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The mass culture of Dunaliella salina for ?-carotene: from pilot plant to
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Hydrobiologia 151/152: 99-105 (1987)
M. A. Ragan and C. J. Bird (eds)
Twelfth International Seaweed Symposium
© Dr W. Junk Publishers, Dordrecht - Printed in the Netherlands
The mass culture of Dunaliella salina for $-carotene: from pilot plant to
production plant
T. P. Moulton, L. J. Borowitzka & D. J. Vincent
Western Biotechnology Limited, 2-6 Railway Parade, Bayswater, W.A. 6053, Australia
Key words: Dunaliella, 3-carotene, mass culture, bioeconomic modelling
Introduction
There has been a considerable amount of interest in
the potential for commercial production of -
carotene from the extremely halophilic green alga
Dunaliella salina (Chlorophyta, Volvocales), and
trial operations have been set up in the USSR (Mas-
yuk, 1966; Masyuk & Abdula, 1969) and in Israel
(Ben-Amotz & Avron, 1980). There is a commercial
operation in California, USA, and incipient com-
mercial operations in Australia and possibly else-
where.
Here we present a continuation of the research
and development carried out at Hutt Lagoon,
Western Australia, and laboratories in Perth
(Borowitzka et al., 1984, 1985) and we demonstrate
part of the decision-making process involved in de-
termining the commercial feasibility of a large-
scale production facility.
The research commenced at the laboratory scale
in 1978 with the Roche Research Institute of Ma-
rine Pharmacology in Dee Why, Australia. It
proceeded to the first pilot-plant stage with Roche
Algal Biotechnology and in 1984 was taken up by
the Australian government, who contracted Wes-
farmers Algal Biotechnology to carry out a second
stage of piloting. The project was taken over in
April 1986 by Western Biotechnology Ltd, who are
building a 25-ha production plant on the basis of
the favorable results from the pilot plant.
99
The site
The hydrogeochemistry of Hutt Lagoon was
described by Arakel (1981) and Arakel & Moulton
(1986). The area has a Mediterranean climate, rela-
tively low rainfall, strong winds and high insolation
in summer (Table 1). The salt lake is separated from
the Indian Ocean by approximately 1.5 km of sand
dunes. It is seasonally dry (approximately 6
months), and floods with winter rains to form a
hypersaline water body.
Laboratory to pilot-plant scale-up
We reported a satisfactory transition from
laboratory-scale cultures to open pond cultures at
Hutt Lagoon (Borowitzka et al., 1984, 1985). Ponds
are maintained at salinities of 250-320 g- L - and
are supplied with nitrogen, phosphorus and trace
elements. Under these conditions, D. salina con-
tains very high levels of P-carotene, and remains the
dominant algal species with little or no predation
by protozoa. Compared with laboratory cultures
under similar conditions of salinity, temperature
and culture medium, pond cultures grow slower
and to lower maximum concentrations of cells and
3-carotene (Moulton et al., 1987, cf Fig. 2 and Ta-
ble 1). This phenomenon has been observed with
various large-scale microalgal operations (e.g.,
Goldman, 1979).
The difference in production of carotenoid be-
tween pond and laboratory cultures was quantified
100
Table 1. Climatic table for Geraldton, W.A. (28°45'S, 1140 36'E), 80 km south of Hutt Lagoon.
Month Mean Mean
daily temp. daily temp. range
(°C) (°C)
January 24.1 10.7
July 15.4 8.9
Annual 19.8 9.8
in an experiment in which samples were taken from
newly established pond cultures and grown in
flasks in laboratory growth cabinets under condi-
tions of salinity, temperature and daylength ap-
proximating those at Hutt Lagoon. Light intensity
in the growth cabinet (900 /zE.m-2.s - l) was ap-
proximately one third the incident light intensity at
Hutt Lagoon. Over three cycles of growth in two
ponds, the pond cultures produced on average 44%
(standard error 10%) of the carotenoid of the
matched laboratory experiments. We do not under-
stand the reasons for the differences between
growth in the laboratory and ponds. Measurements
of the penetration of light through the water
column in ponds show high intensities of light rela-
tive to the laboratory (Moulton et al., 1987). The
exchange of gases was possibly superior in the
laboratory cultures due to the high surface-to-vol-
ume ratio, although the cultures were not stirred or
aerated.
Second-stage pilot-plant scale-up
In the second stage of pilot plant development
(commencing December 1984) we built four
6000-m2 ponds in the same manner as the previous
125- to 625-m 2 ponds; they were constructed with
earthen sides on the relatively impermeable floor of
the salt lake.
The concentrations of -carotene and the growth
rates of D. salina were very similar in the large
ponds and the small ponds, and the ten-fold scale-
up to 6000 m 2 was considered a complete success.
Several features of the 6000-m 2 ponds were more
favorable to -carotene production: the greater ra-
tio of surface area to perimeter resulted in less leak-
Median Evaporation Global radiation Mean wind speed
precipitation (mm) (mW.h.cm -2 ) at 1500 h (knots)
(mm)
1 187 825 15
93 68 325 7
472 1465 567 12
age, erosion of the banks, windrowing of algae and
dilution by rain relative to the volume of the pond.
Three of the 6000-m2 ponds were maintained
through winter 1985 with relatively high salinity,
whereas in the smaller ponds this was not possible
without addition of solid salt due to relatively
greater amounts of water entering the ponds from
run-off. A potential disadvantage of the large
ponds was the difficulty of supplying nutrients
evenly, but in all cases in which measurements were
taken, nutrients added at one point in the pond dis-
persed to all parts of the pond within 2 days. Other
problems with the large ponds were expected; they
were more difficult to sample accurately, and
manipulations such as salinity adjustments, pump-
ing etc. were necessarily slower than with smaller
ponds.
Based on this experience, we expect the planned
scale-up to 5-ha ponds to be achieved with relative-
ly few biological problems.
Pond operating system
Various systems for growing and harvesting algae
in ponds have been suggested, ranging from batch-
wise through semicontinuous to continuous har-
vesting. At an earlier stage of development
(Borowitzka et al., 1984, 1985) we dismissed a sys-
tem based on relatively fast growth of D. salina at
low salinity (approximately 150 g.L - ') followed
by increased salinity (approximately
250- 300 g L- ) to produce higher concentrations
of -carotene. Further experience has confirmed
the infeasibility of doing this.
In theory, batchwise growth and harvesting is es-
sentially more stable than continuous growth and

harvesting because the constituent growth medium
and inoculum can be controlled better. We have
confirmed this in the pilot-plant operations.
Properly managed pond cultures of D. salina are,
however, very stable, and have been maintained for
more than one year at harvestable levels of -
carotene. This encourages the prospect of semicon-
tinuous or continuous harvesting. Laboratory ex-
periments (Moulton et al., 1987) and certain experi-
ments in pilot-plant ponds have demonstrated the
feasibility of maintaining cultures under a regime
of regular harvesting. In other experiments with
semicontinuous harvesting in pilot plant ponds, the
cultures have declined. One problem was remedied
when it was found that certain types of pumps used
to transport unharvested cells back to the growing
pond had injured the cells to the point where they
were unhealthy or inviable. Other problems remain
to be solved.
The question of whether the growth medium can
be recycled after harvesting is vital to the cost of
running a production plant. There does not appear
to be any buildup of growth-restricting compo-
nents in pond cultures maintained for long periods
of time, and two laboratory experiments indicated
that if cells were "harvested" by filtration and the
medium recycled, the cultures remained viable.
However, the feasibility of repeated recycling of the
medium after harvesting has not yet been demon-
strated in ponds.
Harvesting and extraction of -carotene
Various methods of harvesting D. salina have been
reviewed by Borowitzka et al. (1985) and Borowitz-
ka & Borowitzka (1986). We have evaluated several
methods as cost-effective and have taken out a
provisional patent on one.
Extraction of -carotene from the harvested bio-
mass is relatively easy due to the absence of a cell
wall in D. salina. For example, a simple convention-
al solvent extraction from filter-harvested biomass
was patented by Ruane (1974).
The harvested algae may also be spray- or freeze-
dried to form an algal powder that is rich in 3-
carotene. The algal powder can be incorporated
101
into animal feedstuffs or other products as a source
of -carotene. Alternatively, extracted /5-carotene
can be formulated in a variety of ways. Our first
product is a 1.5% (w/v) solution of -carotene in
vegetable oil which can be used for a variety of pur-
poses, including direct incorporation into marga-
rine and encapsulation as a nutritional supplement.
Bioeconomic modelling
Many factors need to be addressed in assessing the
commercial feasibility of producing 3-carotene
from D. salina. We found that by putting all of the
components of the system into a simple computer
model we had a powerful tool for both assessing
the commercial feasibility and directing attention
to the most expensive parts of the system.
In building the model, the first problem was to
estimate the productivity of the ponds in terms of
1-carotene produced per unit area per unit time.
Most of the available data were in the form of
growth curves from ponds set up batchwise at vari-
ous times of the year with various experimental
treatments. To predict the obtainable carotenoid
production, we applied the logistic growth model,
which appeared to fit the observed curve of carote-
noid increase quite well. The form of the equation
was not critical to the accuracy of the predictions,
and other self-limiting relationships, such as the
Gompertz equation, could have been used with
similar results. The differential form of the logistic
equation is:
dN rN (K-N)
(eq. 1)
dt K
where N = concentration or total amount of S-
carotene, r = "intrinsic" growth rate per unit time,
and K = "carrying capacity" or maximum level of
-carotene.
For any value of N (expressed as -carotene con-
centration or total amount in the pond), the rate of
production of -carotene (dN/dt) is obtained. The
maximum production of -carotene per unit time
occurs when N = K/2 and is rK/4 units of -
carotene per unit time.
102
A particular harvesting regime removes an
amount of /3-carotene over a period of time, and
the result is predicted by equation (1). In all cases
discussed below, continuous harvesting is em-
ployed.
Verification of the applicability of the model
came from laboratory experiments in which the ac-
tual yield of carotenoid was somewhat higher than
predicted by the logistic relationship (Moulton et
al., 1987).
In the model, after the harvested volume is re-
moved from the ponds at a particular rate, it is put
through a simulated concentration step which has
an associated concentration factor and loss of /-
carotene. The concentrate is then put through the
step in which /3-carotene is extracted and formulat-
ed into the final product, again with concentration
and loss factors.
The model predicts the cost of production of /3-
carotene. The cost of every component of the
production system was determined and its relation-
ship to other components of the model was built in.
We describe here the general structure of the model
and the most important relationships (Fig. 1). The
- _r lengt growing
Fig. i. Bioeconomic model of p-cot
arrows and theaxproportional
evanshiprre-iee , POND relationships
SRFACE are indicatepac
in[ ea-arene
aro te p oper AREA
) AND
Fig. 1. Bioeconomic model of
arrows and the proportional relationships are indicated by ,x.
model is designed to calculate the necessary size of
production plant given a particular size of annual
production of /3-carotene. It can, of course, assume
a fixed area of ponds and calculate the annual
productivity given the growth rate and loss
parameters.
The components of the model can be divided
into three categories: variable costs associated with
growing the algae, variable costs associated with
harvesting and extracting /3-carotene, and fixed
costs that do not vary with a particular size of
production plant. The variable costs associated
with growing the algae vary predominantly with the
surface area of the ponds; the cost of the earth-
works varies as the square root of the surface area,
and the evaporative make-up water varies in direct
proportion to the surface area. The costs of fertiliz-
ers is modelled to be directly proportional to the
amount of algal biomass and carotenoid produced,
and is hence a fixed cost in the current exercise. The
running cost of harvesting and extraction are
directly proportional to the volume of harvest or
concentrate and the capital costs have been scaled
to the seven-tenths power of the respective volumes.
(The "seven-tenths rule" is commonly used in en-
gineering practice, and its validity was borne out by
our preliminary cost estimates.)
of
ra tion a> T')
d by
Optimization of /3-carotene concentration of
/ ( harvest o
harvesting
An interesting relationship exists between variable
costs of growing algae and variable costs in har-
vesting and extraction. The costs of growing algae
are minimized when the ponds are harvested at
their highest productivity, which is, in theory, when
the -carotene concentration is at half the maxi-
mum level (K/2). However, the costs of harvesting
and extracting /3-carotene are minimized when the
concentration of /3-carotene in the harvest and con-
centrate are maximum, since the variable costs are
associated with the volume of harvest and concen-
trate. The cost of producing -carotene is
-carotene production. The rela-
minimized when ponds are harvested at a rate
which is less than the maximum possible, but at
which the /3-carotene level in the ponds is a com-
 103

c _
o
0

w,
a,.
0: w

z '0
CD
OL
~
vr
Of
C 0
E
CD

CD

3 4 5 6 7 8 9 10
CONCENTRATION OF BETA CAROTENE IN HARVEST
(arbitrary units)

Fig. 2. Relationships among concentration of -carotene in harvest (lower axis) and cost of production (-), pond area (X-X-X),
and harvest volume (· - - ). Maximum -carotene concentration ("carrying capacity") was 10 units. Cost of production was
minimized at 8.5 units of -carotene concentration.

promise between maximum productivity and
reduced harvesting costs. This can be predicted in
the model by varying the level of -carotene at
which the ponds are harvested (N) and maintaining
growth rate, annual production and other
parameters constant (Fig. 2). The varying - cial. For example, cells are often observed to con-
carotene levels determine the pond area, harvest
volume, etc., and result in varying costs of produc-
tion (Fig. 1). The costs associated with harvest vol-
ume (which also affects the extraction step) are
relatively greater than those associated with pond
area, and the optimal -carotene concentration for
harvesting is shown to be 0.85 K, compared to
0.5 K for maximum productivity (Fig. 2).
Although this optimization is significant, it is rela-
tively small compared to the total cost of produc-
tion; the cost saving in moving from harvesting
from a E-carotene level of 0.5 K to 0.85 is 19°%7 of
the total. This is a reflection of the relatively high
proportion of fixed costs in the system.
A corollary of the above analysis is that if the
level of f-carotene at harvest can be raised in any
way other than waiting for further growth, the cost
of production will be reduced. Thus, factors that
increase productivity by increasing the carrying ca-
pacity are more beneficial than those that affect
growth rate, and any method that concentrates cells
in the pond before harvesting is likely to be benefi-
centrate at the surface of the pond and be driven by
wind to the shore of the pond, and if this phenome-
non could be exploited, it could significantly re-
duce the harvesting costs.
The analysis also suggests that certain batch-
mode growth and harvesting regimes would be
more productive than continuous growth and har-
vesting at the relatively high level of -carotene
(0.85 K) in the harvest. This is caused by the fact
that, under logistic growth, the most productive
harvesting regime is one in which the concentra-
tions before harvest and after harvest are symmetri-
cal about 0.5 K. That is, a batch-mode (or semicon-
tinuous) harvesting regime that harvested at a
concentration 0.85 K and reduced the concentra-
tion to 0.15 K would be more productive than con-
tinuous harvesting at 0.85 K.
104
Cost-benefit analysis of carbon dioxide addition
The model can also be used for testing potential
improvements in productivity and efficiency. The
cost and benefit of instigating an improvement can
be added to the model and the effect on the cost of
producing -carotene can be assessed. By this
means, we can avoid the fallacy of considering the
cost and benefit of an improvement in isolation from
its effect in the whole system. For example, we might
speculate that a supply of carbon dioxide can im-
prove the production of 3-carotene by up to 75070.
The cost of CO 2 in Australia is approximately
A$0.50 per kilogram of carbon. If the price of 3-
carotene is assumed to be A$500 kg-', this gives
the price of carbon in (3-carotene (C40H 56) as
A$558 kg-' and implies that a 0.1°70 incorporation
of CO 2 into algal -carotene would be cost-
effective. Assuming that -carotene makes up 10%
of the dry weight of D. salina, the necessary rate of
assimilation of CO 2 (approximately 1%) appears
to be well within the published rates for other
microalgal systems (e.g., Goldman et al. (1981) ob-
served 40-60% assimilation of CO 2 with
Scenedesmus obliquus in freshwater culture). How-
ever, when the costs of administering CO 2 are
modelled along with a 7507o increase in productivi-
ty, the necessary incorporation of carbon from
CO 2 into f-carotene is approximately 1.407o, or ap-
proximately 14%7 incorporation into the total cell
biomass. This calculation suggests that CO 2 addi-
tion is worth investigating, but that it is an order of
magnitude less attractive than in the previous sim-
ple calculation.
Conclusions
The transition from laboratory cultures to outdoor
pond cultures was accompanied by a reduction in
productivity, 44%°7 in one experiment. The scale-up
from small ponds (125-625 m2 ) to 6000-m2 ponds
was not accompanied by a reduction in productivi-
ty, and the larger ponds showed some advantages in
terms of relatively reduced leakage and disturbance
by rain. The scale-up to production-plant size (five
5-ha ponds) is expected to follow this favorable
result.
Bioeconomic modelling proved to be a useful
tool in predicting the cost of production of 3-
carotene at the production-plant scale and in high-
lighting the expensive components of the system.
The most cost-effective harvesting strategy involves
allowing the 3-carotene content of the ponds to in-
crease beyond the level at which the greatest
productivity would be obtained and, in the example
modelled, the optimal level of carotenoid for har-
vesting was at 857o of the maximum level of 3-
carotene. This result was due to the relatively high
cost of harvesting and extracting 3-carotene com-
pared to producing 3-carotene in the ponds.
Another feature of the bioeconomic model was
the relatively high proportion of fixed costs in the
system.
Acknowledgements
The research and development was initially sup-
ported by Roche Algal Biotechnology, and from
1983 by an Australian Government Public Interest
contract to Wesfarmers Algal Biotechnology.
Thanks are due to a team of dedicated scientists
and support staff including Michele Burford, Mark
Coleman, Bill Embry, Mike Hoxey, David Kelly,
Richard Knuckey, Tracy Maguire, Bruce Mackay,
Ian Meares, David Melbourne, Tom Mercz, David
O'Sullivan, Warren Potts, Nathan Sammy, and Ted
Sommer. Kevin Elphick provided the computer
programming and graphics.
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