square meter illuminated area or 26 g. per square meter of surface area occupied by the tank. On a daily harvesting basis, yields would increase by approximately 15%. This is in full accord with our previous experience of this unit (Mayer et al.”). The exact yield, as mentioned, was affected by the stirring pattern and by the stirring speed. Analysis of the results is compli- cated by two factors. As the temperature of the tank changes so does its algal population change. A rapid process of selection operates because of the continuous harvesting of the tank. Consequently the species of algae best adapted to a given set of conditions of temperature and light intensity is selected and this species establishes itself. This principle was also confirmed by small scale growth experiments. Consequently, at different periods of the year different species will be dominant. Furthermore, light intensity also changes throughout the year. Different algal species become light saturated at different light intensities (Fig. 5 ) . Clearly, a t high light intensities, species with high light saturation will be favored, while at low light intensi-

- _

158 MAYER ET AL.

ties this will not be the case. From the photosynthetic studies we were able to confirm this and by applying these results to the finding in the large tank it was to some extent possible to account for varia- tion of algal populations through the year on this basis. At high light intensities, C. pyrenoidosa occurring wild in Jerusalem is the favored organism and this organism is further favored by having slightly higher temperature optimum than the other Chlorella species. Chlamydomonas would also be favored by high light intensities, but suffers from the high temperatures associated with high light intensities. At low iight intensities, probably C. vulgaris and C. ellipsoidea are the most favored organisms. In order to understand the light factor better, cones were also introduced into the tank, in order to diffuse light into it. These were first suggested by Evenari et al.4 and further discussed by Myers and Graham.14 The latter suggested that such cones might assist in overcoming light saturation. This suggestion seems likely to us, as in the winter months the cones did promote growth somewhat. In summer, with high light intensities, selection favored species saturated by high light intensities, so that practically no species with low light saturation were present in the culture. It appears likely therefore that cones as an adjunct to efficient stirring, of the right kind of turbulence, can only assist in mass cultures under very special conditions. The right kind of stirring, as can be seen from the foregoing wilI again be different for different algal species which differ in their requirement for total exposure to the light and the light intermittency. The empirical experiments showed that under any given set of conditions, changing the stirring speed and consequently total exposure and length of individual exposure did in fact influence yields from the tank (Tables IV and V). It was possible to establish for a given species what appeared to be optimal conditions of stirring, so that stirring no longer constituted a limiting factor in growth.

A further factor which we should consider is the question of carbon dioxide supply. Xayer et al." reported for the first time the use of an intermittent supply of pure COz. AIeffertI3 studied this further and confirmed the usefulness of the method. In the present work it was shown that the COz supply can be greatly reduced without affecting yield. Pure COz was metered out automatically a t timed intervals in small amounts, so as to maintain the dissolved COz at a more or less constant desirable level, optimal for the algae. By such means a

BIOTECHNOLOGY AND BIOENGINEERING, VOL. VI. ISSUE 2


considerable saving of gas can be achieved and conditions optimal for growth attained. Such conditions need not be identical for all algal species. The method of stirring used, together with fine diffusers, results in almost complete absorption of the gas and a very high efficiency of utilisation can be achieved. Oxygen is expelled almost quantitatively into the ambient atmosphere.

Although bacteria were present in the culture their number did not reach high numbers. Apparently under the given conditions, bacterial infection would not be a serious complicating factor.

From the foregoing discussion a number of general conclusions about the mass culture of algae by the deep culture method can be drawn. If the culture is an open one, then despite massive inoccula- tions, any local species of alga particularly favored by the conditions for growth will become established. The algal population will change as conditions change, but a fairly constant bacterial population will be present. In addition to nutrient substances in the solution, temperature and light intensity will determine what species becomes established. Different Chlorella species differ greatly especially with regard to light requirement. The light factor can be altered or modified to some extent. Either diffusing cones can be introduced into the tank which is fairly expensive, or more easily and effectively the stirring pattern and speed can be altered. The latter will change both dark-light intermittency and total sojourn in the light. Condi- tions for each algal species must be established experimentally. The overall process of mass culture can be vastly improved by metering out pure COz into the tank. In future work it should be possible to maintain any desired level of COZ in the solution automatically without waste. A search for more suitable algal species for mass culture did not bring to light any species which looks better than Chlorella. The search and screening can be done very simply by checking photosynthetic efficiency using a Warburg technique together with simple growth experiments in Roux bottles in which the algae are harvested regularly. This technique was used here and checked against large scale experiments. No new suitable algal species were found. The marine alga Phaeodactylum was found to be unsatisfactory under our conditions. Apparently it is not very efficient photosynthetically.

Our thanks are due to Messrs. E. Harel, M. Marcus and P. Dror, who par- ticipated in various parts of this work.

190 MAYER ET AL.

The research in this paper has been sponsored by or in part by, Cambridge Research Laboratories, O.A.R. through the European Office, Aerospace Re- search, United States Airforce, under contract No. AF 61(052)-546.

References 1. Burlew, J. S., ed., Algal Culturo from Laboratory to Pilot Plant, Carnegie

Institute of Washington, Publication 600, 1953. 2. Carpenter, E. F., ed., Translations of the Conference on the Use of Solur

Energy, The Scientijc Basis, Vol. 4, Univ. Arizona Press, Tucson, 1955. 3. Conway, E. J., Microdiffusion Analysis and Volumetric Error, Crosby

Lochwood, London, 1957. 4. Evenari, M., A. M. Mayer, and E. Gottesman, “Experiments in the

Culture of Algae in Israel,” in ref. 1. 5. Gafford, R. D., and D. E. Richardson, J . Biochem. Microbiol. Technol.

Eng., 2,299 (1960). 6. Gaucher, T. A., R. J. Benoit. and A. Bialecki, J . Bwchem. Microbiol.

Technol. Eng., 2,339 (1960). 7. Kachroo, P., ed., Proceedings of the Symposium on Algology, ICAR, New

Delhi, 1960. 8. Kanazawa, T., C. Fujita, T. Yuhara, and T. Sasa, J. Gen. A p p l . Microbiol.,

4,135 (1958). 8a. Kolthoff, M., and R. Belcher, Volumetric Analysis, Vol. 3, Interscience,

New York, 1957, pp. 278-279. 9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J . Biol.

Chem., 193,265 (1951). 10. Mayer, A. M., “Achievements and Problems in the Mass Culture of

Algae,” in Proceedings of the Symposium on Algology, ICAR, New Delhi, 1960, p. 167.

11. Mayer, A. M., A. Eisenberg, and M. Evenari, Sci. Monthly, 83,198 (1956). 12. Mackinney, G., Biol. Chem., 140,315 (1941). 13. Meffert, M. E., Zur Methodik der Freilandkultur einzelliger Griinalger und

Vorschlag eines neuen Kulturuerfahrens, Forschungsberichte des Landes Nord- rhein-Westfalen, Nr. 716, 1957.

14. Myers, J., and J. R. Graham, Plant PhyswE., 36,342 (1961). 15. Oswald, W. J., Am. J . Public Health, 52,235 (1962). 16. Pipes, W. O., Appl . Microbwl., 10,281 (1962). 17. Placak, 0. R., and C. C. Ruchhaft, Ind. Eny. CherrL. A4nn. Ed., 13, 12

18. Sorokin, C., Nature, 194,496 (1962). 19. Tamiya, H., Ann. Rev. Plant Physiol., 8,309 (1‘357). 20. Technical Documentary Report, No. AMRL-TL)R-(iS-ll(i, Biologistics

( 1941 ).

for Space Systems, 1962.

Received December 7, 1963

BIOTECHNOLOGY AND BIOENGINEERING. VOL. VI. ISSUE 2

problems of design and ecological considerations in mass culture of algae

Documents