Appl Microbiol Biotechnol (2001) 57:316–322
DOI 10.1007/s002530100786
O R I G I N A L PA P E R
Z.-Y. Wen · F. Chen
A perfusion–cell bleeding culture strategy for enhancing
the productivity of eicosapentaenoic acid by Nitzschia laevis
Received: 30 May 2001 / Received revision: 30 June 2001 / Accepted: 6 July 2001 / Published online: 17 August 2001
© Springer-Verlag 2001
Abstract A perfusion–cell bleeding culture strategy was
developed for enhancing the productivity of eicosa-
pentaenoic acid (EPA) by the diatom Nitzschia laevis. As
the strategy combined the concepts of continuous culture
and perfusion culture, it allowed continuously and simul-
taneously harvesting the algal cells and removing inhibi-
tory compounds during the cultivation. Compared with a
single operation of continuous culture, the perfusion–cell
bleeding culture greatly enhanced the steady-state bio-
mass concentration, biomass productivity, EPA yield,
EPA productivity and glucose utilization efficiency. The
perfusion–cell bleeding culture also allowed higher bio-
mass productivity and EPA productivity than the single
perfusion culture did. At a bleeding rate of 0.67 day–1
and a perfusion rate of 0.6 day–1, the EPA productivity
achieved 175 mg l–1 day–1, which is the highest ever re-
ported in microalgal cultures.
Introduction
The nutraceutical and pharmaceutical significance of
eicosapentaenoic acid (EPA, C20:5ω-3) and doc-
osahexaenoic acid (DHA, C22:6ω-3) have been well
demonstrated in the prevention and treatment of various
human diseases such as arrhythmia, atherosclerosis, car-
diovascular diseases and cancer (Nettleton 1995). As fish
oil products, the traditional sources of EPA and DHA,
possess a number of problems, such as poor taste, insta-
bility and high purification cost, many research efforts
have focused on the use of microalgae as alternatives for
EPA and DHA production (Radwan 1991; Apt and
Behrens 1999).
In microalgal mass culture, heterotrophic culture
mode is preferred, as it eliminates the requirement for
light, and hence offers the possibility of greatly enhanc-
Z.-Y. Wen · F. Chen (✉)
Department of Botany, The University of Hong Kong,
Pokfulam Road, Hong Kong, PR China
e-mail: sfchen@hkusua.hku.hk
Tel.: +852-2299-0309, Fax: +852-2299-0311
ing cell density by using high cell density culture tech-
niques such as fed-batch and perfusion cultures. Our pre-
liminary results have shown that the diatom Nitzschia
laevis is a good EPA producer in heterotrophic condi-
tions (Wen and Chen 2000a).
To make the EPA production by N. laevis cost-com-
petitive over fish oil products, high cell density tech-
niques have been applied to the algal culture and, as a
consequence, the EPA yields have been greatly enhanced
(Wen 2001). However, from a processing point of view,
the EPA productivity achieved is still relatively low. To
obtain high EPA productivity, one strategy is to enhance
the growth rate of the alga so that the maximal cell den-
sity and EPA yield can be reached within a relatively
short period of time. However, the growth rate of the al-
ga is intrinsically limited by the cellular physiology. An-
other method for achieving high EPA productivity is to
use a continuous culture technique, which generally al-
lows higher productivity than batch or even fed-batch
does. In our previous study, an EPA productivity of
73 mg l–1 day–1 had been achieved in continuous culture
(Wen 2001); the EPA productivity obtained was much
higher than other microalgal continuous cultures (Molina
Grima et al. 1993, 1994; Otero et al. 1997a, b). However,
both the glucose utilization efficiency and the steady-
state cell density were still low, presumably due to the
accumulation of inhibitory metabolites in the medium.
To alleviate the inhibition of metabolic by-products, a
continuous culture with cell-recycle technique, frequent-
ly denoted as “perfusion” culture in animal cell cultiva-
tion, can be employed by which the spent medium is re-
moved while the cells are retained in the fermenter.
In this study, we combined the concepts of both per-
fusion and continuous cultures by developing a “perfu-
sion–bleeding” strategy in which cell-free spent medium
was removed while the EPA-containing algal cells were
continuously harvested. The aim of the present study
was to develop an efficient perfusion-cell bleeding strat-
egy for enhancing EPA productivity by N. laevis.

Fig. 1 Schematic diagram of the perfusion–bleeding culture
system. The settler consists of a cylinder part and a cone part. Di-
mensions of the settler: height of the cylinder, 5.5 cm; height of
the cone, 5.5 cm; internal diameter (i.d.) of the cylinder, 5 cm; i.d.
of pipes number 1 and number 3, 3 mm; i.d. of pipe number 2,
5 mm. Pipe number 1 is connected to the settler in the middle part
of the cylinder
Materials and methods
Microalga and subculture
The diatom N. laevis UTEX 2047 was used. The cells were main-
tained in LDM medium supplemented with 5 g l–1 glucose and
30 mg l–1 Na2SiO3·9H2O. The LDM medium consisted of (per li-
ter) 1 g tryptone, 892 ml artificial seawater, 100 ml Bristol solu-
tion, 6 ml PIV metal solution, 1 ml stock solutions of biotin
(25.0×10–5 g l–1) and 1 ml vitamin B12 (15.0×10–5 g l–1) (Starr and
Zeikus 1993). The pH of the medium was adjusted to 8.2 prior to
autoclaving at 121°C for 20 min. The cells were incubated in
500 ml Erlenmeyer flasks containing 200 ml medium in an orbital
shaker (200 rpm, 20°C) in darkness.
Bioreactor system
The perfusion–bleeding experiments were conducted in a 3.7 l fer-
menter (type KLF 2000, Bioengineering, Switzerland) with 2.2 l
working volume. Agitation was provided by three turbine impel-
lers (4.8 cm in diameter) with speed varying in the range
500–700 rpm to maintain the dissolved oxygen (DO) level over
50% saturation. Compressed air (at 600 ml min–1) was supplied to
the cultures through a circular stainless steel sparger. The modi-
fied LDM medium, which contained 20 g l–1 glucose, 120 mg l–1
Na2SiO3·9H2O, 1.6 g l–1 tryptone, 0.8 g l–1 yeast extract, 8 g l–1
NaCl and 0.1 g l–1 CaCl2, was used in the initial batch culture. The
pH was adjusted to about 8.2 before autoclaving at 121° C for
20 min. During the experiment, pH and DO were monitored using
a pH probe and a DO probe, respectively.
Figure 1 shows the schematic diagram of the perfusion–bleed-
ing system. A three-port settling tank was used to accomplish the
perfusion operation. Cell suspension was continuously transferred
by a peristaltic pump into the settler from pipe number 1; algal
cells settled in the settler and the cell-free spent medium was re-
moved from pipe number 3 by another peristaltic pump. The flow
rate of the cell suspension transferred to the settler was adjusted to
double that of spent medium (perfusion rate); thus, the settled cells
could be returned to the fermenter via pipe number 2. During per-
fusion–bleeding cultures, fresh medium was continuously added to
the fermenter, while cells were bled through an overflow tube to
maintain constant culture volume. By controlling the feeding rate
of fresh medium (F) and perfusion rate of spent medium (F2), the
bleeding rate (F1) could be adjusted.
Samples were taken every day during the continuous opera-
tion. For a given operation condition, a steady state was consid-
317
ered to have been established after at least three volume changes,
with the variation in cell dry weight and residue glucose concen-
tration less than 3% in three consecutive samples, without an up-
ward or downward trend.
Analyses
Cell dry weight concentration (DW) was determined by drying as
reported previously (Wen and Chen 2000a). Glucose concentration
was determined by the 3,5-dinitrosalicylic acid method (Miller
1959). The lyophilized cells were analyzed for fatty acid composi-
tion. Fatty acid methyl esters were prepared by transmethylation
with methanol-acetyl chloride (Jiang and Chen 1999) and ana-
lyzed by GC (Wen and Chen 2000a).
Results
Perfusion culture without cell bleeding
Experiments were first conducted with a single perfusion
operation without bleeding cells. As shown in Fig. 2A,
the perfusion operation was started on day 6; the perfu-
sion rate (F) was stepwise increased from 500 to
1,500 ml day–1. In heterotrophic culture conditions, the
cells of N. laevis tended to aggregate (Wen 2001) and so
could settle more easily. In this experiment, it was found
that at F below 1,500 ml day–1, nearly all the cells settled
and the cell dry weight concentration in the spent medi-
um was thus negligible (less than 0.2 g l–1) compared
with the cell concentration in the fermenter. However,
when F exceeded 1,500 ml day–1, significant wash-out of
the cells occurred. Therefore, the upper limit of perfu-
sion rate for no-cell wash-out was determined as
1,500 ml day–1, and F was not increased further during
the perfusion cultivation to avoid losing cells from the
settler (Fig. 2A). Meanwhile DW increased monotonical-
ly, reaching a maximum at day 13, and then leveled off.
It was also found that glucose was depleted after day 10,
suggesting that the culture is likely limited by glucose.
Figure 2B shows that EPA content was relatively stable
with slightly less at the initial culture stage. The trend of
EPA yield was similar to that of DW. The maximal EPA
yield (approx. 700 mg l–1) was obtained at the later stage
(days 12–18), when DW was maintained at 27 g l–1
(cf. Fig. 2A, B).
Perfusion culture with cell bleeding
Figure 3 shows the time courses of perfusion cultures
with cell bleeding. The perfusion was started at day 6 by
feeding the medium with 20 g l–1 glucose (S0=20 g l–1);
no cells were bled during this period. The perfusion rate
was increased stepwise to the highest level of 1,500 ml
day–1 (i.e., 0.6 day–1) at day 10. To avoid washing-out of
the cells, the perfusion rate was maintained at this level
over the following days. During days 6–12, as no cells
were bled, cell concentration (DW) increased monotoni-
cally. When DW attained a high value on day 12, cell
bleeding was started. After another 6 days of operation, a
318
Fig. 2A, B Perfusion culture kinetics of Nitzschia laevis with 20 g
l–1 glucose in feed medium (S0=20 g l–1). A Time course of dry
cell weight (black triangle), glucose (white circle) and volumetric
flow rate of feed medium (line); B EPA content (white square) and
EPA yield (black square)
Fig. 3 Time courses of perfusion–bleeding culture of N. laevis.
Dry cell weight (black triangle), glucose (white circle), volumetric
flow rate of spent medium perfusion and cell suspension bleeding
(lines). The glucose concentration in feed medium (S0) was 20 g
l–1 from days 6–18 and reduced to 15 g l–1 from day 18 onwards
steady state was established as DW and residual glucose
were kept almost constant. At this steady state, however,
it was found that certain amounts of residual glucose ex-
isted in the medium (about 3.5 g l–1), corresponding to a
glucose utilization efficiency of 83%. To reduce the re-
sidual glucose level and increase the efficiency of glu-
cose utilization, at day 18, the glucose concentration in
feed medium was reduced to 15 g l–1 while other culture
conditions were kept constant. It was found that residual
glucose subsequently dropped to nearly zero (Fig. 3). At
this new feed glucose concentration (S0=15 g l–1), a qua-
si-steady state was reached at day 23, before the bleed-
ing rate was switched to a higher level. The switch of
bleeding rate was continued until the end of culture
when DW significantly decreased and residual glucose
increased.
Based on the results as shown in Fig. 3, the kinetic
parameters about cell growth, glucose consumption, and
EPA production as functions of bleeding rate were calcu-
lated.
The changes in cell concentration (X) and glucose
concentration (S) in the fermenter are expressed as:
(1)
(2)
where V is culture volume, S0 is feed glucose concentra-
tion, µ is specific growth rate, Yx/s is cell growth yield on
glucose, F, F1, F2 are volumetric flow rates of feed me-
dium, culture bleeding, and spent medium, respectively.
Here, F=F1+F2 since the culture volume was kept con-
stant.
When the quasi-steady state is reached, X and S are
independent of t, i.e., , thus, Eqs. 1 and 2 can
be derived as:
(3)
(4)
where B is defined as the specific bleeding rate. The spe-
cific dilution rate (D) of feed medium is defined as ,
thus, Eq. 4 can be derived as:
(5)
The biomass and EPA productivity under different bleed-
ing rates are expressed as:
Biomass productivity = µ × X = B × X (6)
EPA productivity = µ × EPA yield (7)
= B × EPA content × X
The calculations of kinetic parameters were based on
above equations. The results are presented in Figs. 4 and
5. As shown in Fig. 4A, the steady-state DW were kept
at relatively high levels at B<0.67 day–1, and decreased
sharply when B exceeded 0.67 day–1. Correspondingly,
the residual glucose concentrations with B showed a re-
verse trend. At B=0.67 day–1, the biomass productivity
reached the maximum of 6.75 g l–1 day–1. Figure 4B
shows that lower bleeding rates (B) resulted in higher
glucose utilization efficiency (E), while cell growth yield
(Yx/s) increased with B in the range 0.22–0.67 day–1, and
leveled off at a higher B level. The poor growth yield at
low bleeding rate may be explained by an increase in
maintenance energy requirement (Chen and Johns 1996)
 319

Fig. 5 Steady-state EPA yield (black square) and EPA productivi-

ty (white square) in perfusion–bleeding cultures of Nitzschia lae-
vis (with perfusion rate fixed at 0.6 day–1). Data are means of three
consecutive samples at steady state and error bars show standard
deviations

percentage of total unsaturated fatty acids (unsatd) kept

Fig. 4 Steady-state cell dry weight concentration (DW) (black tri-
angle), residual glucose concentration (white circle) and biomass
productivity (white triangle) (A); cell growth yield on glucose
(Yx/s) (white diamond) and glucose assimilation efficiency (E)
(black diamond) (B) at different bleeding rates in perfusion bleed-
ing cultures of N. laevis (with perfusion rate fixed at 0.6 day–1).
Data are means of three consecutive samples at steady state and
error bars show standard deviations
almost constant, while the unsaturation degree of fatty
acids (∇/mol) increased and the cellular content of total
fatty acids (TFA) decreased with increasing B from 0.22
to 0.79 day–1.
With respect to EPA production, Table 1 shows that
EPA contents were relatively stable and slightly higher at
intermediate bleeding rates (0.38–0.52 day–1). The
changes in EPA yield and EPA productivity with B are
presented in Fig. 5. The highest EPA yield (321 mg l–1)
was obtained at B=0.38 day–1, while the EPA productivi-
ty reached the highest level (approx. 175 mg l–1 day–1) at
a relatively high bleeding rate (0.67 day–1).

The fatty acid compositions of N. laevis under differ-
ent bleeding rates (B) are shown in Table 1. The major
fatty acids of the alga were C14:0, C16:0, C16:1, and
C20:5 and the cells also contained small amounts (less
than 5% of total fatty acids) of C14:1, C18:1, C18:2,
C18:3(ω3+ω6) and C20:4. The proportions of each indi-
vidual fatty acid against B were quite different. The pro-
portions of C14:0 and C16:0 were relatively stable. In
contrast, the percentage of C16:1 decreased, while that
of C20:5 increased with B. Table 1 also shows that the
Comparison of different culture methods
To compare the performance of the perfusion-cell bleed-
ing culture with that of single perfusion culture and con-
tinuous culture, various parameters obtained from the
three culture strategies are given (Table 2). This shows
that the perfusion–bleeding operation greatly enhanced
the biomass and EPA productivity. The glucose utiliza-
tion efficiency (E) in the perfusion–bleeding culture was
higher than that in the single continuous culture. Com-

Table 1 Fatty acid composi-

tions (% total fatty acids) of Fatty acid B (day–1)
perfusion–bleeding cultures of
Nitzschia laevis at different 0.22 0.38 0.52 0.67 0.79
bleeding rates (with perfusion
rate fixed at 0.6 day–1). Data 14:0 9.42±0.12 9.73±0.84 11.18±0.53 11.77±0.08 11.37±0.11
are expressed as mean ± SD of 14:1 0.93±0.02 0.91±0.09 0.73±0.11 0.56±0.09 0.52±0.18
three consecutive samples at 16:0 25.48±1.08 22.77±1.08 23.25±0.18 23.94±0.50 23.22±0.63
steady state 16:1 39.53±0.19 35.40±1.93 32.41±1.38 28.00±0.67 26.57±0.56
18:1 2.70±0.65 2.70±0.41 3.30±0.50 3.84±0.22 3.99±0.02
18:2 3.38±0.24 2.33±0.09 2.49±0.18 2.79±0.26 3.20±0.19
a unsatd: percentage of unsatur-
18:3(ω-6) 0.70±0.08 0.74±0.03 0.90±0.18 1.10±0.05 1.14±0.11
18:3(ω-3) 0.78±0.15 1.59±0.32 2.26±0.0.32 3.41±0.26 3.96±0.17
ated fatty acids (% of total fatty 20:4 3.32±0.32 2.98±0.42 2.42±0.13 2.14±0.27 1.85±0.30
acids) 20:5 13.70±0.54 19.12±1.57 21.00±0.56 22.43±0.33 24.14±0.36
b ∇/mol: degree of unsaturation
Unstada (% TFA) 65.10±1.14 67.50±1.69 65.36±0.72 64.29±0.55 65.41±0.71
= [1.0 (%monoenes) + 2.0 ∇/molb 1.352±0.041 1.589±0.051 1.656±0.022 1.722±0.006 1.809±0.019
(%dienes) + 3.0 (%trienes) TFA content (% DW) 18.59±0.76 14.54±0.86 13.24±0.32 11.52±0.40 10.36±0.43
+ 4.0 (%tetraenes) EPA content (% DW) 2.54±0.02 2.76±0.11 2.79±0.04 2.59±0.07 2.51±0.10
+ 5.0 (% pentaenes)]/100
320
Table 2 Comparison of the pa-
rameters obtained by different Parameter Unit Culture method
culture methods. The perfusion
rate was fixed at 0.6 day–1 Perfusion Continuous Perfusion–bleeding
(Wen 2001)

Max. biomass g l–1day–1 2.09 2.82 6.75

productivity
Max. EPA mg l–1day–1 55.5 73.04 174.6
productivity

aD
Max. EPA yield mg l–1 721.9 158.2 320.9
refers to the dilution rate
opti
of continuous culture in which EPA content (% DW) 2.59 2.65 2.76
maximal EPA productivity was
Dopti or Boptia day–1 – 0.51 0.67
obtained, while Bopti refers to
the bleeding rate of perfu- E valueb % – 57.3 90.2
sion–bleeding culture in which
maximal EPA productivity was Advantage Alleviating Continuous Continuous
obtained metabolites cell-harvest cell-harvest
b The value of E refers to that inhibition and alleviating
obtained at the optimal dilution inhibition
rate (Dopti) in continuous cul- Disadvantage Continuous Metabolites Complex in operation
ture or that obtained at the opti- cell-harvest inhibition
mal bleeding rate (Bopti) in per- not available
fusion–bleeding culture

pared with perfusion culture, however, the EPA yield
was lower in the perfusion–bleeding culture.
Discussion
Many microalgal species contain EPA and their poten-
tials for EPA production have been widely investigated
(Seto et al. 1984; Yongmanitchai and Ward 1991; Cohen
1994; Kitano et al. 1998; Vazhappilly and Chen 1998;
Cerón García et al. 2000; Wen and Chen 2000b). How-
ever, all these investigations were concentrated on the
optimization of medium components and environmental
factors. The development of efficient culture techniques
for enhancing EPA productivity was rarely attempted.
From an industrial process point of view, development of
process for high EPA productivity is crucial to success.
This is why the present research was conducted.
In heterotrophic culture of N. laevis, there may exist
inhibitory effects on cell growth as a result of metabolic
by-product accumulation especially in dense cultures
(Schügerl 2000). As a matter of fact, a large number of
extracellular metabolic products have been identified in
the cultures of microalgae in general (Vílchez et al.
1997; Srivastava et al. 1999) and diatoms in particular
(Eppley 1977). To eliminate or attenuate the inhibitory
effects, a continuous culture with cell recycling, such as
perfusion culture, may be useful (Chen and Johns 1995,
1996).
The perfusion culture of N. laevis resulted in a high
cell density of the algal culture, which led to a high EPA
yield (Fig. 2). However, EPA was not continuously re-
covered during the cultivation as the cells were only har-
vested at the end of culture. To evaluate the “rate” of
EPA production, here, we define the volumetric EPA
productivity in the perfusion culture (without cell bleed-
ing) as the quotient of EPA yield over culture time (t).
It was found that EPA productivity at day 13 (i.e.,
721.9/13=55.5 mg l–1 day–1) was the highest among all
the data calculated by instant EPA yield over the corre-
sponding culture time. Compared with the EPA produc-
tivity (73 mg l–1 day–1) obtained in continuous cultures
(Wen 2001), the EPA productivity obtained in the perfu-
sion culture was lower. The result suggests that a contin-
uous cell harvest during the cultivation would facilitate a
high EPA productivity. Consequently, a cell bleeding op-
eration was integrated into the perfusion culture in this
study.
The perfusion–cell bleeding culture method was de-
signed so that it could maintain a continuous harvest of
the EPA-containing algal cells by bleeding in combina-
tion with the removal of potential inhibitory metabolites
through perfusion. Such a culture method gave superior
results compared with either a single continuous culture
or a perfusion culture. For example, the maximum bio-
mass productivity of 6.75 g l–1 day–1 was achieved,
which was much higher than that obtained in single con-
tinuous culture or perfusion culture (Table 2). Over the
bleeding rates of B=0.22–0.79 day–1, the residual glucose
was relatively low, corresponding to a high glucose utili-
zation efficiency (Fig. 4B). Whereas in our previous in-
vestigation of continuous culture (Wen 2001), relatively
high levels of glucose were found at a dilution rate great-
er than 0.2 day–1, the glucose utilization efficiency was
rather low. It is understandable that the EPA yield ob-
tained in perfusion–cell bleeding culture should be lower
than that in perfusion culture because the cell concentra-
tion was reduced due to continuous removal of the cells
while the EPA contents in the two culture systems were
nearly identical (Table 2).
Over the bleeding rates ranging from 0.22 to
0.79 day–1, the fatty acid profiles of the alga were similar
 321
Table 3 Comparison of EPA productivity in various algal species and culture conditions. Unless specified, the algae were grown in pho-
toautotrophic conditions

Algal species Culture vessel Culture mode EPA productivity Reference

(mg l–1 day–1)

Phaeodactylum tricornutum Glass tanks Continuous 25.1 Yongmanitchai and Ward 1992
Isochrysis galbana Fermenters Continuous 7.2 Molina Grima et al. 1993
Isochrysis galbana Fermenters Continuous 15.3 Molina Grima et al. 1994
Monodus subterraneus Erlenmeyer flasks Continuous 25.7 Cohen 1994
Monodus subterraneus Flat plate reactors Semi-continuous 58.9 Qiang et al. 1997
Phaeodactylum tricornutum Glass tubes Semi-continuous 5.2 Otero et al. 1997a
Isochrysis galbana Glass tubes Semi-continuous 4.6 Otero et al. 1997b
Porphyridium cruentum Flasks Batch 3.6 Akimoto et al. 1998
Isochrysis galbana Cylindrical fermenters Continuous 23.8 Fernández Sevilla et al. 1998
Nannochloropsis sp. Tubular photobioreactors Continuous 32.0 Zittelli et al. 2000
Phaeodactylum tricornutuma Glass vessels Batch 33.5 Cerón García et al. 2000
Nitzschia laevisb Fermenters Perfusion–cell 174.6 This study
bleeding
a The alga was grown in mixotrophic growth conditions; b The alga was grown in heterotrophic growth conditions

to those given in previous reports (Wen and Chen 2000a,
b). The degree of unsaturation of the fatty acids in-
creased and the cellular total fatty acid content decreased
with increasing B from 0.22 to 0.79 day–1. Similar results
were reported in other microalgal cultures (Otero et al.
1995). The reason might be that, when increasing bleed-
ing rate, the neutral lipids previously stored in the cells
as an energy reservoir are consumed to sustain higher
physiological and metabolic activities. Because neutral
lipids contain mainly saturated and monounsaturated fat-
ty acids (Dunstan et al. 1993), the proportion of polyun-
saturated fatty acids (stored in polar lipids) would in-
crease as the neutral lipids were consumed. Thus, the
consequence of increasing the bleeding rate is that less
lipids/TFA with more unsaturated fatty acids are pro-
duced in the cells.
The present perfusion–cell bleeding culture system
gave superior results in terms of EPA productivity. Un-
der the culture conditions with 0.67 day–1 bleeding rate
and 0.6 day–1 perfusion rate, EPA productivity reached
175 mg l–1 day–1, 2.3-fold and 3.1-fold of that achieved
in the single perfusion and continuous cultures, respec-
tively. As shown in Table 3, the EPA productivity ob-
tained in this work has been the highest so far reported in
microalgal cultures. Based on all the above results, the
perfusion–bleeding operation in this work was consid-
ered to be a superior culture technique for EPA produc-
tion by N. laevis. Further efforts should be devoted to the
development of an efficient downstream process for the
extraction of fatty acids from the algal biomass and EPA
purification from the fatty acid mixture so that a cost-
effective EPA production process by the diatom N. laevis
can be commercialized.
References
Akimoto M, Shirai A, Ohtaguchi K, Koide K (1998) Carbon diox-
ide fixation and polyunsaturated fatty acid production by the
red alga Porphyridium cruentum. Appl Biochem Biotechnol
73:269–278
Apt KE, Behrens PW (1999) Commercial developments in micro-
algal biotechnology. J Phycol 35:215–226
Cerón García MC, Fernández Sevilla JM, Acién Fernandez FG,
Molina Grimal E, García Camacho F (2000) Mixotrophic
growth of Phaeodactylum tricornutum on glycerol: growth
rate and fatty acid profile. J Appl Phycol 12:239–248
Chen F, Johns MR (1995) A strategy for high cell density culture
of heterotrophic microalgae with inhibitory substrates. J Appl
Phycol 7:43–46
Chen F, Johns MR (1996) High cell density culture of Chlamydo-
monas reinhardtii on acetate using fed-batch and hollow-fibre
cell-recycle systems. Bioresour Technol 55:103–110
Cohen Z (1994) Production potential of eicosapentaenoic acid by
Monodus subterraneus. J Am Oil Chem Soc 71:941–945
Dunstan GA, Volkman JK, Barrett SM, Garland CD (1993)
Changes in the lipid composition and maximisation of the
polyunsaturated fatty acid content of the microalgae grown in
mass culture. J Appl Phycol 5:71–83
Eppley RW (1977) The growth and culture of diatoms. In: Werner
D (ed) The biology of diatoms. Blackwell, Oxford, pp 24–64
Fernández Sevilla JM, Molina Grima E, Carcía Camacho F, Acién
Fernández FG, Sánchez Pérez JA (1998) Photolimitation and
photoinhibition as factors determining optimal dilution rate
to produce eicosapentaenoic acid from cultures of the micro-
alga Isochrysis galbana. Appl Microbiol Biotechnol 50:199–
205
Jiang Y, Chen F (1999) Production potential of docosahexaenoic
acid by the heterotrophic marine dinoflagellate Crypt-
hecodinium cohnii. Process Biochem 34:633–637
Kitano M, Matsukawa R, Karube I (1998) Enhanced eicosa-
pentaenoic acid production by Navicula saprophila. J Appl
Phycol 10:101–105
Miller GL (1959) Use of dinitrosalicylic acid reagent for determi-
nation of reducing sugar. Anal Chem 31:426-429
Molina Grima E, Sánchez Pérez JA, García Camacho F, García
Sánchez JL, López Alonso D (1993) n-3 PUFA productivity in
chemostat cultures of microalgae. Appl Microbiol Biotechnol
38:599–605
Molina Grima E, Sánchez Pérez JA, García Camacho F,
Fernández Sevilla JM, Acién FG (1994) Effect of growth rate
on the eicosapentaenoic acid and docosahexaenoic acid con-
tent of Isochrysis galbana in chemostat culture. Appl Micro-
biol Biotechnol 41:23–27
Nettleton JA (1995) Omega-3 fatty acids and health. Chapman and
Hall, New York
Otero A, Aarredondo-Vega BO, Patiño M, Lamela T, Fábregas J
(1995) Production of eicosapentaenoic and docosahexaenoic
acids in semicontinuous cultures of the marine diatom Phaeo-
dactylum tricornutum. J Mar Biotechnol 3:82–85
322
Otero A, García D, Fábregas J (1997a) Factors controlling eico-
sapentaenoic acid production in semicontinuous culture of ma-
rine microalgae. J Appl Phycol 9:465–469
Otero A, García D, Morales ED, Arán J, Fábregas J (1997b)
Manipulation of the biochemical composition of the eico-
sapentaenoic acid-rich microalga Isochrysis galbana in semi-
continuous cultures. Biotechnol Appl Biochem 26:171–177
Qiang H, Zhengyu H, Cohen Z, Richmond A (1997) Enhancement
of eicosapentaenoic acid (EPA) and α-linolenic acid (GLA)
production by manipulating algal density of outdoor cultures
of Monodus subterraneus (Eustigmatophyta) and Spirulina
platensis (Cynobacteria). Eur J Phycol 32:81–86
Radwan SS (1991) Sources of C20-polyunsaturated fatty acids for
biotechnological use. Appl Microbiol Biotechnol 35:421–430
Schügerl K (2000) Integrated processing of biotechnology prod-
ucts. Biotechnol Adv 18:581–599
Seto A, Wang HL, Hesseltine CW (1984) Culture conditions affect
eicosapentaenoic acid content of Chlorella minutissima. J Am
Oil Chem Soc 61:892–894
Srivastava VC, Manderson GJ, Bhamidmarri R (1999) Inhibitory
metabolites production by the cynobacterium Fischerella mu-
scicola. Microbiol Res 153:309–317
Starr RC, Zeikus JA (1993) The culture collection of algae at the
University of Texas at Austin. J Phycol [Suppl] 29:90–95
Vazhappilly R, Chen F (1998) Eicosapentaenoic acid and doc-
osahexaenoic acid production potential of microalgae and their
heterotrophic growth. J Am Oil Chem Soc 75:393–397
Vílchez C, Garbayo I, Lobato MV, Vega JM (1997) Microalgae-
mediated chemicals production and wastes removal. Enzyme
Microb Technol 20:562–572
Wen ZY (2001) A high yield and productivity strategy for
eicosapentaenoic acid production by the diatom Nitzschia lae-
vis in heterotrophic culture. PhD thesis, The University of
Hong Kong, Hong Kong
Wen ZY, Chen F (2000a) Production potential of eicosapentaenoic
acid by the diatom Nitzschia laevis. Biotechnol Lett 22:727–
733
Wen ZY, Chen F (2000b) Heterotrophic production of
eicosapentaenoic acid by the diatom Nitzschia laevis: effects
of silicate and glucose. J Ind Microbiol Biotechnol 25:218–
224
Yongmanitchai W, Ward OP (1991) Growth and omega-3 fatty ac-
id production by Phaeodactylum tricornutum under different
culture conditions. Appl Environ Microbiol 57:419–425
Yongmanitchai W, Ward OP (1992) Growth and eicosapentaenoic
acid production by Phaeodactylum tricornutum in batch and
continuous culture systems. J Am Oil Chem Soc 69:584–590
Zittelli GC, Pastorelli R, Tredici MR (2000) A modular flat panel
photobioreactor (MFPP) for indoor mass cultivation of Nanno-
chloropsis sp. under artificial illumination. J Appl Phycol
12:521–526