BIOTECHNOLOGY AND BIOENGINEERING

VOL. IV, PAGES 377-390 (1962)

Mean Light Intensity-A Useful Concept in

Correlating Growth Rates of Dense Cultures
of Microalgae

A. E. RABE* and R. J. BENOIT, Research and Development, General

DynamicslElectric Boat, Groton, Connecticut

Summary
An analysis of the distribution of light within some culture vessels developed at.
General Dynamics/Electric Boat is presented. The concept of a mean effective
light intensity, E , is introduced and procedures are developed for computing i?
for any culture vessel geometry and incident light intensity distribution. A
correlation is performed indicating the significance of E in assessing the growth
rate of dense, continuous cultures of microalagae.

INTRODUCTION
Photosynthesis and growth of microalgae, especially strains of
Chlorella, have been studied for many years using dilute cultures
and low light intensity. The geometry of the culture vessels used
in fundamental studies was freely changed to accomodate experi-
mental goals. By 1953, however, the practical application of mass
culturing of microalgae was under intensive investigation.' The
study of continuous cultures of high equilibrium density involves
special problems of engineering design and the geometry of the appara-
tus used has become relatively complex. This paper presents an
analysis of the distribution of light within some culture vessels
developed at General Dynamics/Electric Boat laboratories, and of
the effect of light distribution upon the growth rate of steady-state
cultures. The growth rate is dependent upon other factors, of
course, but it is felt that the composition of the medium, the carbon
dioxide supply, and the temperature were optimal for the strain of
* Present address: Engineering Experimental Station, E. I. duPont de
Nemours & Co., Inc., Wilmington, Delaware.
377
378 A. E. RABE AND R. J. BENOIT

Chlorella2 used. The degree of stirring or mixing achieved was

different in the various culture vessels discussed here. We believe
that this difference was not significant however, even though it is
possible that turbulence was suboptimal. Furthermore, there is
no evidence that the culture vessels included in this analysis contained
any materials of construction toxic or inhibitory to Chlorella. Lastly
it should be noted that the growth rates used in the analysis are the
best rates that could be achieved in extended factorial programs with
the various culture vessels. This is not to say that the growth rates
achieved are the best ever shown for Chlorella pyrenoidosa 71105,
for Sorokin3has shown much more rapid growth of the strain in dilute
suspensions of synchronized cultures; rather, the growth rates
achieved are characteristic for steady-state continuous cultures
of Chlorella diluted at a rate which gives maximum yield for the
culture vessels in question.

DISCUSSION
In moderately dense cultures, i.e., cultures of concentration greater
than 0.1% packed cell volume* the attenuation of light intensity
is rapid. It has been estimated4 that incident light intensity of
30,000 footcandles diminishes to 250 footcandles in 10 cm. of a 0.1%
PCV culture; similar incident and exiting light intensities are
achieved in a culture depth of about 1.5 cm. when the cell concentra-
tion attains 1% PCV. In general, where the design of practicable
culture vessels is of concern, the incisive queston becomes: What
efective light intensity prevails in the culture? This effective light
intensity should be the time averaged luminous flux density (il-
lumination) to which algal cells throughout a continuous culture
are subjected. Defining this intensity as the mean light intensity
( E ) ,it is reasonable to expect a correlation between algae prolifera-
tion, henceforth called culture growth rate (GR-I), and I?, for cultures
not limited by other factors.
GR = f(E) (1)
where GR is the generation time of a continuous culture in hours,
i.e., the time required for the culture to double in algal cell concentra-
tion. The reciprocal of this value, which is used in the correlation,
*For Chlorella, 0.1% PCV corresponds roughly to 250 mg. dry matter per
liter of culture.
BIOTECHNOLOGY AND BIOENGINEERING, VOL. I V , ISSUE 4
 MEAN LIGHT INTENSITY 379

is the “number of doublings” occurring per hour. is the mean light

intensity, a space and time averaged illuminance in footcandles.
n’eglecting, for the moment, the fact that certain wavelengths
of visible light are attenuated more rapidly in an algal culture in
accordance with well-known absorption spectra, it is desired t o
derive a n equation expressing the following relationship :
= f(Eo,l,c) (2)
where Eo is the incident illuminance on the lighted surface of the
culture vessel in footcandles; I is the characteristic path length of
the light energy passing through the culture in inches; c is the algal
cell concentration (percent packed cell volume).
Beer’s Law may be cited as an example of a fundamental relation-
ship which can be used to calculate E .
OD = - log(I/Io) = E ~ C (3)
where OD is the optical density (dimensionless) and I is the luminous
intensity a t any depbh, I, in the culture in lumens per steradian.
I is defined a t d F / d W , i.e., the incident luminous flux emanating
from a point source and measured over unit solid angle, W . This
unit is also called one candle. The expression “candle power,”
still in common usage, is to be avoided as iuminous intensity is
not to be confused with power. lois the luminous intensity a t
1 = 0 in lumens per steradian and E is the extinction coefficient (spec-
tral effects, ex, not considered), (l/mm.) (l/%l’CV).
From elementary physics it can be demonstrated that E is related
t o I by:
E = d F / d A = I COS(O)/T’ (4)
where E is the illuminance; the incident luminous flux falling on the
area, d A , in footcandles; 0 is the angle of incidence of the source
normal to the area, dA; r is the distance from the point source of
light t o the center of area d A , in inches.
Introducing equation 4 into equation 3, for the case of orthogonal
and collimated or diffuse light, and writing in exponential form:

where e is the natural logarithm base 2.7183. . .

The mean light intensity, B, can now be calculated for certain
culture chambers having simple geometries. For example, in the
case of a concentric annular culture chamber (neglecting end effects),
380 A. E. R A B E A N D R. J. B E N O I T

with light supplied in the center tube, the significance of Eo and 1

are quite clear. The incident light can be considered to be collimated
and the distance 1 becomes the annular spacing. Where irregular
culture and/or lamp geometries are encountered, estimates of the
“average” incident intensity and of the average effective light path
length must be made. I n the analysis presented here, systems were
broken up into zones to obtain estimates of Eo and 1.
A typical calculation for a simple geometry is presented. The
algal culture is housed in a rectangular tank; the incident light is
orthogonal and collimated.
The illuminated surface of the culture is designated lo,and the oppo-
site surface of the culture is designated L. The concentration of cells
in the culture is designated c. Mean light intensity, E, is defined
by the following equation:

Assuming: (1) validity of Beer’s Law in describing light attenua-

tion in a culture, ( 2 ) the incident light intensity, Eo, is uniform, (3)
the algae concentration, c, is constant in the continuous culture,

- Eoe-2.3rzc/2.3(L - Eo)ec 1 L

lQ
(7)
It is reasonable to expect that a correlation of culture growth rate,
GR-I, as a function of El exists for nonlimited cultures subjected
to light intensities below inhibiting i n t e n ~ i t i e s . ~ ,(The
~ “solarizing”
intensity is the maximum level of illuminance to which cells may be
subjected without inhibiting g r o ~ t h . ~Except
,~ a t very low culture
densities, this value varies with algal cell concentration. For Chlorella
pyrenoidosa 7 1105 in reasonably dense cultures, the discussion places
this value at 30.000 foot candle^.^) The correlation has been calcu-
lated in a somewhat qualitative sense, and is presented and discussed
in the next section. A few preliminary remarks on the applicability
of this concept should be presented first.
I n all except extremely dilute cultures, the validity of Beer’s
Law is questionable.4 For moderately dense cell suspensions, de-
finitive experimental data are required to establish the actual degree
of light attenuation occurring. Information on optical density (OD)
BIOTECHNOLOGY A N D B I O E N G I N E E R I N G , VOL. IV, ISSUE 4
 MEAN LIGHT INTENSITY 381

versus culture depth, obtained for a number of concentrations,

can be correlated analytically to obtain expressions which function
as Beer’s Law in the above calculation. It is not improbable that
several expressions, each valid over certain specific depth and/or
concentration zones would be obtained. Obtaining actual light in-
tensity measurements with a linear-response photocell for a number
of culture thicknesses would circumvent problems arising as a result
of complex spectral effects.
An interesting question arises with regard to the applicability
of the correlation to any particular fluid particle. Specifically,
the question may be stated: For a fluid element of algal culture
randomly selected at time, to, what minimum time must elapse before
the time-averaged light “seen” by the element falls in the range
0.9 < I? ’< l.lI?, with, e.g., 90% probability? To derive the prob-
abilistic. formula required to solve this problem, lacking more de-
finitive data concerning the (average) path length of the fluid element
and its (average) velocity, it might be assumed that these are inde-
pendent, and further, that the fluid element can rebound in any
direction (three dimensional freedom, with confining walls) with equal
probability. Although the problem is beyond the scope of this
treatment, sufficiently convincing qualitative information has been
obtained to indicate that this minimum time is of the order of seconds,
perhaps fractions of a second in well-stirred cultures. For example,
in a culture vessel (where AZ = ll/z in.) specifically designed for
intense agitation, peripheral eddy velocities of about 30 ft./sec. have
been measured. Assuming reasonable time for eddy break-up, it
can be concluded that the vast majority of algae will be ‘Iseeing)’
a light intensity not far removed from i?, within fractions of a second.
Even in unstirred cultures, where rising gas bubbles are relied upon
to mix the culture, gas bubble momentum, imparted to the culture
fluid accomplishes thorough exchange of the fluid medium between
vessel walls in a very few seconds. Specific effects of turbulence are
under detailed study by Professor Henry Tsuchiya and colleagues
at the University of Minnesota.

CORRELATION
General Dynamics/Electric Boat has been engaged in the study of
photosynthesis as related to algal cell propagation since 1956.4,7,9,10
Much of the experimental program has dealt with the study of dense
(e > 0.1% PCV = 250 mg. dry cells/liter) continuous cultures of
 w
TABLE I n
w
CONTINUOUS CULTURE DATA ON CHLORELLA 71105 GROWN IN PHOTOSYNTHETIC
GAS EXCHANGERS AT GENERAL DYNAMICS/ELECTRlC BOAT DIVISION

Data
-

P
3 0.01 14 0.45 1.125 50, OM) 1,lSO
3 0.04 0.29 725 50.000 1,490
F
3 0.071 0.23 575 50,OM) 1.680 Pc:
2 0.0971 0.19 475 50. OM) 1,850 tr:
>
z
Unit No. 1 (Code: 0 )The flcurescent lamps are concentrically housed in a tier of five water coolant
u chambers. The overall assembly is approxlmately 1' x I' x 2.5' hlgh.
Front Slde

W
M
Z
4 0.0746 0.14 350 7.300* 1,700

e 4 0.0746 0.25 625 14,OM)* 1.960

0
r
4 0.0746 0.29 725 21.600* 2,690

c Unit No. 2 (Code: A1 The s t a k of incandescent lamps are mounted m a r the radial axlr of a dome
r
n
m shaped glass retainer. The cylindrical assembly is approximately 2' In dlometer ad 2 1/2' hlgh.
C
m
'SURFACE-AVERAGED VALUES
 PropagationRate lncldent Llght Mean Llghl
Of Culture Culture Denrlty ( c ) lntenslty lntenslty IE)
NO. Of IGR-1)
Runs (vol/vol-hr) 1% PCV) [mgs [dry) cells/llter) (footcandler ) (footcandles]

4 0.05 0.78 1,950 8. ooo 1.160

5 0.1 0.34 850 8,000 2.120

1 0.1 5 0.04 100 8. ooo 4.61 0

1 0.0247 1.9 4,750 8. ooo 590

4 0.04 0.83 2,080 8. ooo 903

4 0.071 4 0.63 1,580 8. OOO 1,120

4 0.1 0.31 775 8. OOO 1,850

Front 4 0.04 0.41 1.030 8. OOO 1,070

4 0.0714 0.29 725 8. OOO 1,350

4 0.1 0.12 300 8. OOO 2.630

Unit No. 3 [Code: 8) A 1500-watt Incandescent lamp Is concentrically housed In a water coolant
chomber. The overall assembly Is 1' x 1 ' x 2/3' wide.

W
00
W
384 A. E. RABE AND R. J. BENOIT

Chlorella. Considerable experience was gained in the operation of

a variety of photosynthetic gas exchangers, with different geometries.
Continuous culture data obtained in three vessels are presented in
Table I.
To obtain an estimate of the extent of light attenuation with
culture depth, the data of Myers and Graham" were correlated. The
following empirical expressions were obtained4:
for 1 < 2.95 in., c 4 0.0575% PCV,
OD = 7.251~- 0.0411 (8)
for 1 < 2.95 in., c > 0.0575y0 PCV,
OD = 5.051: - 0.2171 (9)
It is of interest to note that the Myers and Graham dilute culture
data (eq. 8) do not follow Beer's Law, differing by the term 0.0411.
At higher concentrations the magnitude of this residual term increases
to 0.2171.
The method of applying eqs. (8) and (9) to test the hypothesis
is straightforward. The various culture vessels diagrammed in
Table I are divided into convenient zones and treated as in the sample
calculation. I n the case of collimated and orthogonal light, incident
on a planar surface when the cell concentration exceeds 0.057570
PCV, for culture depths less than 3 in., eq. (9) may be rewritten:
E = E~ 10-(5.051~ - 0.2171)
(10)
But

Performing the indicated integration,

= Eo(l - e-2.3AL)/2.3AL

+
where, A = (5.05~ 0.217) and L is the culture depth in inches.
In the case of a concentric annular chamber with the inner cylinder
housing the light source, the incident light rays are orthogonal but
not collimated. Equation (9) reduces to:

Performing the integration indicated by eq. ( l l ) ,from lo, the inner

cylinder radius, to L, the outer cylinder radius, yields:
BIOTECHNOLOGY AND BIOENGINEERING, VOL I V , ISSUE 4
 MEAN LIGHT INTENSITY 385

e- 2.3A
(2.3AZ)
E=-
L - lo {- 1
In IIl - 2.3AZ + ___-
2.2!

--(2.3AI )
3.3!
+--(2.3A
4\4!
I)
-n.n!I )
. . . + -(2.3A
Unfortunately this unwieldy equation converges rather slowly.
Consequently, it is simpler to perform a graphical integration of
eq. (11) and establish E by use of Simpson’s Rule.
The propriety of generally applying the expressions derived from
the Myers and Graham light intensity correlation can be questioned.
In the strictest sense, eqs. (8) and (9) apply only to data which they
correlate, i.e., Myers and Graham data. Those data were obtained
(1) using light from a 1,000 w. projector lamp filtered through a 1.9
cm. lucite cell containing 0.1M CuSOi solution, and ( 2 ) measuring
the exiting intensity with a lead sulfide photocell with a nearly
linear relative response over the visible or photosynthetic spectral
range. In the Electric Boat experiments, relatively dense cultures
were studied. Light incident on the culture surface was unfiltered
and its intensity was measured with an uncorrected selenium cell.
Because the relative response of the uncorrected selenium cell
decreases rather markedly in both the red and violet, Electric Boat
Eo values would have been considerably higher if measured with a lead
sulfide cell. Also, the attenuation of light intensity in an algal
culture would be markedly different for light having passed through
a blue filter than for unfiltered light since the red light, most strongly
absorbed by green cells, is removed by the filter. Thus values of E
measured at various culture depths (and used to compute E ) would
differ depending upon the photometric technique used and the spec-
tral composition of the incident light. An exponential attenuation
would be found; the rate of attenuation could differ markedly.
Finally, use of Myers’ data for dilute cultures to describe dense
cultures severely strains the conscious limits of reasonable extrapola-
tion.
It is equally true, however, that whichever photometric method
is used, the very nature of the Computation of E tends to suppress
differences. If large values of ?l are calculated using one approach,
comparatively large values must also occur using another, and vice
versa. This is particularly true if all culture vessels considered
have similar culture thicknesses. Accordingly, in a restricted sense,
386 A. E. RABE AND R. J. BENOIT

Myers’ optical density data should permit a reasonable test to be

made of the prospective correlation between growth rate and mean
light intensity, E.
To serve as a yardstick of the merit of the GR-’ = f(E) cor-
relation, a second independent variable, algal cell concentration,
was introduced. The existence of the functional dependence:
GR-’ = f(c) is based on, generally, the analogies existing in chem-
ical kinetics for continuous reactions.
After preparing several different scatter diagrams of c vs. GR,
and vs. GR, the most appropriate linear relationships were found to
be of the form:
GR-’ = log(c) + bl (15)
where log(c) = base 10 logarithm of the algal cell concentration and
a& = empirical constants. Also,

GR-’ = a2 log (E) + 6 2 (16)

where a& = empirical constants.
Plots of these scatter diagrams are shown in Figures 1 and 2 ,
respectively.
Values of E , calculated in accordance with eq. (11) by substituting
for E either eq. 10 or 13, are shown in Table I. These values depend
not only upon Eo and c but also upon culture chamber geometry, as
indicated by eq. (12).
The data included in Table I were subjected to a statistical regres-
sion analysis with GR-’ the dependent variable, and c and E the
independent variables. The followingequation was obtained :
GR-’ = 0.1325 log(E) - 0.00538 log(c) - 0.356 (17)
Of the total variance inherent in the dependent variable, GR-’,
about 65% is assigned to the independent variables B and c. I n
view of the manifold problems associated with the analysis and the
crudeness of some assumptions, the degree of variability success-
fully associated seems remarkable. The coefficient of multiple
correlation, R, is 0.8.
To compare the two independent variables, as to their relative
importance in assigning or “explaining” the total variability in
GR-1 it is necessary to normalize eq. (17). (Simply comparing
the magnitude of the numerical coefficients of log I? and log c, to
assess relative significance is improper since they describe different
BIOTECHNOLOGY A N D BIOENGINEERING, VOL. IV, ISSUE 4
 MEAN LIGHT INTEXSITY 387

0.15-

-
I
2 0.13 -
-
1-
K
0 0.11 -
I
w
z
z
0.09 -
0
t
5 0.07 -
w
2
(3

0.05 -
PP
0.03-
0

Fig. 1. A statistical scatter diagram of reciprocal generation time (no. of cell

doublings per hour) and algae cell concentration (% pcv).

physical entities.) The normalizing procedure is accomplished

by using the existing variances of all data variates to adjust the
numerical coefficientsof eq. (17). This yields:
GR-'/"GR-' = 5.65 log(-!?)/%g(E) - 0.0412 log(c)/"log(c) (18)
Thus it is demonstrated, that mean light intensity is 5.65/0.0412,
or about 140 times as important as cell concentration, in accounting
for growth rate of an algal culture over the ranges of the factors
included in the data. I n view of the minor role of cell concentration,
the scatter diagrams of GR-1 vs. E(Fig. 2) was subjected to least
squares analysis to obtain the relationship :
GR-' = 0.133 log(E) - 0.356 (19)
Some explanation for the relatively insignificant role of cell con-
centration in influencing culture growth rate is required. That
the correlation itself is negative (negative sign of the log(c) term in
eq. 17) is not surprising considering the traditional cell concentration
versus time relaOionship which governs batch cultures. (See Fig.
388 A . E. ItABE AND R. J. RENOIT

ii'F
W
0.11

0.09 1
0
0:

"-
2
LL
0.05

0.03
I , , , , , ,,,, , , , ,[(r03,

.3
I , ,

0.01
20 I00 1000 8000
MEAN LIGHT INTENSITY - E, footcandles

Fig. 2. A statistical scatter diagram of reciprocal generation time (No. of cell

doubiings per hour) and the mean light int,ensity, E (footcandles). Coded dat,a
points are referenced in Table I. The equation for the data is, GR-' = 0.133
log ( E ) - 0.356.

3.) The negative dependence obtained in the correlation (zone 5

of Fig. 3) is expected a t higher cell concentrations.
The general shape of Figure 3a obtains for batch cultures subjected
to a variety of levels of light intensity, COz concentration, nutrient
concentration, etc.. The length of the six zones and, particularly,
the slope of the linear segments of zones 2 and 4 of Figure 3b, will
vary widely among specific cases, however. This indicates that cell
concentration is actually a secondary or derived variable and that
the prime source of variability may be attributed to some other
primary factor such as light intensity. This conclusion is substan-
tiated by the correlation presented. It is difficult to judge the use-
fulness of eq. (19), in designing practicable photosynthetic gas
exchangers a t this time. Certainly the postulated relationship is
worthy of testing with all available continuous culture data.
Further determinations of light intensity versus culture depth,
using consistent photometric equipment, are essential as support
data.
BIOTECHNOLOGY AND BIOENGINEERING, VOL. IV, ISSUE 4
 MEAN LIGHT INTENSITY 389

z
0
_L_t_ I
5
a
t
2
W
0
t
8

I I
I

-- I \ I I

'i \!
dc I
dt

I
TIME

Fig. 3. The general relationship of algal cell growth to time in a batch culture.
( a ) Algal cell concentration as a function of time in batch culture: 0induction
period; @ exponential growth, dc/dt = time (or cell concentration); @ linear
growth; @ decreasing growth, -dde/dt a time (or cell concentration); @ 110
growth; @ decrease in total cell concent~r:ttion. ( h ) Algal rell growth rate as a
Function of time in x h t c h system.

(:ONCLUSIONS

(1) As the result of it correlatioii aiialysis, the prime importaiict:

of light distribution on growth rate of dense, continuous microalgae
cultures is shown for all cultures examined.
390 A. E. RABE AND R. J. BENOIT

(2) It is significant that the mean light intensity (E’) concept

presents a valid means for correlating algal cell growth rate.
(3) Culture density is shown to be negatively correlated with
growth rate over the range of densities included in the analysis.

This paper analyzes data gathered in research projects a t General Dynamics/

Electric Boat, Research and Development Laboratories under company sponsor-
ship, and under the sponsorship of National Aeronautics and Space Administration
contract NASW 95. The authors are indebted t o a number of Electric Boat
co-workers who obtained the data used t o demonstrate the correlation. In
particular, Dr. E. A. Zuraw and Mr. Donald E. Leone are cited for their careful
and significant labors in advancing the technology of dense, continuous cultures
of microalgae. I n addition, data of Professor Jack Myers of the University of
Texas were used to derive certain expressions. We gratefully acknowledge the
premier scientific contributions of Professor Myers and his generous and frank
discussions on the central problems of continuous culture of microalgae.

References

1. Algal Culture fr0m LabWUtOry to Pilot Plant, J. 8. Burlew, Ed., Carnegie

Institute of Washington, Publication 600. Washington, D.C., 1953.
2. Sorokin, C., and J. Myers, A High-Temperature Strain of Choreh. Sci-
ence, 117,330 (1953).
3. Sorokin, C., and R. W. Krauss, Plant Physiol., 33,109 (1958).
4. Rabe, A. E., and R. J. Benoit, Engineering Research on a Photosynthetic
Gas Exchanger. Part II. Engineering Considerations, General Dynamics/Electric
Boat, Groton, Connecticut, U41342-023, May 7,1962.
5. Kok, B., Biochim. Biophys. dcta, 21,234 (1956).
6. Burk, D., G. Hobby, and T. A. Gaucher, Closed-Cycle Air Purifiatton with
Algae in Man’s Dependence on the Earthly Atmosphere, K. E. Schaefer, Ed.,
Macmillan, New York, 1962, p. 401.
7. Gaucher, T . A., R. J. Benoit, and A. Bialecki, J . Biochem. Microbial.
l’echnol. Eng., 2,339 (1960).
8. Kok, B., Biochim. Biophys. dcta, 21,245 (1956).
9. Zuraw, E . A., e t al., Photosynthetic Gas Exchange in the Closed Ecosystem
for Space. Part I . Pilot Photosynthetic Gas Exchange Studies, General Dynani-
ics/Electric Boat, Groton, Connecticut, U411-61-131, August, 1961.
10. Wallman, H., et al., Research and Development of a Liquid-Gas Contactor ~ O T
Photosynthetic Gas Exchangers, General Dpnamics/Electric Boat, Groton, Con-
necticut, U413-62-108, August, 1962.
11. Myers, .J., and J. R. Graham, Plant Ph.’husiol.,34,345 (1959).

Received November 26, 1962

BIOTECHNOLOGY A N D BIOENGINEERING, VOL. IV. ISSUE 4