Industrial product

manufactured by

using Bioreactor

BIOREACTOR:

1

• An apparatus for growing organisms (yeast, bacteria, or animal cells) under

controlled conditions.

• Used in industrial processes to produce pharmaceuticals, vaccines, or antibodies

• Also used to convert raw materials into useful byproducts such as in the

bioconversion of corn into ethanol.

• Bioreactors supply a homogeneous (same throughout) environment by constantly

stirring the contents.

• Bioreactors give the cells a controlled environment by ensuring the same

temperature, pH, and oxygen levels

Types of Products produced by bioreactor:

• Natural Products

– Drugs

• Penicillin is early example

• Taxol

• Mupricin

• Cyclosporin A, etc.

– Foods

• Fermented beverages

• Fermented dairy products

• Transgenic Products

– Gene for a therapeutic protein inserted in foreign expression system

• Factor IX

• a-1-antitrypsin

• EPO

• Antibodies

• antithrombin III

• tissue plasminogen activator (TPA)

• Interferons, etc.

2

Expression Systems- these type of product are expressed in following type of

cells.

• Bacterial Cells

• Fungal Cells

• Plant Cells

• Insect Cells

• Mammalian Cells

            Numerous types of bioreactors are currently used at laboratory or industrial scale.

1. Mixing Vessels

– Stirred tank bioreactor

• Simple batch, fedbatch

• Simple CSTR (seldom used because of low cell concentration)

– Roller bottles or multiple parallel plate bioreactors

– Bubble column bioreactor

– Airlift bioreactor

– Fluidized bioreactor (3 phase)

2. Plug flow type

– Hollow fiber bioreactor

– Packed bed bioreactor

Stirred tank bioreactor

Similar to CSTR; this requires a relatively high input of energy per unit volume.

Baffles are used to reduce vortexing. A wide variety of impeller sizes and shapes is

available to produce different flow patterns inside the vessel; in tall fermenters,

installation of multiple impellers improves mixing.

Typically, only 70-80 % of the volume of stirred reactors is filled with liquid; this

allows adequate headspace for disengagement of droplets from exhaust gas and to

accommodate any foam which may develop. Foam breaker may be necessary if foaming

3

is a problem. It is preferred than chemical antifoam because the chemicals reduce the rate

of oxygen transfer.

The aspect ratios (H/D) of stirred vessels vary over a wide range. When aeration

is required, the aspect ratio is usually increased. This provides for longer contact times

between the rising bubbles and liquid and produces a greater hydrostatic pressure at the

bottom of the vessel.

Care is required with particular catalysts or cells which may be damaged or

destroyed by the impeller at high speeds.

Bubble column

In bubble-column reactors, aeration and mixing are achieved by gas sparging; this

requires less energy than mechanical stirring. Bubble columns are applied industrially for

production of bakers’ yeast, beer and vinegar, and for treatment of wastewater.

4

A height-to-diameter ration of 3:1 is common in bakers’ yeast production; for

other applications, towers with H/D of 6:1 have been used. The advantages are low

capital cost, lack of moving parts, and satisfactory heat and mass transfer performance.

Foaming can be problem.

Homogeneous flow: all bubbles rise with the same upward velocity and there is

no back-mixing of the gas phase.

Heterogeneous flow: At higher gas velocity. Bubbles and liquid tend to rise up in the

center of the column while a corresponding down flow of liquid occurs near the walls.

Airlift reactor

Airlift reactors are often chosen for culture of plant and animal cells and

immobilized catalyst because shear level are low. Gas is sparged into only part of the

vessel cross section called the riser. Gas hold-up and decreased liquid fluid density cause

liquid in the riser to move upwards. Gas disengages at the top of the vessel leaving

heavier bubble-free liquid to recirculate through the downcomer. Airlift reactors

configurations are internal-loop vessels and external-loop vessels. In the internal-loop

vessels, the riser and downcomer are separated by an internal baffle or draft tube. Air

may be sparged into either the draft tube or the annulus. In the external-loop vessels,

separated vertical tubes are connected by short horizontal section at the top and bottom.

Because the riser and downcomer are further apart in external-loop vessels, gas

disengagement is more effective than in internal-loop devices. Fewer bubbles are carried

into the downcomer, the density difference between fluids in the riser and downcomer is

greater, and circulation of liquid in the vessel is faster. Accordingly, mixing is usually

better in external-loop than internal-loop reactors.

5

Packed bed

Used with immobilized or particulate biocatalysts, for example during the

production of aspartate and fumarate, conversion of penicillin to 6-aminopenicillanic

acid, and resolution of amino acid isomers. Damaged due to particle attrition is minimal

in packed beds compared with stirred reactors.

Mass transfer between the liquid medium and solid catalyst is facilitated at high

liquid flow rate through the bed. To achieve this, packed are often operated with liquid

recycle. The catalyst is prevented from leaving the columns by screens at the liquid exit.

Aeration is generally accomplished in a separated vessel because if air is sparged directly

into the bed, bubble coalescence produces gas pockets and flow channeling or

misdistribution. Packed beds are unsuitable for processes which produce large quantities

of carbon dioxide or other gases which can become trapped in the packing.

Fluidized bed

To overcome the disadvantages of packed bed, fluidized bed may be preferred.

Because particles are in constant motion, channeling and clogging of the bed are avoided

and air can be introduced directly into the column. Fluidized bed reactors are used in

6

waste water treatment with sand or similar material supporting mixed microbial

populations, and with flocculating organisms in brewing and production of vinegar.

Trickle bed

Is another variation of the packed bed. Liquid is sprayed onto top of the packing

and trickles down through the bed in small rivulets. Air may be introduced at the base;

because the liquid phase is not continuous throughout the column, air and other gases

move with relative ease around the packing. Trickle-bed reactors are used widely for

aerobic wastewater treatment.

7

8

There are three basic transfer phenomenon that occur in bioreactor and have great

influence on the process these are-

1. Heat transfer (conduction, convection, radiation)

2. Mass transfer

3. Momentum transfer

Bioreactors operate in different mode

• Batch: Media and cells are added to the reactor and it is run until a predetermined

set point (i.e. time, concentration). The bioreactor has a constant volume (the

initial volume).

• Fed-Batch: The bioreactor is a batch process in the beginning and after a certain

point a feed input is introduced and the volume of the vessel increases.

• Continuous: The bioreactor starts with an initial volume and media is constantly

introduced and product is constantly taken out. The inputs and outputs are at the

same rate, so the volume always remains the same.

9

Bioreactor Components:

• Bioreactors consist of:

- Vessel - Cooling jacket

- Agitator - Ports for input and output of material

- Sparger - Condenser

- Baffles - Back pressure valve

- Valves - Load cell

- Probes - Inlet filters and exhaust filters

Temperature

Dissolved Oxygen

pH

Pressure Gauge

INDUSTRIAL EXAMPLE OF PRODUCT MANUFACTURED BY USING

BIOREACTOR

1. Insulin

Commercial manufacturers: Novonordisk, Elililly

10

Use: hypoglycemic

Fermentation microorganism: In case of Novonordisk, insulin and analogues are

ontained by microbial fermentation of genetically modified yeast, E.coli bacteria and

mammalian cells. For each product, NovoNordisk develops specific genetically modified

cell lines. The main waste product from Novo Nordisk’s insulin production is a nutrient-

rich organic material known as yeast sludge, which is recycled either as pig feed (yeast

cream) or as raw material for biogas generation. The biomass undergoes heat treatment to

ensure that all microorganisms are killed

Reactor considerations:

Type of bioreactor: continuous stirred tank reactor. An external heat exchanger jacket is

needed.

substrate feed: glucose as carbon source, ammonia as nitrogen source, mineral salts.

The fermentation is an aerobic process; hence, air is sparged for oxygen delivery.

Impellers are used for enhancing oxygen and nutrient transfer. Insulin is produced

extracellularly.

- Glucose concentration < 0.5 g/L - Prevents formation of ethanol.

5000 kg insulin per year - Assume 20 % loss due to purification

- kLa within 10% of 2088 hr -1

Prevents oxygen limited reaction

Prevents anaerobic metabolism

General Bioreactor specifications: total volume: 11.8 m3

11

Di

1.3

m

Initial Reactor Volume 8500L Max Oxygen Demand 8mmole/gh Target maximum kLa 2088h-1

Tank Diameter 1.5m Impeller Diameter 1.3m Tank Height (HL) 4.81m HL* 5.51m HL** 6.06m Rpm 45 Qg 0.036m3/s Ni 2 Corrected Power (Pg) 6789W

Pg/V 776.21W/m3

Design kLa 1998h-1

Pm 8.06W/kg Geometry Correction Factor (φ)

0.13

These are final design specifications.

Reactor employed by Novonordisk

12

2. Kefir : is a fermented milk drink that is prepared by inoculating cow, goat or sheeps

milk with kefir grains. Kafir grains are combinations of bacteria and yeast in a matrix of

protein, lipids and sugars. This symbiotic matrix form grains that resemble cauliflower.

Kefir grains contain microorganisms such as saccharomyces lactobacillus caucasicus,

leuconnostoc and lactose fermenting yeast.

Kefir is produced on industrial scale by using CSTR.

1. Antibiotics :

Penicillin- major steps in the commercial production of penicillin.

1. Production of inoculums

2. Preparation and sterilization of medium

3. Inoculation of the medium in the fermentor.

4. Forced aeration with sterile air during incubation.

5. Removal of mould mycelium after fermentation.

6. Extract and purification of the penicillin.

13

7. Penicillin G is now prepared by air lift fermentation by batch mode.

Continuous fermentation is not common practice in antibiotic industry.

Strain- Penicillium chrysogenium ATCC 48271 was used.

A 21 B Braun airlift fermenter with working volume of 200ml was used. Sterile

air is sparged through a sintered plate located near the bottom of the central

concentric tube. Thetre was no mechanical stirring, only the air nozzle was forced

through centered tube and the flow directed to the annulus tube side. Aeration

cause circulation of media flow is gentle without serious shear forces. The

temperature is maintained at 260C.

Incubation control necessitates the precise control of several influential parameter.

1. Temperature 5. agitation

2. pH 6. Dissolved oxygen

3. pressure 7. Air flow

4. nutrients 8. Redox condition

These are important process variables and growth conditions.

3. Vitamine B2 :

14

Commercial Manufacturers: Roche, BASF, Hubei Guangji.

Use: nutritional supplement.

Fermentation parameters:

Fermentation microorganism: Each producer uses its own variant of the process with

different strains of micro-organism and different raw materials. Scientists at Roche have

now developed an economical biotechnological alternative to the manufacturing process

employed to date for the production of vitamin B2. The organism used is soil bacterium

Bacillus subtilis, the vitamin B2 output of which has been specifically increased.  It was

chosen by the process of mutagenesis and selection. Bacillus subtilis  was chosen because

many of the substances that it and closely related organisms produce in foodstuff

technology are GRAS (generally regarded as safe). The biotechnologically derived

vitamin B2 is identical to the traditionally manufactured product in terms of efficacy and

other criteria. From the point of view of ecology this new manufacturing process offers

some important advantages: firstly, the major raw material used in the biotechnological

process is renewable natural glucose; secondly, as significantly smaller amounts of

solvents and auxiliary substances are used, smaller amounts of air pollutants and waste

materials arise; and thirdly, energy consumption is reduced.

Substrate: The required raw material include sucrose, glucose, ground lentil, ammonium

succinate, sodium dithionate, n-butyl alcohol, soybean oil etc. Higher yields may be

possible by addition of glycine and pancreatic extract. Roche produces riboflavin via a

combined method in which bioconversion of D-glucose to D-ribose by microorganisms is

followed by chemical synthesis using ribose as one of the starting substances.

Type of bioreactor: The size of the bioreactor is influenced by the spacetime yield

which is the amount of product made per unit volume per unit time. It also determines the

length of time required for the fermentation process. A slow organism may take many

days to complete the vitamin B2 fermentation. In this case, the manufacturer will need

numerous fermenting vessels, resulting in additional capital costs compared with

producers using faster organisms. As the process is aerobic, a well stirred reactor with air

sparging and impellers is used. The material of construction is stainless steel along with

15

SS connecting pipes , SS pump for sterile agitation and aeration. Gadgets and recording

devices for monitoring of fermentation, Automatic control for : pH , Temp , DO ,

supplements with sterile nutrient etc are employed. With suitable aeration and agitation

over a period of 36 hours after inoculation, enough cell mass is developed.

Fermentation time: 168 hours including 24 hours set up time

Aeration rate: aeration rate being 0.33 volumes of air per volume of medium per minute.

Temperature: 28o C

pH: the usual pH is around 7.0. When the fermentation is completed the pH is adjusted to

4.5. 

The fermented broth is concentrated to 30% solids and dried on double drum dryers. To

obtain the pure product the fermented broth is heated to 121°C for 1 hour, centrifuged to

remove insoluble solids. Riboflavin recovered from the solvent by solvent extraction and

finally precipitated by using reducing agents like hydrosulphite.

4. Ethanol/ Fuel alcohol :

Commercial manufacturers: phoenix industries, India

Fermentation microorganism: Saccharomyces cerevisiae

Substrate: either one of the below: i) sugars (sucrose, molasses, glucose)

ii) starches (corn starch)

iii) cellulose (wood, straw, bran)

Fermentation process:

16

i) Batch process: Low productivity of 1-4g/L/h. Open tanks are acceptable. Anaerobic

fermentation produces alcohol and CO2 while aerobic fermentation produces little

alcohol. In anaerobic process, some dissolved O2 is necessary to provide for cell

replenishment and a concentration of about 10% of saturaton is optimum. The substrate

concentration is 12% to 16% sugars with essential inorganic additives with trace

elements. Inoculation of substrate with 10% to 15% by volume of yeast produces

maximum ethanol concentration of 8-12 g/L after 14-20 hours. The process is subject to

substrate as well as product inhibiton (above 12% w/w)

pH: 3.5.

ii) Continuous process: CSTR gives high productivity of 6 to8 g/L/h. The equipment

size is also reduced, so is the labor required. Assembly of the CSTR is more complicated

as complete sterile conditions must be mentained. To further increase the productivity

(11-20g/L/h), a cell recycle CSTR can be used.

5. Acetone/Butanol :

Commercial manufacturers: Mosam chemicals, Mumbai.

Use: solvents, biofuel.

Fermentation Parameters:

Fermentation microorganism: bacteria belonging to the genus Clostridium. The relative

proportion of different products varies with the species and the strain of Clostridium

used.

Substrate: molasses plus maize steep liquor, wood hydrolyzates.

Type of bioreactor: the fermentation is anaerobic. The reactor is gassed with CO2. The

fermentation is carried out at 33-35°C, with initial pH of 5.8 to 6.0. However, the pH

drops in the first 18 hoursincreases again due to

the production of acetone andbutanol from these acids. Afetr the process is complete,

Acetone, butanol and ethanol are recovered through continuous distillation and

17

fractionation. Carbon dioxide produced is recovered and converted into liquid CO2 or dry

ice. The residue left after the distillation may be dried and utilized as animal feed.

6. Citric acid :

Commercial manufacturers: Bombay lubricants oil company, Starchem India

Use: Citric acid is an important chemical used in food and candies, beverages, medicines,

flavouring extracts,manufacture of ink, effervescent slats, dyeing, mirror silvering, and

detergent industries. 

It is also increasingly used in the removal of toxic and corrosive gases in air.

Fermentation Parameters:

Fermentation microorganism: mainly, Aspergillus niger. Also, variety of yeasts such as

Candida, Hansenula etc. have been found to produce citric acid.

Substrate: A variety of carbohydrate sources such as beet molasses, cane molasses,

sucrose, commercial glucose, starch hydrolysates etc., have been used for citric acid

production. Among these, sucrose, cane and beet molasses have been found to be the

best. For citric acid production the raw material is diluted to 20-25 per cent sugar

concentration and mixed with a nitrogen source and other salts. The pH of the medium is

maintained around five when molasses is used and at a lower level (pH 3.0) when sucrose

is used. he production of citric acid by A.niger is largely influenced by the concentration

of trace metals such as iron,manganese, copper and zinc in the medium. An appropriate

concentration of these elements is essential for good acid production. However, an excess

is detrimental. To optimize the level of these trace metals, the raw materials are treated

with either ferrocyanide, charcoal, chelating agents or catien exchange resins. Addition of

methanol at 3-4 % concentration has been found to enhance the yield of citric acid. This

fermentation is an aerobic fermentation and, therefore, adequate aeration is essential for

successful citric, acid production.

18

Type of bioreactor: the fermentation can be carried out in either in i) shallow pans

(surface culture method) or in ii) large tanks (submerged culture method). The

fermentation process is aerobic. The shallow trays provide a large surface area for oxygen

transfer and hence, additional air sparging is unnecessary. This results in reduced energy

consumption. The temperature is maintained at 25-30 degree Celsius, and the

fermentation process takes 7 to 10 days to complete. However, this method is not very

suitable for commercial scale manufacturing due to large space requirements and longer

fermentation times.

In submerged culture method, inoculated medium is contained in large tanks instead of

shallow pans and a supply of sterile air is forced through these tanks to make oxygen

available; the fermentation process is carried under vigorous stirring and mixing and

completes rapidly by this method.

Solid state fermentation can also be used. In this, the mold is grown over carrier material

such as bagasse etc., which is impregnated with the fermentation medium.

7. cGMP production :

Manufacturer : GE life sciences.

Use : in clinical production of human cell therapeutics.

Type of bioreactor : WAVE bioreactor/ Cellbag reactor.

Process parameters: The gentle wave action of cellbag reactor limits shear stress on the

cells in comparison to traditional stirred tank bioreactors. For volumes up to 25 L, only

one Cellbag bioreactor is needed. This makes the system easy to use and considerably

less laborious than using static cultures where multiple bags or flasks are often needed.

Use of presterilized disposable Cellbag bioreactors minimizes the risk of contamination

making the WAVE Bioreactor system suitable for cGMP production. The ability to

automatically monitor and control multiple parameters also contributes to low

contamination risks, shorter processing time, and lower costs. Since cell cultures can

reach target concentrations in one Cellbag bioreactor, the costs of disposables can be a

fraction of those incurred when using other methods.

19

8. T- lymphocytes :

Manufacturer : GE life sciences

Production parameters :

The WAVE Bioreactor System can be used for cultivation of human T cells and cell

densities of more than 1 × 107 cells/mL at very high viabilities are readily achieved.

Because Cellbag bioreactors are disposable, presterilized, and completely contained,

they are ideally suited for applications where the prevention of cross-contamination is

critical. Additionally, the volume in a Cellbag bioreactor can be increased by a factor of

10, reducing inoculum requirements and the need for transfers. For example, a culture in

Cellbag-10L can be started with as little as 0,5 L of volume and fresh media can be

added to match growth to bring the final batch volume to 5 L. Using the perfusion

capabilities of the WAVE Bioreactor system, very high cell densities can be generated

while maintaining a closed system.

20

9. Vitamine B12 :

Manufacturer : N.R. chemical corporation.

Use : as a dietary suppliment.

Fermentation :

Pink pigmented gram negative bacterial FM-02T strain is used for production of vit B12.

The strain uses methanol as carbon source. When cultured on rotary shaker at 200 rpm

and 30 degree C growth reached maximum in 3 days and amount of vit produced reached

max in 2 days. When growth conditions were optimized in shaking culture, the yield was

not high . hence cultivation method was improved to obtain the cells in larger no.

Exponential fed batch cultivation was found to be significantly favorable for bacterial

growth as well as Vit. B12 production.

Cultivation method Methanol conc. Vit. B12 produced

%, vol/vol micro-gm/litre

Batch 1 108

1 130

2.4 116

Exponential fed batch 0-0.05 400

0-0.95 863

0.4-1.38 2560

21

10. Growth hormone :

Manufacturer : Hangzhou Hongda C&P Co., Ltd.

Use : miracle anti aging drug.

Fermentation :

Recombinant E. coli strain is used. A glucose fed batch fermentation process using a

complex medium was used. In this process a yield of 2000 mg/L of met-ala-glu-hGH

(MAE-hGH) was obtained. The fermentation process comprised a glucose-limited growth

phase followed by a second phase with increased glucose feed and exhaustion of

phosphate from the medium. The second phase is characterized by an MAE-hGH

production, whereas further biomass formation is blocked. High concentrations of

glucose led to reduced specific expression of MAE-hGH—the specific and total yield in

batch glucose fermentations is only about 30% of the yield in optimized fed batch

fermentations. Furthermore it was shown that the specific production rate of MAE-hGH

was independent of the specific growth rate and it was further demonstrated that the

decrease in expression efficiency in glucose batch fermentation was a result of an

inhibitory effect of acetic acid. In batch fermentations this inhibitory effect was enhanced

by a salt effect caused by increased consumption of acid and base used to control pH.

11. Recombinant Chymosin :

Commercial Manufacturer: MaxirenR-DSM Food Specialities(Formerly Gist-

Brocades)

ChymaxR: Pfizer

ChymostarR:Danisco

ChymogenR: Genecor / Chr. Hansen

22

Use: Chymosin used in cheese production. It is first artificially produced enzyme

registered and allowed by United States Food And Drug Administration.60% of US hard

cheese is made with genetically modified chymosin.

Fermentation parameters:

Fermentation microorganism:

Gist brocades – maxiren –K.lactis

Pfizer – chymax-E.coli

Genecor/Chr.Hansan-Chymogen-A.niger

Substrate:Maltose

Type of bioreactor:

Process: Fed Batch

NLF 22 bioreactor: NLF 22 bioreactor has impeller and draft tube with diameter of 6.9

cm and 10.9 cm respectively. Bottom of NLF is spherically shaped. The height to

diameter ratio is 1:95 . The temperature is regulated through an integrated water jacket.

NFL is steam sterilisable in situ by heating water, filled into bioreactor to 120 0C. Culture

is aerated baffle free at low cell density and are intermittently sparged with air/water.

Scale:50 qm

Temperature: 35oC

pH:5.5

12. Cephalosporin-C :

Acremonium chrysogenum NCIM 1069 was used for the biosynthesis of cephalosporin-C

(CPC) in batch mode of cultivation. The culture was maintained on potatodextrose- agar

(PDA) after incubation for 7 days at pH 7.2 and temperature 28ºC.

23

Bioreactor used- The biosynthesis of CPC is studied in a 2L-stirred tank bioreactor

(STBR) using synthetic medium at optimal conditions.

The culture was propagated in the seed medium containing (gL-1) sucrose, 30; potassium

dihydrogen orthophosphate, 1.5; magnesium sulphate, 0.3; and trace metal solution 15mL

which was sterilized separately.

Batch fermentations were carried out in shake flask and in a STBR at 28ºC for 144 hrs on

the synthetic production medium. The production medium contained (gL-1) sucrose, 30;

ammonium sulphate, 7.5; dipotassium hydrogen orthophosphate, 15.6; potassium

dihydrogen

orthophosphate, 15.3; DL-methionine, 3.0; sodium sulphate, 1.7 and oleic acid, 1.5. The

pH of the medium was adjusted to 7.2 before sterilization.

It was observed that with the progress of fermentation increase in the viscosity of broth

occurs and thereby decreasing oxygen mass transfer. then attempted to increase the

oxygen

transfer rate to the broth by coimmobilizing mold with algae called Chlorella

pyrenoidosa in a symbiotic mode.

13. MonoSodiumGlutamate (MSG) :

Substrate :

Small seed cultures are grown on glucose, potassium phosphate, magnesium sulfate,

yeast extract and urea as a source of nitrogen. Larger cultures use cheaper sources of

sugar including cane or beet molasses and starch hydrolyzates from corn or cassava.

The sugar source roughly parallels the geographic location of the process. Ammonia and

ammonium sulfate are used as nitrogen sources. Ammonia can also be used to control

pH during fermentation. Cheap sources of vitamins and other nutrients include corn

steep liquor, a by-product of cornstarch manufacture that is replete with amino acids,

nucleic acids, minerals and vitamins.

Type of bioreactor:

The process is most commonly run as a fed-batch type,

24

where sugar is added during the fermentation process. The reason for using fed-batch

rather than batch, where all components are present at the beginning of the process, is

that high sugar concentrations, more that 20% in total would otherwise need to be added

to the medium. Such high levels can lead to incomplete oxidation of the sugar to lactic or

acetic acids or to osmotic inhibition of growth with subsequent decrease in yields.

To begin the process, stocks of Corynebacterium glutamicum are used to inoculate shake

flask cultures. The resulting cells are transferred to a small seed culture tank that is

grown and used to inoculate a larger tank, and so on. The intermediate seed

culture volumes are variable but generally in the range of 200-1000 liter, then 10,000-

20,000 l and finally the production tank of about 50,000-500,000 l. The process is

carefully controlled at each step such

that cell density, nutrient composition, temperature, pH, aeration, agitation rates

and sugar flow rates are as consistent as possible from batch to batch. Oleic acid

(0.65 ml/l) might be added at the beginning of the fermentation to encourage

glutamate excretion; pH is set around 8.5 with ammonia, and maintained at 7.8

the process. After 14 hours of growth, the temperature is increased from about 32-

33°C to 38°. Sugar is fed in as the

fermentation proceeds up to about 36 h. During the course of the fed-batch process

about 160 g or more of glucose per liter, or its equivalent, is fed into the

bioreactor. Glutamate concentrations are monitored at intervals and, after the process

is complete, the broth is pumped from the bioreactor to recovery tanks. Yields of

glutamate from a large-scale fermentation are in excess of 100 g/l. That means that a

100,000 liter bioreactor yields about 10,000 kg of glutamate.

25

14. Phenolic polymers using peroxidase immobilized into alginate matrix :

            The gas or the liquid (media) are introduced under the layer of biocatalysts with a

velocity at least equal with the minimum fluidization velocity. But, the circulation

velocity of the heterogeneous phase biocatalysts media has to be lower than that needed�

for solid phase pulling-out (the loss of biocatalysts is avoided by enlarging the section of

the bioreactor top).

            The main advantages of these bioreactors are as follows.

-         intense mixing of the media, thus leading to high rates of mass transfer and

of biosynthesis;

-         high rate of heat transfer, therefore lower surfaces needed for

heating/cooling;

-         the possibility to easily control and  regulate the process.

On the other hand, the fluidization induces some negative phenomena: the

mechanical lysis of biocatalysts, due to the high shear forces or to the collision between

the particles, the non-uniform distribution of the residence time, high consumption of

energy.

A bioreactor used for phenolic polymer production is of 4 m height. The

bioreactor has been operated in continuous regime, the overall conversion reached over

60% and the activity of biocatalysts remained at a rather constant level for a long time

26

period (the initial decrease in the biocatalysts activity was due to the deposition of the

polymer layer on the particles surface).

Figure. The activity of immobilized peroxidase measured at the bioreactor outlet.

15. Desacylation of the glycopeptide antibiotic A40926 :

Desacylation of the glycopeptide antibiotic A40926 is done in a cascade of three airlift

bioreactors with immobilized Actinoplanes teichomyceticus cells.

Figure 3. The cascade of the airlift bioreactors used for antibiotic desacylation

27

            The circulation of the broths can be described by the plug-flow model. The

antibiotic conversion was higher than that reached in the single airlift bioreactor or in the

conventional stirred bioreactor, being of 95-99%.

Bioreactor specifications: in this bioreactor The broth circulation and, consequently, the

mixing, are induced by the gradient of density existing between the gassed/aerated and

non-gassed/non-aerated regions into the bioreactor.

            Among the gaslift bioreactors, those having the internal circulation of the media

are considered the most adequate for the immobilized biocatalysts. These bioreactors

could be individually used, or disposed in a cascade system.

16. Lovastatin :

Commercial Manufacturer: Biocon India Limited

Use: HMG Co-A Reductase Inhibitor used in Hypercholesterolemia

Fermentation parameters:

Fermentation microorganism:

Lovastatin produced by using the solid substrate fermentation(SSF) cultivating

Aspergillus. Terreus.

Substrate:

Lovastatin production medium, optimized for A. terreus , was used . This medium

contained 4%lactose, 0.3% soybean meal, 0.2% KNO3, 0.3% KH2PO4, 0.05%

MgSO4.7H2O, 0.05% NaCl,and 0.125% trace elements in solution (in g/l: MnSO4, 1.6;

ZnSO4.7H2O, 3.4; CoCl2.6H2O,2; FeSO4.7H2O, 5) and had a pH of 6.5.

A concentrated version (1.5×, 2×, etc.) of liquid lovastatin production medium with or

without glucosewas inoculated with spores of A.terreus (108 spores/ml) and used to

impregnate the solid support with a given moisture content. The artificial solid support

used here was polyurethane foam (PUF).

28

Type of Bioreactor:

Six grams of solid medium was used to pack glass column fermenters (150-mm

length×21-mm ID) that were aerated with pre-humidified air and incubated in a

Raimbault-type set-up. 6 g of solid medium represented approximately 18 loosely-packed

PUF cubes (initial moisture content (IMC) of 80% and impregnated with 2× medium)

containing 0.73 g of dry PUF.

Aeration rate: Medium incubated in column fermenters with aeration rate of .16 l/g/h

Initial moisture content(IMC): 80% IMC.

pH:6.5

CONCLUSION:

To produce pharmaceuticals, antibiotics, vaccines, antibodies, vitamins and many

neutraceuticals etc. there is need to develop large scale bioreactor. Type of bioreactor

used for the production of particular product needs to satisfy the optimum conditions

require for better yield.

29