Influence of light intensity on the kinetic and yield parameters of

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Influence of light intensity on the kinetic and yield parameters of
Process Biochemistry, Vol. 32, No. 2, pp. 93-98, 1997
Copyright O 1996 Elsevier Science Ltd
Printed in Great Britain. All rights reserved
0032-9592/97 $17.00 + 0.110
FLSEVIER
PII:
S0032-9592(96)00045-3
Influence of light intensity on the kinetic and
yield parameters of Chlorella pyrenoidosa
mixotrophic growth
Ma. E. Martinez, a F. C a m a c h o , b J. M. Jim6nez" a n d J. B. E s p i n o l a t'
~Instituto de Biotecnologia, Departamento de Ingenieria Quimica, Universidad de Granada, Granada, Spain
bDepartamento de Ingenieria Quimica, Universidad de Granada, Granada, Spain
(Received 5 January,1995; revised version received 17 April 1996; accepted 28 April 1996)
Abstract
The influence of light intensity on growth of the freshwater microalga Chlorella pyrenoidosa has been
studied. A mineral culture medium supplemented with glucose at concentrations of 0"1, 0'5 and 1 g litre
was used. The different growth phases observed have been described. In the exponential mixotrophic phase,
the specific growth rate did not depend on light, since the algae were able to use glucose as an energy
source, a fact demonstrated by the increase in the specific rate of glucose consumption on reducing the light
intensity. When the organic substrate was completely consumed, and after an adaptation period, an autotrophic phase followed. The results show that it is advisable to add glucose only to light-limited cultures.
The highest biomass/substrate yields are attained at 0-1 g litre t. Copyright © 1996 Elsevier Science Ltd
Nomenclature
• k6oo
(?
( 7o
[ ;m
c ;/Co
i
J(
I,-F
I s
/,'o
i
z/K
ttm
Absorbance of the cell suspension at
600 nm
Biomass concentration (glitre 1)
Biomass concentration at t = 0 (g litre ~)
Biomass concentration on consumption of
the organic substrate (g litre 1)
Adimensional biomass concentration
Average light intensity in the culture (lux)
Light intensity (lux)
Light saturation intensity (lux)
Parameter of eqn (4)
Coefficient of extinction (litre g i c m - i )
Light path length (cm)
Probability in the analysis of variance
Regression coefficient
Specific rate of organic-substrate consumpt i o n ( g g 1h-1)
Residual glucose concentration (g litre i)
Initial glucose concentration (g litre 1 )
Culture time (h)
Yield in biomass/substrate (g g-1)
Parameter of eqn (4)
Maximum yield of biomass/substrate
Yield of biomass/substrate in darkness
(gg ')
Specific growth rate (h J)
Maximum specific growth rate (h ~)
Introduction
The high values of the extinction coefficients of microalgal suspensions I result in limited growth rates due to
light restriction in most of the geometric configurations
of the photobioreactors used to culture microaigae at
moderate biomass concentrations. Efforts have been
made to avoid this limitation, but serious problems
emerge in trying to provide a culture with non-limiting
and uniformly distributed light. One result in laboratory cultures is overheating, 2 since the normal tendency
is to situate the light source inside the culture. Another
effect is contamination in open outdoor cultures, since
these systems usually tend to be shallow with large
surface areas exposed to the light)
Many algae cannot only grow photosynthetically, but
also by using organic substrates for biosynthesis and
cell maintenance. 4 7 Therefore, the addition of certain
organic compounds to the photobioreactor can supply
the additional energy necessary in situations of light
limitation, s.~
The present paper examines the influence that light
intensity exerts on the kinetic and yield parameters in
the growth of the unicellular alga Chorella pyrenoidosa
in a mineral medium supplemented with glucose.
93
Ma. E. Martinez et al.
94
Materials and Methods
Results and Discussion
The freshwater microalga used was C. pyrenoidosa,
Chick 8H Emerson.
Figure l(a) and (b) shows, in semi-logarithmic coordinates, two growth curves and the corresponding curves
of organic-substrate consumption under the experimental conditions, Io and So, specified in the figure
legend.
At the higher intensity of light (see Fig. l(a)),
several differentiated phases appeared. The first was a
phase of exponential growth which corresponds to mixotrophic growth, in strict terms. A stationary phase
ended when practically all the organic substrate was
consumed; afterwards an exponential autotrophic
phase appeared, and finally a deceleration corresponding to light limitation. With less intense light (see Fig.
l(b)), the exponential phase of mixotrophic growth
preceded the stationary phase which, after 26 h, was
followed by the phase in which growth was light
limited.
These two situations cover all the experiments performed. A comparison of curves shows that the slopes
of the straight-line phases corresponding to mixotrophic exponential growth are similar. In the
stationary phase, the biomass formed was greater in
the experiment of So = 0.5 g litre-1. The last phase, of
deceleration, could not be due to nutrient limitation,
Cultures
All experiments were carried out in a batch-culture
system consisting of three photobioreactors, each of
1 litre holding capacity, jacketed for the circulation of
water, equipped with a thermostat and stirred magneticallyJ ° The mineral culture medium used was the
A medium proposed by Rodriguez L6pez, 11 supplemented with D-glucose as organic substrate at initial
concentrations (So) of 0.1, 0.5 and 1-0glitre -1. The
complete culture medium was sterilized by filtration
with membranes of 0-2 #m pore size. As an inoculum, a
suspension of cells was taken from a preculture grown
in solidified mineral medium with agar for 4 days at
room temperature under constant illumination from a
Westinghouse Plant-Gro fluorescent lamp. The pH of
the culture medium was adjusted to 6.5 and the temperature used was 30___0.5°C, optimal values for the
alga used. All the cultures were supplied a mixture of
air and carbon dioxide at 5% v/v, sterilized by filtration
with a membrane of 0.2/~m pore size, at a rate of 0-5 v
v-1 min-1. Culture illumination was provided by two
Westinghouse Plant-Gro fluorescent lamps, specially
designed for plant growth, most of the light being emitted in the red and blue zones of the spectrum. The
different intensities of light used (2000, 1400, 800 and
400 lux) were achieved by regulating the distances
between the reactors and the lamps, or by using metal
black (light-absorbing) netting. Light was measured by
a Gossen-Mavolux luxometer.
= 1 0 0 ~
o
(a)
~ ]o
~0
Analytical methods
:~ 0'10
Cell concentration was measured indirectly by the
absorbance of the culture at 2 = 600 nm. Two relationships of absorbance vs dry weight (expressed in
g litre-i) were used at the beginning of the culture in
the presence of glucose:
I0
20
30
40
50
Time (h)
60
70
80
= I00
~
C/Co
(b)
C(g litre-1) = 0"73 A6oo
used in the interval 0-0.5glitre -I and when the
glucose had been eliminated from the culture medium:
o
C(g litre 1) = 0"49A6oo
OI
used in the interval 0-0"3 g litre- 1
For monitoring the concentration of the glucose in
the culture, two methods were used: Somogyi ~2 for the
interval of 0.01 at 0.08glitre -I and Miller 13 for the
interval of 0.08 at 0.5 g litre- 1.
0
10
20
L
30
I
410
50
Time (h)
610
I
70
80
Fig. 1. Variation of the adimensional concentration of biomass C/Co (e) and residual glucose S (A) with culture time.
Experiment (a) lo = 2000 lux, So = 0-1 g litre- 1. Experiment
(b) I,, = 400 lux, S,, = 0"5 g litre- ~.
Influence of light intensity on mixotrophic growth of Chlorella
95
Table 2. Mixotrophic growth: specific rates of glucose consumption
Table 1. Mixotrophic growth: specific growth rates
t,,
(lux)
S,,
(g litre- 1)
~
(h- 1)
r2
2!.)22
1983
1907
1386
1 370
1255
M4
809
~02
372
395
369
0.1
0'5
1.0
0"1
0"5
1.0
0.1
0-5
1-0
0.1
0-5
1.0
0.1107
0.1070
0.1094
0.0932
0.0970
0"1000
0-0880
0.0874
0.1027
0.1047
0"1058
0-1195
0.997
0.995
0.997
0-990
0.998
0.986
0.973
0.995
0.985
0-971
0-990
0-990
L, (lux)
So
(g litre -1)
q~ × 103
(g g - ' h-l)
p-r 2
2000
0"1
0"5
1.0
0.856
0.761
0.996
1400
0'1
0"5
1.0
400
0' 1
0.5
1"0
43-85
68.74
51.34
mean value 54.64
138.76
157.46
141.00
mean value 145.74
52.78
223.53
265.90
mean value 180"74
~iven that it begins at two different biomass concentrations, while the same mineral medium was used in each
tase. Limitation by light appeared at a lower biomass
toncentration at Io = 400 lux.
0-990
0.940
0.987
0.870
0.950
0.950
(Tt
and carrying out the integration, gives
Phase of exponential mixotrophic growth
qsCo
So - S = - [e ('mr) - 11
Pm
,~pecific growth rate
Table 1 presents the values of specific growth rate
tluring the mixotrophic exponential phase, calculated as
d(LnC)/dt at the beginning of the culture. Each value
i~ the mean of the results obtained in four experiments.
A two-way analysis of variance of the initial glucose
toncentration, So, and the light intensity, Io, indicated
t at the significance level of 0.05) the independence of p
lrom So, P = 0-2357 and with Io, P = 0.6449. Thus, the
specific rate was independent of the combined effect of
the two variables (P = 0.9777). The mean value of p in
the variation interval studied is 0.1021 h 1. The independence of # from Io appears to indicate that the
intensity of saturation for the mixotrophic growth of C.
t,yrenoidosa is less than 400 lux. This value is lower
~han the saturation intensity (926 lux) reported for
~utotrophic growth of Chlorella.10 This displacement of
it may be due to a reduced need for light energy on
the part of the alga when presented with the possibility
,,f using both light and organic substrate as energy
~,ources.
,;pecific rate of organic-substrate consumption
Given that during the phase of exponential growth, q~,
~he specific rate of organic-substrate consumption
~emained constant and that the biomass concentration
varied over time according to the equation:
C = Coe""
then, changing eqn (1) with the definition of qs
(1)
(2)
This equation enables the calculation of qs, adjusting
by means of a non-lineal regression of the values of S
and e (~m').
With the theoretical values of S, given by eqn (2),
the lines S - t were drawn in Fig. 1. Table 2 shows the
specific rate of glucose consumption and the coefficients p - r 2. For one given level of light intensity (Io),
the values of qs do not appear to vary significantly
with the initial glucose concentration. The most discordant value was that obtained with a lower
regression coefficient, and therefore the specific rates
have been averaged and these average values
included in Table 2.
The average values indicate, as a response of the
cell, an increase in the rate of metabolic consumption
of the organic substrate, on decreasing the light
energy received. These qs values continue to rise on
decreasing Io, as shown by Camacho et al., 14 who, on
using the same microalga in darkness, obtained the qs
value of 365 mg of glucose g t of biomass h - i when
the initial glucose concentration in the culture
medium was 0"25 g litre- 1.
Stationary phase
Biomass yield
The yield in biomass was determined during the
stationary phase observed at the end of the mixotrophic exponential growth phase, by means of the
expression:
96
Ma. E. Martinez et al.
Table 3. Biomass concentration on depleting the glucose, Cm
So
(glitre -I)
0.1
0.1
0.1
0.1
0.5
0.5
0.5
0.5
1.0
1.0
1.0
1.0
it,, (lux)
Cm
(glitre l)
C,,
(glitre l)
372
814
1386
2022
395
809
1370
1983
369
802
1255
1907
0.072
0.089
0.120
0.107
0.200
0.208
0.323
0.364
0.349
0.330
0.397
0.433
0.013
0.014
0.013
0.011
0.015
0.018
0.015
0.014
0.015
0.017
0.019
0.013
This constancy indicates that light contributes the
energy needed for growth and cell maintenance, while
the glucose is used for the formation of biomass. At an
Io < 1500 lux, lower values for Y signal that luminous
energy is inadequate and that the cells have also used
the glucose for growth and cell maintenance.
This latter situation also explains the fall in the yield
values on increasing the glucose concentration to 0.5
and 1.0 g litre-Z at the same L, value, since in these
cases the cell concentrations reached on arriving at the
stationary phase were higher than at 0-1 g litre i (see
Table 3).
Thus, the variation in the mean yield values with
light intensity can be adjusted to a function of the type:
~+#L
Y = - K+L,
Cm -- Co
Y= - So
(3)
C m represents the biomass concentration on
depleting the glucose. The values Cm, Co and So are
indicated in Table 3, grouped according to the So
value. The Cm increases on raising the Io and So.
Figure 2 represents the biomass yield against the light
intensity. A different curve appears for each initial concentration of glucose. The greater yields (Y) were
reached at an initial glucose concentration of 0-1 g litre -I and these increased for each So as the light
intensified. This increase was less pronounced at the
glucose concentration of 1 g litre- 1.
At low light intensities, the influence of the glucose
concentration on yield was less pronounced, the three
curves tending towards a single residual value at Io = 0
of approximately 0"3 g dry biomass g-1 glucose.
At a glucose concentration of 0.1 g litre 1, the mass
yield, Y, tends towards a constant value at Io > 1500 lux.
where
,2[
Y
-~'
'~Lfi 1"0
"u
/
.J"
(4)
which predicts the constancy of Y at Io values high
enough for the light to be the only energy source.
The previous equation reproduces the saturation
effect observed at the glucose concentration of 0.1 g litre -I, and also the linear variation found at 0"5 and
1 glitre 1, since this equation is transformed in the
linear function:
C¢
/;/
(5)
Y = - - + ' - - - Io
K
K
when K >>lo.
In both equations, o¢/K represents the yield in darkness, and fl the maximum yield attainable at high light
intensity. Table 4 shows the values obtained in the fit
of the experimental values Y-l,, by non-linear regression to eqn (4). This fit has made it possible to draw
the continuous lines in Fig. 2, and indicates that the
value of the yield at Io = 0 is 0.33 and that the maximum yield value is 1.24. Both values, ~/K and fl, are
independent of the glucose concentration. The light
intensity, Io, at which the maximum value, fl, is reached
is greater as the initial glucose concentration increases.
This fact is illustrated by the increase of K with S,,,
since this constant K in eqn (4) represents the value of
Io at which 63% of the maximum yield value is
reached.
The values of K calculated indicate that, at least at
the concentrations of 0.5 and 1 g litre- l, the maximum
yield is unattainable in practice.
Table 4. Equation (4) parameters
r.-.---,,
0.2
•
I
500
Io
I
1000
I
1500
I
2000
S,,
~t/K
(g g-~)
K (lux)
0"1
0"5
1"0
0'33
0"33
0"33
417
3733
23353
(g litre l)
2500
I n c i d e n t l i g h t i n t e n s i t y (lux)
Fig. 2. Variation in the biomass/substrate yield with the
intensity of light at the glucose concentrations (*) 0.1, (tt)
0.5, (A) 1 g litre- 1.
fl
SSQ
1"24
1'24
1'24
2"31 x 10 -3
1"24 x 10 2
2"60 × 10- 3
(g g-l)
Influence of light intensity on mixotrophic growth of Chlorella
It is useful to analyse the appearance of these value
limits for yield in a mixotrophic culture. The value of
~/K in the present work is practically equal to the average yield of a heterotrophic culture in darkness
10"3 g g - l ) obtained by Camacho et al. t4 This indicates
that the limit of mixotrophic growth at zero light intensity is heterotrophic growth. On the other hand, only a
yield value of 1.24 g g-~ is known, published by Endo
~,t al., 4 for the growth of Chlorella regularis S-50 with
lght, CO2 and acetic acid.
Table 5.
Tamiya model
lt--
Exponential model
/o
(l. 1
The specific growth rate appears to vary linearly with
',he light intensity of up to approximately 800 lux, with
~aturation beginning above this level. Table 5 shows
the fit by a non-linear equation to two kinetic models,
lhe hyperbolic one of Tamiya 15 and the exponential
one) 6 Both models, represented jointly in Fig. 3,
acceptably fit the experimental variation /~-/. From
/ = 1500 lux, the /~ values of Tamiya's model increase
more rapidly with I than do those of the exponential
0.12
la
0.1l]
~ I~ = 926 lux
although the difference with respect to those of the
Tamiya model is less. In addition, the difference
between #m of the Tamiya model and p., for mixotrophic growth is also less.
This agreement appears to indicate that, upon the
depletion of the glucose in the culture, and after an
adaptation period coinciding with the stationary phase
observed, the cells grow at rates equivalent to those of
purely autotrophic cultures, that is the initial mixotrophic period does not irreversibly distort the
photosynthetic apparatus of the cells.
For a comparison of the mixotrophic and autotrophic specific growth rates in Fig. 3, values have been
given jointly. At moderate light intensities, the mixotrophic growth rates exceed the autotrophic rates;
nevertheless, as I increases, both rates tend to equalize,
implying that the culture derives no benefit from supplemental organic substrate at high intensities of light.
References
Tamiya model
S
0.06
~rj
SExponential model
e ~ ' o Autotrophic growth
~ O.(N
./(
0
#r, = 0"099 h
Mixotrophic growth
~" 0.08
0.02
model. The value of the parameters ,um and I~ obtained
in the fit to the Tamiya and exponential model are
similar to those obtained by Camacho et al. ~° in the
autotrophic culture of Chlorella pyrenoidosa:
(6)
valid for the photobioreactors used, with a value of
k a . L = 14.7 litre g--J, as demonstrated by Camacho et
It = ltm(1--e l/Is)
ll,n = 0"076 h J
L = 708 lux
SSQ = 1"599 x 10 4
p - r e = 0"935
,Specific growth rate
I= - (1 - e - k - ' L c )
k , . L .C
p.,l
L+I
itm =0.116 h I
L = 1011 lux
SSQ = 1"606 x 10 --4
p - r e = 0.935
Exponential phase of autotrophic growth
Figure 3 presents the average specific growth rates
t orresponding to the autotrophic exponential phase,
1or the experiments in which such a phase was
detected, against the average light intensity at the
beginning of the autotrophic exponential phase, calculated by the equation:
97
./"
I
500
I
I
1000
1500
Light intensity (lux)
2000
Fig. 3. Variation in the specific growth rate with light inten~ity.
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