Designing of Bioreactor for the production of Scopadulcic...

Chapter IV

Designing of Bioreactor for the production of Scopadulcic acid B

4.1 INTRODUCTION

Designing of bioreactor is the key step towards commercial

production of secondary metabolites through plant biotechnology.

Bioreactor offers optimal conditions for the large scale production of

metabolites for commercial manufacturers. Much progress has been

achieved in the past on optimization of these systems for the production

and extraction of valuable plant ingredients with medicinal properties

(Tabata, et al., 1988).

The heart of the fermentation process is the bioreactor. It is a

container in which a favorable environment is maintained for the operation

of a desired biological process. The unique feature of a bioreactor lies in

the type of cells being grown and also in the agitation and aeration of the

system. The bioreactor may be used for growing eukaryotic cells like plant

cells, mammalian cells, and also microbial cells. The type of bioreactor

used and its design is dependant on the properties of the cultures used

Research over the last two decades has established efficient

protocols for isolated cell culture and a large scale bioreactor system.

Bioprocess optimization involves biocatalyst design, medium design and

process design. Initial optimization studies were usually carried out in small

scale, under conditions as close as possible to the actual large scale

fermentation (Shueler and Kargi, 2002). Shake flask cultures are simple

type of batch bioreactors which were initially used for bacterial and yeast

culture. Later it was developed for fungi and animal cell (Ziv, 2005). They

are easy to handle and economic and are available in different volumes

from 25ml - 5 litres. In this type, a nutrient charge and a cellular inoculum

are mixed, shaken and allowed to grow. As the cells grow, the medium

becomes continually depleted of nutrients (Shuler, 1986). Under batch

conditions, several compounds in the medium become exhausted and the

154 Chapter IV

environmental conditions become limited (stationary phase of growth

cycle) leading to production of secondary metabolites.

4.1.1 Plant cell bioreactors

Routier and Nickell (1956) first discussed the use of plant cell

cultures for the commercial production of compounds normally extracted

from plants. In addition to the production of secondary metabolites, plant

cell cultures have been used for biotransformations (Alfermann et al.,

1980, Veliky and Jones, 1981). Later with the production of ginseng

biomass (Furuya et al., 1984) and berberine in large scale, bioreactors of

large volume and precise control parameters were evolved (Scragg and

Fowler, 1985).Various types of bioreactors with gas sparged mixing were

suitable for the production of cluster of buds, meristems or protocorms. A

simple glass bubble column bioreactor for proliferation of ornamental and

vegetable crop species resulted in biomass increase of 3-6 folds in 3-4

weeks. Micro bioreactors or microtiterplates of 5 µl working volume for

analytical purpose integrated with optical sensors were used in medical,

pharmaceutical and industry (Deutz et al., 2000). Bubble column

minibioreactors were designed with a working volume of 200ml for

optimisation and production of a protein from Staphylococcus carnosus

(Dilsen et al., 2001).

An internal loop bioreactor was used for the embryogenic cultures

of asparagus, celery, and cucumber. Disposable pre sterilized plastic

bioreactors (2-5 l) were used for the proliferation of meristematic clusters

of several ornamental, vegetable and woody plant species (Ziv, 2005).

4.1.2 Commercial bioreactors

Commercially produced Stirred Tank Bioreactors (STR) were

initially used in Japan for the cultivation of Tobacco cells (Nicotiana

tabaccum). During 1970s introduction of airlift bioreactors as an alternative

Designing of Bioreactor for the production of Scopadulcic acid B 155

to stirred tank bioreactors came into use. Stirred system provided a

homogenous environment with controlled closed and reproducible cultures

(Collins, et al., 1998).

Unlike microbial suspension culture, plant cell suspension consists

of groups or aggregates of many thousands of cells up to 2mm or more in

diameter. A fine suspension has cells with 200-500 per diameter (Dixon

and Gonzales, 1994). Culturing period of plant cells are very low, when

compared to microbial cells. But plant cells have the ability to control their

own pH hence, pH control is not normally necessary. To increase the

productivity of slow growing cultures, a high cell density is advantageous

as in Coptis japonica (Scragg et al., 1986).

Since immobilized plant cells were in an intermediate state

between a homogenous suspension and highly structured tissue of the

whole plant, immobilisation provided an environment favourable to viable

cell maintenance. Bioreactor cultures have several advantages compared

with agar based cultures. They can be controlled in a better way. The

growth regulator supply, aeration and medium supply, filtration of medium

and scaling up of cultures can be optimised.

A wide literature survey revealed that Murashige and Skoog

medium was the most common medium used in fermenters especially in

batch cultures. The growth pattern of Nicotiana tabaccum was similar to

that of microorganisms (Hashimoto and Azechi, 1985). The size of

inoculum is an important factor which determines the production of

secondary metabolite. The size of tobacco culture inoculum was one-tenth

the size of total culture medium. Tanaka (1982) reported that as the cell

concentration increased to 30 g/l of the media concentration, there was an

increase in yield of the product. This was achieved by adding sugar into

the culture medium. Aeration and agitation are two important factors which

promoted cell division. Since there is a tendency for cell aggregation in

156 Chapter IV

continuous system aeration may be adopted for maintaining cell

homogenicity

4.1.3 Objectives

The following objectives were considered for bioreactor studies

1. Preparation and standardization of Luffa sponge matrix for

immobilization by shake flask cultures.

2. Designing of a three bed bioreactor for the production Scopadulcic

acid B from Scoparia dulcis immobilized on Luffa sponge.

3. Production of Scopadulcic acid B using the bioreactor.


4.2 REVIEW OF LITERATURE A number of bioreactors have been developed and are successfully

being employed in secondary metabolite production from plant cells. The

design of plant cell bioreactor is being decided by the characteristics of

plant cell suspension. For suspension cultures, air lift and stirred

bioreactors were commonly used as they provided efficient mixing of the

cells in suspension.

4.2.1 Bioreactors for plant suspension cultures

Propagation in bioreactors through the organogenetic pathway was

achieved in Banana, Boston fern, Strawberry, Potao, Coffee, Pineapple,

Orchids, Narcissus and Cyclaen (Ziv, 2000). A pilot scale culture of Coffea

arabica was used as a model in performed novel loop fluidized bed

reactor cell line (Dubius et al., 1995). Liquid cultures have been used for

plant culture in both agitated vessels, and in bioreactors for somatic

embryogenesis (Scragg, 1992). A bubble column reactor was designed for

Catherantheus roseus (Smart and Fowler, 1984) Bioreactors of 5 litre size

was developed for plant suspension culture of Lithospermum erythrorhizon

(Tanaka, 1987), Mentha sp. (Tal and Goldberg et al., 1983), Morinda

citrifolia (Wagner and Vogelmann, 1977).

A two step procedure for in vitro multiplication of Rubus

chamaeomorus shoots using bioreactors were designed by Debnath

(2007). This plant was noted for its anticarcinogenic and antimicrobial

properties. It is a constituent of traditional medicines. Somatic

embryogenesis was induced for large scale production of secondary

metabolite in Siberian ginseng (Eleutherococcus senticosus) an

endangered medicinal plant. Embryogenic callus obtained from leaf

explant in 1/2 -1/3 MS medium produced phenolics and flavenoids in

addition to the secondary metabolite. They were found to be in higher

158 Chapter IV

concentration in fully mature embryos than in various other stages

(Shohanel et al., 2006).

Bioreactors were designed for organized cultures such as roots,

shoots, and embryos. Reports of novel designs and modification of existing

bioreactors for hairy root culture include a 10 litre modified Stirred tank

bioreactor (STR) for roots of Atropa belladonna (Akita and Takayema,

1988), Trickle bed bioreactors for Carthamus tinctorius (Dilorio et al., 1992)

and Bubble column bioreactor for Lithospermum erythrorhizon (Sim and

Chang, 1993). Mist Bioreactors, a new type of bioreactors were successful

in the production of shoot cultures in Musa and Nephrolepis (Weathers and

Giles, 1988) and Dacus carota (Kondo et al., 1989). Air lift bioreactors

Medicago sativa (Stuart et al., 1987) and Stirred tank bioreactor were used for

embryo cultures of Datura stramontium (Hilton et al., 1988). Bioreactor

system was also applied for embryonic and organogenic cultures of

several plant species.

4.2.2 Plant cell bioreactors for industrial applications

Nicotiana tabaccum cells were grown as cell suspensions in a semi

continuous culture in 30 litre jar fermentor (Matsumoto et al., 1971). The

first commercial process for producing a natural plant product by plant cell

culture was developed in Japan for the production of shikonin (Curtin,

1983). Berberine, an isoquinone alkaloid was scaled up to 400 l scale by

Mitsui Petrochemical Industry, Japan (Zenk et al., 1985). Now a number of

industries have come up for the production of secondary metabolites.

In bioreactors, the concentration of nutrients in the medium is

affected mainly by absorption rate and by cell lyses. The availability of

growth regulators in bioreactor cultures was effective in controlling the

proliferation and regeneration potential than in agar culture (Ziv, 2005). In

the case of many plants cultivated in the bioreactors, continuous aeration


mixing and circulation causes shearing damage and cell wall break down.

Foaming was reduced when 1/2 strength of medium was used (Ziv, 1995).

Four variations of gel entrapment methods were usually used for

plant cell culture. They included preformed polyurethane foams for

Capsicum frutiscens (Mavituna and Park 1985), nylon pan scrubbers for

Beetroot and Hop cultures (Rhodes 1982), polyacrylamide polymers for

Nicotiana sp. (Rosavear, 1981), Catherathus roseues (Bordelius and

Nilsson, 1980), alginate entrapment for Dacus carota (Velky and

Jones,1981) and Morinda citrifolia (Bordelius et al., 1980).

Natural plant products like dry fruits of Luffa cylindrica were initially

used for entrapping microbial cells. They acted as a carrier for immobilizing

by trapping yeast cells. A new biosorbant was developed by immobilizing

unicellular micro algae Chlorella sorokinara within Luffa sponge disc to

remove metal ions from aqueous solution (Akhatar et al., 2003). It was also

successfully employed as a matrix for immobilizing Aspergillus niger-26

which produced poly methyl galacturonase (Slokoska and Angelova, 1998).

Large cells and aggregates of cells in suspension cultures of Coffea arabica,

Catheranthus tinctorius, and Angelica sinensis (Liu. et al., 1998) were

immobilized on the sponge. Yeast cells were successfully immobilized on

Luffa sponge by Ogohonna et al., (1994).

From 1998 onwards, works on plant cell culture using Luffa sponge

gained importance. Recently the trend was to use Luffa sponge as an

alternative matrix to coir in tissue culture technique to stabilize root

induction as in Philodendron ‘Xanadu’ propagation (Gangopadhya et al.,

2004). The principle of this method was that at first a mechanical

entrapment was formed and later cells got fixed due to adsorption and

adhesion or even aggregation by their natural tendency. It was also a

simple and rapid method of immobilization.

160 Chapter IV

4.2.1 Bioreactors for Immobilized Culture

Immobilized plant cells have a number of advantages over normal

cultures as it allows continuous operation, separates biomass from

medium, protects shear and stimulates secondary product formation. The

selection and design of bioreactor depended on how the cells were

immobilized and also on the nature of the plant cells. Flat bed immobilized

systems consisted of a simple culture vessel with the cells fixed to a matrix

made up of substratum of polypropylene. A separate reservoir of nutrient

medium was supplied to the cultured cells by dripping from a reservoir on

to the polypropylene foam with recirculation by means of peristaltic pump

(Lindsey and Yeoman, 1983 b).

In this context the feasibility of using Luffa sponges (Luffa

cylindrca) as a carrier for immobilization was investigated for the

production of scopadulcic acid B from suspension cultures of Scoparia

dulcis. A simple, novel bioreactor system was designed, where cells were

immobilized on vegetable sponge matrix of Luffa cylindrica.


4.3 MATERIALS AND METHODS 4.3.1 Immobilisation of cells on Luffa sponge in shake flask

cultures 4.3.1.1 Source of Luffa discs. The mature dried fruits of Luffa cylindrica were collected from local

market and were used for immobilization. It was an annual climbing herb

with leaves, 5-7 lobed, flowers white, and fruits oblong and cylindrical.

Fruits were smooth and became fibrous when old (Gamble, 1984a).

4.3.1.2 Pre-treatment of discs The outer fruit walls were removed to get dry fibrous sponges

(Plate4.1a, 4.1b). They were cut into discs of 2.5 cm diameter (Plate 4.1c)

and 1-2 cm thick and soaked in boiling water for 30 minutes. It was

thoroughly washed under tap water and left in distilled water for 24 hrs.

The water was changed three times. The Luffa discs were dried in an oven

at 700 C and autoclaved for 20 minutes at 1210C (Akhtar, et al., 2003).

4.3.1.3 Production of Scopadulcic acid B by the cells immobilized on Luffa sponge

The oven dried discs were weighed under sterile condition. 10ml of

two week old cell suspension in MS medium supplemented with NAA

(5mg/l) and BA (1mg/l) was used as inoculum for mother suspension. The

discs were gently transferred to 250 ml medium containing viable cells for 2

hours They were then removed and washed carefully with fresh medium to

remove excess cells. 100ml of MS medium supplemented with NAA (5mg/l)

and BA (1mg/l) were used for the production of secondary metabolite using

S. dulcis. The Luffa discs with immobilized cells were placed carefully in

250ml conical flasks containing MS medium supplemented with NAA (5mg/l)

and BA (1mg/l). They were kept on rotary shaker at 110 rpm for 30 days.

SDB content was analyzed after 30 days. SDB was estimated as

mentioned under section 2.3. A control was used without any hormones in

the medium. The experiment was conducted in 5 replicates.

162 Chapter IV

a b

c d

Plate 4.1 Luffa cylindrica

4.1a. Dry fruit of mature Luffa cylindrica. 4.1b. Fibrous net work of Luffa cylindrical after removing the outer fruit wall 4.1c. Transverse section of sponge of Luffa cylindrica 4 1d. Fibrous net work of Luffa cylindrica covered with immobilized plant cells x1000

(Nikon Eclipse E 400 microscope)


4.3.2 Designing and working of the Bioreactor

An immobilized three bed glass bioreactor with an external

recirculation was designed for enhancing the production of scopadulcic

acid B from the suspension cells of Scoparia dulcis.

The reactor was of vertical type (20cm × 4 cm) consisted of three

separate units A, B, and C fitted one above the other (plate 4.2a). An

inlet for inflow of culture medium and outlet for collecting spent medium

at the top of the column A were provided for the bioreactor. A reservoir of

1 litre capacity was connected to peristaltic pump through a peristaltic

tube. Luffa discs with the immobilized cells prepared as mentioned under

section 4.3.1.3 (two weeks old) were inserted into column A, B and C

using sterile forceps and needle under laminar airflow hood (Plate 4.2 b

and 4.2c) The MS liquid medium supplemented with NAA (5 mg/l) and

BA (1 mg/l) was sparged through column C, B and A. The Luffa sponges

embedded with cell biomass were completely soaked in the medium. The

collected product containing medium was recirculated through an

external loop back into the reservoir using the peristaltic pump. The

aliquots of samples were collected at regular intervals of 24 hrs for the

detection of SDB. SDB was estimated as mentioned under section 2.3.

164 Chapter IV

a b

c

Plate 4.2 Parts of the Bioreactor

4.2a Glass columns with three cartridges designated as A, B, C 4.2b A cartridge filled with Luffa matrix 4.2c Catridges A, B, C filled with Luffa matrix


Plate 4.3 Three bed bioreactor with cells of Scoparia dulcis immobilized on Luffa sponge for the production of Scopadulcic acid B.

All connections were made air tight and the units were held firmly

with the help of clamps. A peristaltic pump was used to pump the medium

from the reservoir and flow rate was adjusted to 4 ml / minute. The pH of

the medium was adjusted to 5.6 (Plate 4.3).

166 Chapter IV

4.4 RESULTS 4.4.1 SDB production in shake flask cultures

Microphotographs (plate4.1d) of cells indicated a uniform growth

along the surface of fibrous thread indicating that the immobilized cells

were not localized at a single point. The fibrous network of Luffa sponge

was completely loaded with immobilized plant cells. After 30 days the

sponges were removed, weighed and net weights of immobilized cells

were recorded. Quantity of the immobilized plant cell biomass was

determined as the difference between constant dry weights of Luffa disc

before and after immobilization. The average fresh weight of cells adhered

to the matrix was 3.18 g. In the shake flask cultures, single discs were

used to immobilize cells and five trials were conducted. Maximum SDB

content of 52.42mg/l was observed after 30 days of incubation (Table 4.1).

Table 4.1 –SDB Production in Shake flask culture

F.wt. (g) of

Luffa sponge before

inoculation

F.wt. (g) of Luffa sponge after 30 days

F.wt. (g) of of cells immobilized on

Luffa

SDB mg/g cells Immobilized on Luffa after 30

days

Control trials 3.211 4.831 1.620 --

T1 2.518 4.407 1.889 46.87

T2 3.592 9.535 5.943 52.42

T3 4.608 5.829 1.221 51.71

T4 5.670 9.261 3.591 51.28

T5 3.515 4.680 1.665 51.97


4.4.2 SDB production in Bioreactor When Luffa sponges inoculated columns were used for SDB

production in bioreactor, a maximum amount of SDB (350.57 mg/g cells)

was obtained in the first batch operation by 19th day (Figure 4.1).

Performance evaluation of the bioreactor was done continuously for almost

50 days (Fig.4.2). The maximum productivity of the reactor could be

maintained up to 25th day. Subsequently, the productivity decreased

almost in a proportional manner with a result of half productivity at 30th

day. The half life period was almost calculated as 10 days. Batch wise

operation of the bioreactor was done in three cycles (Fig.4.3). The second

cycle could yield 175.2mg/g, while the third batch could give 120 mg/g.

TLC, UV, HPLC, and IR analysis confirmed the presence of SDB. TLC

of SDB content obtained from bioreactor showed an Rf at 1.5 (Plate 4.4). UV

analysis indicated maximum absorption spectra at 282.2nm. HPLC

confirmed retention peak at 9.0 minutes (Figure 4. 4). IR also showed

functional groups corresponding to SDB (Figure 4.5).

0 5 10 15 20 25

0

50

100

150

200

250

300

350

400

450

SD

B (m

g/g

of c

ells

imm

obili

sed)

Time (Days)

Production of SDB in Bioreactor

Fig 4.1 Production of SDB in Bioreactor for 21 days

168 Chapter IV

0 5 10 15 20 25 30 35 40 45 50 550

50

100

150

200

250

300

350

400S

DB

(mg/

g of

cel

ls im

mob

ilise

d)

Time (Days)

Performance of the Bioreactor

Fig 4.2 Half life period of SDB production in Bioreactor

0 5 10 15 20 25 30 35 40 45 50 55 60 650

50

100

150

200

250

300

350

400

SD

B (m

g/g

of c

ells

imm

oblis

ed)

Time (Days)

Ist Stage 2nd Stage 3rd Stage

Fig 4.3 Production of SDB in Bioreactor in various cycles


Plate 4.4 TLC of SDB produced from Bioreactor

Fig 4.4 HPLC of SDB produced in the bioreactor with cells of Scoparia dulcis immobilized on Luffa sponge in MS medium with NAA(5mg/l and BA(1mg/l).

170 Chapter IV

Fig. 4.5 IR of SDB produced in the bioreactor with cells of Scoparia dulcis immobilized on Luffa sponge in MS medium with NAA(5mg/l and BA(1mg/l).

4.5 DISCUSSION

Plant cell bioreactors can be used as a tool at commercial level to

increase the productivity of a culture for secondary metabolite production.

They provide a rapid and efficient plant propagation system for many

agricultural and forestry species, and there by avoiding intensive manual

handling.

Immobilization technique using sodium alginate was experimented

for secondary metabolite production in S. dulcis. In Amaranthus tricolor

when cells were immobilized in calcium alginate they produced only low

quantity of oxalic acid (Knorr and Teutonico, 1986). Similar reports were

seen when biosynthetic activity of alginate immobilized and free

suspended cells were compared for accumulation of capsacin. The rate of

increase in fresh weight and dry weight of alginate immobilized cells was

less than that of freely suspended cells in culture conditions favoring cell


division. This was reflected even in the rate of protein synthesis (Lindsey,

1986a).

A novel method of immobilizing cells was adopted where free cells

were directly deposited on sterile Luffa sponge matrix. The use of Luffa

sponge disc as a biosorbant for the removal of metal ions from aqueous

solutions by immobilizing unicellular Chlorella, a microalgae was highly

successful (Akahtar et al., 2003). Similarly the use of yeast cells,

(Oghonna et al., 1994) and fungal cells (Iqbal and Zafar, 1993) along with

Luffa sponge were also reported. In the study, when Luffa sponges were

used in bioreactor, the production of SDB was found to be enhanced at an

earlier period (19 days) than in shake flask conditions. Microscopic studies

revealed that the fibrous network of the Luffa sponge discs were

completely covered by plant cells. In free cultures, the cells were in small

aggregates. In the method of Luffa sponge immobilization, cells appeared

in reticulate open net work of Luffa sponge matrix which provided free

access and enhanced surface area to the medium for diffusion. Luffa

sponge immobilization of cells was found more effective for SDB

production than sodium alginate immobilized cells. The production of

secondary metabolites was observed within 19-21 days in this method

compared to shake flask method (30 days). The TLC, UV, HPLC and IR

studies confirmed the production of Scopadulcic acid B through the Luffa

bioreactor (Table 4.1).

The production of SDB in the Luffa bioreactor was more efficient

than in shake flask culture. In shake flask studies, the amount of

Scopadulcic acid B obtained was less than expected. The flasks were kept

in the shaker and hence good mixing and aeration, were achieved. The

less productivity obtained even under these conditions indicated that there

might be the possibility of increased shearing stress as a result of which

172 Chapter IV

plant cells might have undergone partial lysis and hence might have

resulted in less productivity.

In shake flask cultures, of the five trials conducted, maximum

production was in T 2 with 52 mg/g cells of immobilized cells on the 30th

day. In the bioreactor, the production of SDB was enhanced on 19th day

(350.57mg/g of immobilized cells) in the first batch where as in the second

batch it was less than 176mg/g of immobilized cells and in the third batch it

was slowed to less than 120 mg/g of immobilized cells.

Thus there was more production of Scopadulcic acid B in the

bioreactor. The Luffa sponge packed in the reactor offered a good matrix

for the immobilization of the cells. As it was by physical adsorption the

accessibility to the nutrient medium was more. The cell aggregation was

less and there was no resultant shear due to lack of mixing. The medium

was passed into the reactor from the bottom. Hence the incoming medium

was getting expressed to the immobilized cells layer by layer achieving

effective utilization by cells. The medium was put into recirculation which

might have resulted in a cumulative increase in Scopadulcic acid B within

19 days. Comparatively good spots were obtained on TLC (plate 4.4).

HPLC analysis of the extract of the medium from the bioreactor confirmed

that the purity of the SDB produced was comparatively better than all other

cases mentioned earlier (Fig. 4.4). The amount of SDB produced was

highest and there were only few peaks other than that of SDB(9min). IR

also showed all representations of the characteristic structural features of

SDB (Fig. 4.5).

The present work assumes significance of using Luffa sponge as a matrix for immobilizing free cells from suspension cultures of S.dulcis. This innovative technique of employing Luffa in the bioreactor has an ample scope for scaling up the production of SDB to commercial levels. The demand for SDB as antimicrobial, antiviral and


for other clinical applications may be met with this new, enhanced

production method.

The main advantages of using Luffa sponge matrix in production of

SDB is that it is inexpensive, easily available, simple to install, physically

strong, highly porous and reusable. Recovery of Scopadulcic acid B is

possible as early as by the 19-21st day. A small amount of medium comes

in contact with large amount of biomass ensuring increased cell to cell

contact. Another advantage is that the product containing supernatant is

free from cells or debris, which is usually a problem in shake flask cultures.

A disadvantage of this type of reactor is that after sixty days of

continuous production, a sheath of cell debris was formed which adhered

to the outer surface of the sponge. This hindered the free circulation of the

medium and oxygen supply. The Luffa matrix is a plant product and hence

there is a tendency for its decomposition. This drawback can be overcome

by using its disposable cartridges which may be cost effective and may be

easy to assemble.

Immobilization of cells and tissues, which synthesize important

metabolites, has great future in large-scale product synthesis. There is a

need to identify highly active cell lines for specific biotransformation of

commercial importance. By using suitable bioreactors specific for plants, a

continuous production of phytochemicals, which can overcome the

inherent limitations in handling highly fragile cells, is possible.

There is an ample scope for the application of the immobilized

three bed bioreactor with disposable cartridges filled with pre sterilized

Luffa sponge for the efficient and enhanced production of Scopadulcic

acid B. This method is comparatively cost effective and environmental

friendly. The raw material is easily available or can be produced locally

with low financial or technical inputs.

Designing of Bioreactor for the production of Scopadulcic...

Documents

Transcript of Designing of Bioreactor for the production of Scopadulcic...