Bioprocess Development for Large Scale Production of Anticancer Exo-Polysaccharide by Pleurotus ostreatus in Submerged Culture
S. El Sayed
In the recent years, mushroom derived active metabolites have emerged as an important class of bioactive compounds with several therapeutic applications. Most of the production methods were based on cultivation of mushroom on solid substrate in controlled temperature green house. For the production of bioactive compounds for therapeutic application, production should be carried out under sterile and well controlled condition. Thus, the interest of cultivation of mushroom in bioreactors for bioactive compound production was increased during the last few years. In the present study, mushroom cells were cultivated in submerged culture for the overproduction of anticancer exo-polysaccharides (EPS). Cell cultivation was optimized in fully controlled stirred tank bioreactor in batch and fed-batch culture to improve the process and to increase the anticancer EPS production. In the first part of this study, significant improvement in EPS production was achieved upon transferring the process from shake flask to 15 L bioreactor. Further development in the process was conducted through optimization of some process parameters in bioreactor batch culture. In fed-batch culture, among different feeding strategies, optimized glucose feeding based on using the in-line data for oxygen and carbon dioxide obtained from out-gas analyzer was the best. The maximal yield of EPS obtained was 2.1 g L-1 in optimized fed-batch culture. The obtained EPS was almost two fold higher than those obtained in batch culture.
Since, centuries, mushrooms have been eaten and appreciated for their flavor,
economical, ecological and medicinal values. Beside the known nutritional value
of mushrooms, as one of the rich sources of protein, carbohydrates and lipids
(Morais et al., 2000; Sanchez,
2004), they have been used in traditional medicine in many cultures all
over the world. Many mushrooms contain different types of antimicrobial active
compounds with potential applications against different types of bacteria (Smania
et al., 1999; Mothana et al., 2000),
fungi (Smania et al., 2003) and viruses (Brandt
and Piraino, 2000; Awadh et al., 2003). These
beside the traditional use of different types of mushroom extracts for the treatment
of many other non-microbial related diseases based on their activities as hypoglycemic
(Mizuno, 1999; Wasser et al.,
2002), antioxidant (Lin et al., 2004), anti-inflamatory
(Zhang et al., 2002), hepato-protectant (Chen
and Yu, 1993) and immune-modulator (Lin, 2004).
Moreover, many attractive polysaccharides of anti-tumor activity have been also
isolated from different types of mushrooms (Mizuno, 1999;
Lindequist et al., 2005; Wang
et al., 2005).
Traditionally, mushrooms are usually cultivated on solid substrate under controlled
temperature and humidity in green house. The main disadvantages of this method
are: long cultivation period up to several months, the difficulties to cultivate
many wild mushrooms, low yield of active metabolite, long process of bioactive
compound purification and ease of contamination by other microorganism in this
open cultivation system. Thus, submerged cultivation of mushrooms is viewed
as a promising alternative system for efficient production of mushroom medicinal
product since it can also fulfill the cGMP requirements for bioactive compounds
production. Of different medicinal mushrooms, Pleurotus ostreatus, an
edible mushroom belonging to the family Basidiomycetes, is potential source
for anticancer polysaccharide when cultivated in either solid or submerged cultures.
This polysaccharide can be produced extracellularly in submerged culture. In
our previous work the structure of this type of polysaccharide was identified
by IR and 13C NMR. The spectral pattern showed a typical highly branched
chain of peptidoglycan with 1→3 and 1→6 linkage (Daba
et al., 2005). The isolated polysaccharide posses immune-stimulatory
effect and anticancer activity against different types of cancer cells. In this
context, the current study was focused on the improvement of the production
process of this type of polysaccharide and bioprocess optimization in 15 L bioreactor
using different production processes.
MATERIALS AND METHODS
Microorganism and cultivation conditions: Basidiomycetes fungus Pleurotus ostreatus NRRL 366 was kindly provided by the agriculture research service Peoria, USA. The strain was maintained and reactivated monthly in Petri dishes containing a sterile solid potato dextrose agar medium (PDA, Oxoid, UK). Cells were incubated at 26°C for 14 days and stored in a refrigerator at 5°C.
Medium for cell growth and EPS production in bioreactor: The medium used in bioreactor experiments was composed of (g L-1): glucose, 20.0; KH2PO4, 0.46; K2HPO4, 1.0; MgSO4.7H2O, 0.5; peptone, 2.0 and yeast extract, 2.0. Glucose was sterilized separately and added to the cultivation medium before inoculation. The pH was adjusted to 5.5 after sterilization.
Inoculum preparation and cell cultivation: Pleurotus ostreatus
was initially grown on PDA medium in a petri dish for 14 days at 28°C. The
surface mycelia with spores were harvested in sterile saline solution and used
as inoculum. Cells were gently homogenized to prevent the formation of large
aggregates in submerged culture. Inoculum for bioreactor was in form of 50 mL
mycelium/spore suspension with optical density of 1 OD Shake flask cultivations
were performed using 250 mL Erlenmeyer flask with working volume of 50 mL. The
inoculated flasks were shaken at 200 rpm on a rotary shaker (Model, 4230 Innova,
New Brunswick, NJ, USA) with eccentricity of 2.0 cm and cultivation temperature
26°C. The bioreactor cultivations were carried out in 15 L stirred tank
bioreactor (Biostat-C, Sartorius BBI Systems, Melsungen, Germany) with working
volume of 9 L. The stirrer was equipped with two 6-bladded rushton turbine impellers
(di (impeller diameter)= 85 mm; dt(tank diameter)=214
mm, di/dt=0.397). Agitation was adjusted at 200 rpm and
temperature was controlled at 30°C throughout the cultivation time. Aeration
was performed using compressed air, sterilized using hydrophobic microbiological
air filter and supplied continuously to the bioreactor with rate of 1 v v-1
m-1. Air flow was adjusted and controlled using mass flow controller
(F102D, Bronkhorst High-Tech B.V., Nijverheidstraat, The Netherlands) coupled
with the control console CDU of the bioreactor. Foam was suppressed by the addition
of the antifoam agent Struktol (Schill+Seilacher Gruppe, Hamburg, Germany).
During the cultivation process, the dissolved oxygen concentrations were analyzed
using a polarographic electrode (Ingold, Mittler-Toledo, Switzerland).
During cell cultivation, the out-gas of the bioreactor was analyzed continuously using O2 and CO2 out-gas analyzer (Sartorius BBI, Melsungen, Germany). The in-line data of the out-gas analyzer was continuously recorded using MFCS supervisory control system. In fed-batch experiment (with continuous glucose feeding using CO2 out-gas data), peristaltic pump (Watson Marlow, Wilmington, MA, USA) was connected to the control system.
Sample preparation and cell dry weight determination: Samples in form of two flasks or 20 mL of broth in case of bioreactor culture were taken at different time intervals and collected in pre-weighed centrifugation tube of 50 mL (Falcon, USA), centrifuged at 5°C with 5000 rpm for 20 min. Supernatant was frozen at -20°C for sugar and EPS determination. The cell pellets were washed twice by distilled water, centrifuged again and dried in an oven at 60°C for determination of cell dry weight.
Determination of glucose: Glucose was determined in the fermentation media by enzymatic method using a glucose determination kit (Glucose kit Cat. No. 4611, Biocon Diagnostic GmbH, Burbach, Germany).
Extraction of polysaccharide from mycelial culture: After sample centrifugation,
the resulting culture filtrate was mixed with equal volume of absolute ethanol,
stirred vigorously and kept overnight at 4°C. The precipitate exo-biopolymer
was centrifuged at 10.000 g for 20 min. After discarding the supernatant, the
precipitate of pure EPS was washed separately with ethanol, acetone and ethyl
ether then lyophilized (Bae et al., 2000). After
complete lyophilisation cycle of about 4 days, the obtained precipitate was
Cultivations in shake flask culture: Cells were cultivated in shake
flask cultures to evaluate the potency of P. ostreatus for EPS production
in submerged culture. As shown in Fig. 1, cells grew reaching
the maximal cell mass of about 4.5 g L-1 after about 225 h cultivation.
The production of EPS was firstly observed in culture after 50 h cultivation
and increased gradually in parallel to cell growth. The maximal polysaccharide
production of 0.69 g L-1 was obtained after 240 h. As shown also
in Fig. 1, glucose concentration was gradually decreased in
culture throughout the cultivation time. However, during the growth phase (the
first 225 h of cultivation) the glucose consumption rate was about 0.053 g/L/h.
The glucose consumption rate was decreased thereafter to only 0.021 g/L/h for
the rest of production time. It is also noteworthy to mention that the pH value
of culture was dropped gradually from 5.5 at the beginning of cultivation to
3.6 after 144 h and increased again gradually reaching 3.9 at the end of cultivation
time. The growth morphology of mushroom in shake flaks culture was mainly in
pellet form. The pellet size was obvious in culture after only 48 h cultivation
and increased gradually with time.
||Cell growth and EPS production by P. ostreatus in shake
EPS production in batch culture in stirred tank bioreactor: The production
of EPS was conducted in pilot scale 15-L stirred tank bioreactor under fully
aerobic condition as described in materials and methods part. Fig.
2, summarizes the cell growth, EPS production and other changes in culture
during this cultivation process. As shown, cells grew exponentially with rate
of 0.036 g/Lh reaching maximal cell mass of about 6 g L-1 after 168
h. During that time glucose was consumed by cells with rate of 0.08 g/L/h and
totally consumed after 240 h. In parallel, EPS was produced in culture after
48 h and accumulated in culture with production rate of 5.3 mg/L/h reaching
maximal volumetric production of about 1.12 g L-1 after 216 h and
kept more or less constant for the rest of production time. On the other hand,
the DO in culture was dropped significantly during the growth phase reaching
about 20% saturation and increased again gradually as cell reached stationary
||Cell growth and EPS production by P. ostreatus in batch
culture in stirred tank bioreactor under controlled pH conditions
The out-gas analysis shows also that significant decreased in oxygen percent
in out gas concomitant with an increased in carbon dioxide content during the
growth phase. This also indicates the active cell metabolism during this phase.
As cells entered the stationary phase, oxygen ratio in outgas decreased gradually
with significant reduction of carbon dioxide concentration in out-gas. However,
cell morphology of culture was different from those obtained in shake flask.
In bioreactor culture, cells were aggregated during the early phase and form
small and lose pellet. As cultivation time increase, the pellet size increased
gradually. In general, the pellets in bioreactor culture were smaller with more
hairy surface compared to those obtained in shake flask cultures.
Fed-batch cultivation with intermittent glucose addition: As shown in the previous experiment, glucose was limited in culture after 240 h cultivation. Thus, the termination of EPS production may be attributed to the glucose limitation. Therefore, fed-batch cultivation was designed with addition of glucose of 90 g at 216 h (time at which glucose concentration reached less than 2 g L-1 in batch culture). Figure 3 shows the time course of cell growth and EPS production in fed-batch culture. As shown, cells grew exponentially during the first 168 h during the batch phase. As glucose added to culture no further growth was observed. On the other hand, the production of EPS was increased gradually after glucose feeding with same production rate and reached 1.74 g L-1 at the end of cultivation time. The DO in culture reached 20% saturation at the end of growth phase and kept more or less constant for the rest of cultivation time. On the other hand, the percent of oxygen in out gas increased gradually after cell entering the stationary phase. However, the increase of oxygen percent in outgas was less than those value obtained in batch culture. This indicates the higher cell physiological activity compared to those in batch culture after entering the stationary phase. Meanwhile, the data of carbon dioxide concentration in out-gas analyzer showed also that the decrease of the percent of carbon dioxide in out-gas after entering the stationary phase was less than in batch culture. These all together support the idea that the cell activity in fed-batch culture was higher than batch culture in stationary phase.
Fed-batch cultivation with on-line glucose feeding strategy using out-gas
analysis data: In the present experiment, EPS was produced in fed-batch
culture under controlled feeding conditions based on the value of carbon dioxide
||Cell growth and EPS production in fed-batch culture. Arrow
show the time at which glucose was fed to the bioreactor in single shot
As shown in the previous experiments, oxygen consumption is decreased (indicated
by increased fraction of oxygen in out-gas) and carbon dioxide production decreased
(indicated by the decrease of CO2 fraction in out-gas) under glucose
limitation. However, cultivation of mushroom is fully aerobic process and the
high CO2 in out-gas indicates high metabolic activity. Thus, it was
taken as key parameter for glucose feeding. The glucose feeding pump was cascaded
to the value of outgas to keep the carbon dioxide concentration in out-gas at
the maximal value achieved before glucose limitation. As shown in Fig.
4. The value glucose feeding started after 192 h as cells entered the stationary
phase. Feeding rate was varied between 0 and 0.08 g/L/h (the value of glucose
consumption rate in batch culture during the growth phase). Based on this feeding
strategy, EPS production increased gradually and reached about 2.1 g L-1
at the end of cultivation time. However, neither glucose nor oxygen was limited
throughout the cultivation time. Thus, we can conclude that glucose was the
limiting nutrient for EPS production but not for growth since growth was terminated
after 168 h (as in case of batch culture).
||Cell growth and EPS production in fed-batch culture in CO2
stat culture. Glucose was fed to maintain constant concentration of carbon
dioxide in out-gas of the bioreactor
Water extract of many mushrooms used in traditional Chinese medicine and other
folk medicines have long history in various diseases treatment. Nowadays, based
on the discovery of many novel molecules in mushrooms, increased interest has
been observed on using the medicinal mushroom extracts for cancer treatment.
Long cultivation period, extensive purification steps and low yield are the
main drawbacks of using mushroom fruit bodies for the production of anticancer
bioactive compounds. Thus, during the last few years the cultivations of mushroom
cells in submerged culture for exo-polysaccharide (EPS) production was very
attractive topic for many researchers (Yang and Liau, 1998;
Cho et al., 2006). In this study, we developed
new production process of EPS by oyster mushroom P. ostreatus in submerged
culture. As shown from the results of the early experiment in shake flask, the
production of EPS was associated with cell growth and terminated as cell entered
the stationary phase. However, glucose was not limited in culture in that time.
This incomplete utilization of glucose in culture may due to the decrease of
glucose consumption as growth was mainly in form of large pellet. During the
growth phase the pH dropped significantly in culture. This decrease of pH was
due to acid formation as also reported by other authors (Rajarathnam
et al., 1992). This may also due to insufficient oxygenation in shake
flask and the growth of cells in form of large pellet. In general, the growth
of fungal cells in large pellet increases the tendency for acid formation and
large fraction of cells is growing under anaerobic conditions (El
Enshasy et al., 1999). As the production process was transferred
to stirred tank bioreactor under controlled pH of 5.5, the cell mass increased
by about 15% this was concomitant with an increased in EPS production by about
25%. In this culture, glucose was fully consumed after 250 h. However, the better
cell growth and EPS production may attributed to the better mixing characteristics
and oxygenation in stirred tank bioreactor compared to shake flask (El
Enshasy, 2007; Maier and Büchs, 2001). As glucose
was limited after 220 h, both of growth and EPS production were terminated accordingly.
Generally, mushroom use glucose as their primary carbon source for both growth
and also contributed significantly in EPS production, but when glucose concentration
is high in the medium, mycelia growth is inhibited (Boyle,
1998). It have been also reported that to minimize the glucose repression
effect on different types of mushroom growth, glucose should not exceed the
range of 20-30 g L-1 (Azuma and Kitamoto, 1994).
When cultivation was conducted in fed-batch culture by different modes, significant
increase in EPS production was observed. In first fed-batch experiment, glucose
was added in one shot of 90 g to increase the glucose concentration in culture
up to 10 g L-1. Glucose addition resulted in the continuous production
of EPS in culture and reached maximal concentration of 1.74 at the end of cultivation
time without any significant increase cell mass production compared to batch
culture. Thus, we can conclude that glucose was the limiting substrate for EPS
production but not for growth.
On using this alternative new method of glucose feeding based on the out-gas analyzer data for CO2 concentration of the out-gas from the bioreactor to keep it at the same value when cell entered the stationary phase, EPS production was increased significantly. This increase in EPS production was not due to further cell growth. Thus, we can conclude that fed-batch cultivation increased the volumetric EPS production through the increase of cell productivity.
In the present study, the optimized submerged culture conditions for mycelia growth and EPS production by P. ostreatus in large scale was addressed. From the series of experiments, we determined that glucose was not the limiting substrate for cell growth but was limiting substrate for EPS production. By scaling up the process from shake flask to pilot scale stirred tank bioreactor significant increase in EPS was observed. Further improvement in EPS production process was achieved by cultivating the cells in fed-batch culture. Among different feeding strategies applied, cascading the glucose feeding rate to the value of carbon dioxide in out-gas was the best feeding strategy and yielded 2.1 g L-1 EPS at the end of cultivation time (this value was almost double of those obtained in batch culture). Thus we can conclude that fed-batch culture for EPS production using mono-glucose feeding controlled by out-gas analysis data is the most suitable system for large scale production of antitumor EPS by P. ostreatus.
The Authors wish to thank Mr. Ahmed Abdel Fattah, Mr. Ramadan Mohamed and Mr. Aboul Soud for their technical assistance.
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