Process Biochemistry, %Iol. 31, No. 6, pp.

601-604, 1996
Copyright © 1996 Elsevier Science Ltd
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ELSEVIER S0032-9592(96)00006-4

Heterotrophic Growth of Chlamydomonas

reinhardtii on Acetate in Chemostat Culture
F e n g C h e n a* & M i c h a e l R. J o h n s b
a Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong
bDepartment of Chemical Engineering, The University of Queensland, Brisbane, QLD 4072, Australia

(Received 22 September 1995; revised manuscript received 11 December 1995 and accepted 6 January 1996)

The possibility of using heterotrophic chemostat culture to achieve relatively

high cell densities of Chlamydomonas reinhardtii by avoiding the inhibition of
growth by the substrate, acetate was investigated. Continuous culture gave
significant improvement in cell dry weight concentration and productivity
compared to batch culture. It is postulated that this was due to low acetate
concentration in the continuous culture. The highest cell concentration obtained
was 1.5 g/litre at a feed acetate concentration of 3.4 g/litre. At acetate
concentrations up to this level, the modified Monod model successfully
predicted the steady-state cell concentration and cellular productivity. However,
no further increase in cell concentration was achieved, despite the use of higher
feed acetate concentrations (i.e. 5"1 g/litre), in which, steady-state acetate
concentrations were below the inhibition level (i.e. 0.4 g/litre). This inhibitory
effect was probably due to sodium rather than to acetate per se in the cultures.

INTRODUCTION Heterotrophic cultures may provide a cost-

effective, large scale alternative method of
Microalgae are produced on a large scale as cultivation for some microalgae, which utilise
health food, and are used in agriculture, waste- organic carbon substances as their sole carbon
water treatment and aquaculture and for the and energy source. 8 This culture technique can
production of chemicals such as phycobilipro- overcome the common problems encountered
teins and fl-carotene. 1-4 They produce other in the photoautotrophic system and achieve
chemicals of significant interest, including anti- improved cell density and productivity. 9
cancer agents, xanthophylls, polyunsaturated Several microalgae grow heterotrophically
fatty acids and astaxanthin. 23
' only on substrates such as acetate which inhibit
A significant limit to the widespread use of growth in batch culture. ~° Attempts to use fed-
microalgae is their mass culture in photoauto- batch culture by minimising acetate
trophic growth processes. Problems include the concentrations failed due to the accumulation
difficulty of maintaining monoculture, light limi- of sodium in the media, which inhibited
tation and lack of control of important growth. 11 Subsequent use of a Hollow Fibre
environmental factors. 4 These problems have Cell Recycle System was successful. 9 However,
attracted much recent work. 5-7 this system is expensive and difficult to operate
at large scale.
*To whom correspondence should be addressed. Continuous culture maintains low acetate and
601
602 E Chen, M. R. Johns

sodium concentrations in the bioreactor and is a Media and fermenters were sterilised by
more straightforward growth technology. The autoclaving at 121°C for 20 min. On cooling, the
heterotrophic growth of C. reinhardtii on acet- medium was inoculated with 5% (v/v) of an
ate in chemostat culture has not been reported inoculum grown at 25°C on CR-M1 medium 1°
and only a few reports of chemostat cultures of containing 0.85g/litre acetate and 0.2g/litre
heterotrophic Chlorella are available. 12'13 This urea under a cool white fluorescent tube (1800
paper reports the application of heterotrophic lux) on a 16/8 on/off cycle. The fermenter was
chemostat culture of Chlamydomonas reinhardtii permitted to grow in batch mode until cell
on acetate as a means for increasing cell con- density was sufficient to permit feed medium to
centration and productivity. be supplied. Culture was continuously dis-
charged via an overflow tube to maintain a
constant culture volume (1 litre). Steady-state
MATERIALS AND METHODS
conditions were considered to have been estab-
lished when cell concentrations from at least
Organism
three samples collected after a period of three
Chlamydomonas reinhardtii Dangeard (CS-51)
residence times, varied by no more than 10%.
was obtained from CSIRO Marine Labora-
tories, Hobart, Australia and maintained at 4°C
Analyses
on nutrient agar slopes prepared from Bristol's
Acetate and cell concentration in the culture
solution ~4 with 0-1% bacto-peptone and 1.5%
fluids was determined by HPLC and optical
agar.
density measurement at 750 nm, respectively. 1°
Cell dry weight was estimated from optical
Medium
density by a calibration curve.11
Modified Sager & Granick media ~5 referred to
as CR-M2 medium consisted of (per litre) 0.3 g
Modelling
KzHPO4, 0.3 g MgSOa'7H20, 0.053 g CAC12"2
Steady-state cell and acetate concentrations and
H20, 0.01 g FeCI3"6H20, 0.5 g sodium citrate
cell productivity were predicted using the
and 1 ml of trace metal solution comprising
Monod model modified for an inhibitory sub-
(per 100 ml) of 125 mg H3BO3, 125 mg
strate as presented by Chen and Johns) ° At
ZnSO4"7H20, 38 mg MnSO4"H20, 25 mg high culture acetate concentrations (>0-4g/
COC12"6H20, 25 mg Na2MoO4" 2H20 and 8 mg
litre), the Monod model was modified to
CuSO4"5H20. Nitrogen was supplied as urea incorporate an inhibition term. Also mainten-
(0.5 g/litre), since it was found to be superior to
ance energy was used to account for diversion
nitrate in heterotrophic batch cultures.ll
of substrate from cell growth to cell mainten-
Sodium acetate was used as sole carbon and
ance activities. The parameter values used for
energy source. An attempt to use the pH-buf-
the simulations were 1Yog=0-55 g/g, m=0.011 g/g/
fered medium, CR-M11° containing 1.7g/litre
h and #m=0"084 h-1
acetate without automatic pH control was
unsatisfactory, since large changes in culture pH
occurred.
RESULTS AND DISCUSSION

CULTURES A chemostat culture of C. reinhardtii was oper-

ated at a variety of dilution rates using CR-M2
The stirred, glass fermenter used has been medium with feed acetate concentrations of
described by Chen and Johns. 1° The culture 0.85 g/litre and 1.7 g/litre. The results for 1-7 g/
temperature was maintained at 35°C by a water- litre acetate are presented in Fig. 1. A
bath and automatic pH control, with sterile 1 M steady-state cell concentration of 0.8 g/litre was
HCI, was used to maintain culture pH at pH obtained and acetate concentration was negli-
7-2. Sterile air was supplied to maintain dis- gible at all dilution rates tested. The modified
solved oxygen concentrations above 50% Monod model fitted the data well. The 0.85 g/
saturation during cultivation and the fermenter litre acetate cultures responded similarly to the
was maintained in darkness to prevent photo- dilution rates, and could be represented by the
synthetic growth. modified Monod model.
 Heterotrophic growth of Chlamydomonas reinhardtii 603

1.0 0.10 3.0

09 L -o.o9 2.7
,-, 0.8 -- - ~ 0.08 2.4
2.1
0.06 ~
0.05
0.04 a~ X 1.2 so* • .. m. A" "" ~"

0.03 0.9 ,o s o "r'l- fro ~-~ FI •

0,02 0.6
0.3
~ A ~ - "-'4~"'A, --'--~1 I 0.01
0 0 I I I I I I I
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08
°0 0.01 0.02 0.03 0.o4 0.05 0.o6 0.07 0.)8 D (1/h)
D (l/h)

Fig. 1. Effect of dilution rate on steady-state acetate and Fig. 3. Comparison between the simulations and the
cell concentrations and cell productivity of C. reinhardtii steady-state cell concentrations at various feed acetate
in chemostat culture with a feed acetate concentration of concentrations. Model: (--) 1.7g/litre; (--) 2.55 g/litre;
1.7 g/litre. (A) Acetate concentration; (rn) cell concentra- (----) 3.4 g/litre; (--) 5.1 g/litre; Data: (D) 1.7 g/litre; (m)
tion; (m) cell productivity; ( - - ) model prediction. 2.55 g/litre; (•) 3.4 g/litre; (A) 5-1 g/litre.

The values of the true growth yield, Yg, and feed medium, led to only poor cell concentra-
the maintenance coefficient, m, for C. reinhard- tions, well below the predicted values. The
tii were estimated from Fig. 2 using the inhibition was not due to acetate, since steady-
modified Monod model. 1° The values are state acetate concentration in the culture was
0.55 +0.03 g/g and 0.011 +0.001 g/g/h for Yg and less than 0-4 g/litre acetate. At this concentra-
m, respectively. These values are identical to tion and culture pH, acetate is not inhibitory to
those previously obtained. ]° the growth of C. reinhardtii) °
These values were used to predict the steady- The effect of feed acetate concentration on
state cell concentrations achieved by the use of cell concentration and the observed cell yield
feed acetate concentrations in the range (Y) is compared at a dilution rate of
2.55-5.1 g/litre for various dilution rates. The 0.025 ___0.002/h in Fig. 4. Whereas the observed
comparison between the simulated results using cell yield is relatively constant for acetate con-
the modified Monod model and the cell concen- centrations of 3.4 g/litre or less, it fell markedly
trations measured from actual chemostat at the highest acetate concentration, which is
cultures performed at three feed acetate con- indicative of severe growth inhibition.
centrations within this range are presented in The observed inhibition was probably due to
Fig. 3. Whereas, good agreement was achieved high sodium concentrations associated with the
at acetate concentrations up to 3.4 g/litre, the use of sodium acetate in the feed, since the use
use of 5.1 g/litre acetate concentration in the of perfusion culture using salt-permeable mem-

4.0 1.6

3.5 1.4 L

3.0 1.2

2.5 ~ 1.0 -

~ 2.0 ~ 0.8 -

•-" 1.5 ~n90.6 -

1.0 X 0.4~

0.5 0"2 V
0 I I I I I
10 20 30 40 50 60 00 3 6
1/D (h) Sf (g/litre)

Fig. 2. Determination of true growth yield coefficient, Fig. 4. Effect of feed acetate concentration on steady-
Yg, and maintenance coefficient, m, of C. reinhardtii for state cell concentration and observed growth yield at a
growth o n acetate (Sf=0.85g/litre and 1-70g/litre) in dilution rate of 0.025 + 0.002/h. (D) Cell concentration; (m)
chemostat culture. observed growth yield.
604 E Chen, M. R. Johns

brane bioreactor relieved the inhibition.9 The cal assistance provided by Mr Peter Abeydeera
sodium concentration in the 5.1 g/litre cultures for the HPLC analysis.
was estimated to be approximately 2 g/litre by
assuming that the acetate ions were preferen-
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