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Bioprocess Technology 827
CHAPTER
33 Bioprocess Technology
Bioprocess technology makes use of microbial, animal and plant cells and components of cells such
as enzymes to manufacture new products and destroy harmful wastes. Application areas com-
monly associated with bioprocess technology include the production of biofuels, ethanol, amino
acids and other organic acids, antibiotics, design and operation of fermentation systems, develop-
ment of food processing systems, application and testing of product separation technologies,
design of instrumentation to monitor and control biological processes, and many more. The
purpose of this chapter is to lay the foundation of basic concepts and practical uses of bioprocess
technology in agriculture, medical, veterinary and industrial applications.
Keywords : Bioprocess technology; bioreactor; downstream processing; batch processing; me-
tabolites.
INTRODUCTION
Bioprocess technology works at the frontiers of biological and engineering sciences to Bring
Engineering To Life through the conversion of biological materials into other forms needed by
mankind. Bioprocess technology is a specialization of Chemical Engineering or of Agricultural
Engineering. It deals with the design and development of equipment and processes for the manu-
facturing of products such as food, feed, pharmaceuticals, nutraceuticals, chemicals, and polymers
and paper from biological materials. Application areas commonly associated with bioprocess tech-
nology include the production of biofuels, design and operation of fermentation systems, develop-
ment of food processing systems, application and testing of product separation technologies,
design of instrumentation to monitor and control biological processes, and many more. Like other
engineering disciplines, bioprocess engineers are trained in the application of engineering sciences
and problem solving techniques. What separates them from other engineers is their understanding
of how every engineering field relates to living materials.
Bioprocess development for biopharmaceuticals involves all aspects of generating a safe, effec-
tive, and stable product. It begins with the biological system, continues with product isolation and
purification, and finishes when the product is placed in a stable, efficacious, and convenient form.
The product is initially derived either directly or indirectly from a living organism. Thus, process
development starts with the development of the biological system. It is usually a living organism
that expresses the desired protein; but it might be an enzyme for protein modification or an antibody
for immuno-affinity purification. rDNA and hybridoma technology allows the biological system to
be optimized for maximal formation of the product, for facilitation of downstream processing, for
828 Biotechnology in Medicine and Agriculture
high product quality, and for improved interaction with the production equipment (Figure 33.1).
In this phase of bioprocess engineering, many disciplines must be applied, including molecular
biology, genetics, biochemistry, analytical chemistry, and bioprocess engineering. Thus, engineers
become full partners with experts trained in the bioscience disciplines in developing and scaling up
manufacturing technology for biopharmaceuticals. As the task of process development proceeds
through isolation, purification, and formulation, multidisciplinary approaches continue to be advan-
tageous. Bioprocess engineers combine their skills with those of biochemists and analytical bio-
chemists to develop the optimal process. The multidisciplinary task of bioprocess engineering is
usually applied to developing processes for the production of biopharmaceutical products, but it
can also be applied to the discovery of new products. For example, automated screening methods
that use purified cell receptors can be developed. More efficient methods for producing and
isolating families of precisely modified proteins can be devised, and the technology required to
produce protein pharmaceuticals efficiently with mammalian cells can be applied to develop cell-
based assays to screen for product activity or toxicity.
Figure 33.1 Steps in the development of a complete bioprocess for commercial manufacture of a
new recombinant-DNA-derived product.
The bioprocessing of renewable and nonrenewable resources requires the application of multiple
disciplines in a cross-disciplinary approach. As in the case of biopharmaceuticals, products are
derived either directly or indirectly from living organisms. Unlike those in the biopharmaceutical
sector, however, bioprocess engineers dealing with renewable and nonrenewable resources are
involved with high processing volumes and often design and operate large installations. The
processes themselves must still be reliable, robust, and cost-effective. The goal is to generate
products from renewable resources or to produce microbial and other biocatalysts for use in
processing renewable or nonrenewable resources to obtain value-added products. Economies of
scale and scalable equipment are important, if not critical, process characteristics. This sector of
the biotechnology industry also produces specialty products (enzymes, amino acids, organic acids,
and animal-health products) for which the processing technology and regulatory issues are similar
to those associated with high-volume biopharmaceutical products, but the ratio of market price to
processing cost can be much lower. Production efficiency, as well as product quality, is paramount.
A key function of bioprocess engineering is the design, operation, and improvement of manufac-
turing technology to yield bioproducts at high volume and acceptable costs. Success will require
bioprocess engineers to become partners with molecular biologists and geneticists, biochemists,
carbohydrate chemists, wood chemists, food scientists, analytical chemists, and microbial physi-
ologists. With the rapid growth of biologically based technologies, bioprocess engineers are
uniquely qualified to solve the problems of today and tomorrow. The demand for bioprocess
engineers continues to grow. They provide a bridge between the research laboratory and the
economic, large-scale implementation of biotechnologies and food production systems.
CONCEPTS
Bioprocess technology evolved as a means of producing food, beverages, and medicines. More
than 8,000 years ago, it was used to make leavened bread. Some 5,000 years ago, moldy soybean
curd was used to treat skin infections in China. The malting of barley and fermentation of beer was
used in Egypt in 2500 BC. Louis Pasteur proved in 1857 that yeast is a living cell that ferments
sugar to alcohol; in 1877, he showed that some bacteria kill anthrax bacilli. In 1923, Banting and
Best showed that insulin from animals could be used to treat people suffering from diabetes. In
1928, Alexander Fleming showed that growing colonies of Penicillium notatum inhibit Staphylococ-
cus cultures. Beginning in 1939, Florey and Chain rediscovered that Flemings Penicillium could
lyse bacteria, but the yield of penicillin was small; in addition, the penicillin it did produce was
unstable. They realized that producing penicillin on a large scale would require isolation and
purification procedures that minimized product loss. Early bioprocess engineers found solutions to
this problem when researchers in the U.S. Department of Agriculture (USDA) laboratory in Peoria,
Illinois, discovered that mold on a cantaloupe (P. chrysogenum) could be grown in large tanks in
submerged cultures. During World War II, government incentives encouraged several pharmaceu-
tical companies to develop cost-effective manufacturing processes for penicillin. Chemical engi-
neers, industrial chemists, and microbiologists quickly devised methods of countercurrent extrac-
tion, crystallization, and lyophilization to recover penicillin in an active and stable form and estab-
lished the viability of submerged fermentations. The benefits of biotechnology might be an anomaly
if it were not for engineering, specifically bioprocess technology, the discipline that puts biotech-
nology to work. To quote Louis Pasteur, bioprocess engineering is to biotechnology as the fruit
is to the tree. Neither can exist without the other. The realization of the benefits of penicillin
required the development of methods of transforming microbial growth on the surface of a moldy
cantaloupe to cultures grown in large stirred tanks fed by sterile air. It took engineers to design the
tanks, impellers, pumps, compressors, columns, pipes, and valves that have made biotechnology
products available to large numbers of people. The lifesaving benefits of insulin required engineering
for the extraction and purification of insulin from cow and pig pancreas, and later, the large-scale
propagation of bacteria engineered to make human insulin, as well as methods of recovery,
refolding, and purification to obtain an active molecule. Biochemical manufacturing and bio-
separations have made it possible to purify products derived from biotechnology on a large scale.
PRINCIPLES
The basic principles of bioprocess technology are based on understanding of the microorganisms
or biocatalysts involved. This ensures:
(i) consistent product quality and product safety, regardless of scale;
(ii) addressing environmental consequences of the manufacture and use of the products; and
(iii) continually improving and developing processes in response to the competitive pressures
generated by consumer markets.
The engineering operations that cut across those requirements include the development of
bioreactor technology, the processing of impure dilute product streams into purified and concen-
trated form, and the ability to apply engineering economics in decision-making processes during
the design, development, operation, and improvement of bioprocess facilities. That requires engi-
neers who can solve engineering problems on the basis of an understanding of the biochemistry
and genetics of living organisms. A sustained policy is needed to foster development of a funda-
mental knowledge base for the manufacture of a broad spectrum of bioproducts and the training
of bioprocess engineers who will grow with the growing industry.
BIOPROCESS TECHNOLOGY
Purpose: Bioprocess technology has been used for the following purposes:
(i) In the formation of complex molecular structure such as antibiotics and proteins where
there is no practical alternative.
(ii) In the exclusive production of one specific form of an isomeric compound.
(iii) Because micro-organisms may efficiently execute many sequential reactions.
(iv) Because bioconversions may give high yields.
Requirements for Bioprocessing

Raw materials
Current bioprocess technology uses an extremely limited range of raw materials just a few agri-
cultural commodities in many of the existing industrial bioprocesses. The need for raw materials
for bioprocesses, however, could become a major factor in commodity grain markets if
bioprocesses find a place in large scale fuel or chemical production.
Biocatalysts
The substances that actually cause chemical changes in bioprocesses are the enzymes produced
by a living cell. Biological transformation of the substrate involves several sequential and interrelated
chemical reactions, each catalyzed by a separate enzyme, however whole cells (e.g., microorgan-
isms such as bacteria, yeast or fungi) are used as biocatalysts. Bioprocesses used for the synthesis
of complex molecular structures (e.g., antibiotics or proteins such as insulin) require entire system
of enzymes. Sometimes animal cells and tissues are employed for vaccine production and related
activities but the catalytic capabilities of plant cells except for some algae, have not yet been
employed commercially. Biocatalysts generally exhibit great sensitivity to changes in temperature,
pH and even concentrations of certain nutrients or metal ions. The success of a bioprocess depends
on the extent to which these factors are controlled in the medium where interaction between
biocatalyst and substrate take place. Biocatalysts that can withstand high temperatures and high
pressure may also be useful industrially. Wider availability of thermo tolerant biocatalysts is impor-
tant for all industries using bioprocesses. The advantages of thermo tolerance include:
(i) Reduced susceptibility to contamination
(ii) easier removal of conversions
(iii) more complex and rapid conversions
(iv) easier recovery of volatile products
Water
Water is the dominant component of the medium for virtually all current bioprocesses and is needed
in many of the ancillary services such as heating, cooling, cleaning and rinsing. Clean water of
consistent composition is therefore required in large quantities from reliable permanent sources.
When assessing the suitability of a water supply it is important to consider pH, dissolved salts and
effluent contamination. Products must usually be purified from the dilute aqueous solution. The
environment is closely controlled and this necessity markedly influences their design.
Supply of Nutrients
The medium must provide for the nutritional needs of living cells in addition to establishing a suitable
environment. A primary requirement is a source of carbon. In addition to supplying the energy
needed for metabolism and protein synthesis, carbon sources contribute structural elements re-
quired for the formation of complex compounds. Other important nutrients required by living cells
are nitrogen and phosphorus which are sometimes oxygen; nitrogen and phosphorus are incorpo-
rated into structural and functional molecules of a cell and may also become part of product
molecules. A number of other nutrients such as vitamins and metal ions, though required only in
very small amounts, are nevertheless essential.
On a large scale the normally used sources of nutrients to create a medium which will meet as
many as possible of the following criteria:
(i) It should produce the maximum yield of product or biomass per gram of substrate used.
(ii) It should produce the maximum concentration of product or biomass.
(iii) It should permit the maximum rate of product formation.
(iv) There should be the minimum yield of undesired products.
(v) It should be of a consistent quality and be readily available throughout the year.
(vi) It should cause minimal problems during media making and sterilization.
(vii) It should cause minimal problems in other aspects of the production process particularly
aeration and agitation, extraction, purification and waste treatment.
Energy sources
Energy for growth comes from either the oxidation of medium components or from light. Most
industrial microorganisms are chemo-organotrophs, therefore, the commonest source of energy
will be the carbon source such as carbohydrates, lipids and proteins. Some microorganisms can
also use hydrocarbons or methanol as carbon and energy sources.
Carbon source
The main product of a bioprocess will often determine the choice of carbon source, particularly
if the product results from the direct dissimilation of it. It is now recognized that the rate at which
the carbon source is metabolized can often influence the formation of biomass or production of
primary or secondary metabolites. Fast growth due to high concentrations of rapidly metabolized
sugars is often associated with low productivity of secondary metabolites. The purity of the carbon
source may also affect the choice of substrate. The method of media preparation, particularly
sterilization, may affect the suitability of carbohydrates for individual bioprocesses. It is often best
to sterilize sugars separately because they may react with ammonium ions and amino acids to form
black nitrogen containing compounds which will partially inhibit the growth of many micro-
organisms.
In the most common practice carbohydrates are used as the carbon source in microbial fermen-
tation processes. The most widely available carbohydrate is starch obtained from maize grain. It
is also obtained from other cereals, potatoes and cassava. Maize and other cereals may also be used
directly in a partially ground state, e.g., maize chips.
Barley grains may be partially germinated and heat treated to give the material known as malt,
which contains a variety of sugars besides starch. Malt is the main substrate for brewing beer and
lager in many countries. Malt extracts may also be prepared from malted grain.
Sucrose is obtained from sugar cane and sugar beet. It is commonly used in fermentation media
in a very impure form as beet or cane molasses which are the residues left after crystallization of
sugar solutions in sugar refining. Molasses is used in the production of high-volume flow-value
products such as ethanol, SCP, organic and amino acids and some microbial gums. Molasses or
sucrose also may be used for production of higher value/low-bulk products such as antibiotics,
special enzymes, vaccines and fine chemicals.
Nitrogen sources
Most industrially used microorganisms can utilize inorganic or organic sources of nitrogen. Inor-
ganic nitrogen may be supplied as ammonia gas, ammonium salts or nitrates.
Organic nitrogen may be supplied as amino acid, protein or urea. In many instances growth will
be faster with a supply of organic nitrogen, and a few microorganisms have an absolute require-
ment for amino acids. However, amino acids are more commonly added as complex organic
nitrogen sources which are non-homogeneous, cheaper and readily available. Other proteinaceous
nitrogen compounds serving as sources of amino acids include corn-steep liquor, soya meal, peanut
meal, cotton-seed meal and yeast extract.
Supply of Oxygen
Since most of the microorganisms currently used by industry perform their conversions aerobi-
cally. The fundamental problem in supplying sufficient oxygen is oxygens low solubility. Solubility
of oxygen decreases as the concentration of dissolved solutes increases. However, solubility of
oxygen in the growth medium can be increased by:
(i) Increasing oxygen pressure of the gas phase by increasing reactor pressure.
(ii) Using oxygen rich gas for aeration.
(iii) Changing in process design and operation.
For small-scale culture, e.g., less than 1L, it is possible to supply adequate amounts of oxygen
by growing the microbe in an Erlenmeyer flask which is agitated constantly, provided that the
volume of growth medium does not exceed 10-20% of the flask volume. For large-scale culture
and/or high cell densities the oxygen demand of the culture can be met by blowing air through the
culture.
At laboratory-scale cultures may be aerated by means of the shake-flask technique where the
culture is grown in a conical flask shaken on a platform contained in a controlled environment of
the chamber.
At Pilot- and industrial-scale fermentations broth or culture is aerated by stirred or agitation
method.
Some bioreactors or fermenters are so designed that adequate supply of oxygen is obtained
without agitation and such bioreactors or fermenters are called bubble columns and air-lift fer-
menter.
Bartholomew et al. (1950) represented the transfer of oxygen from air to the cell, during
fermentation, as occurring in a number of steps:
(i) The transfer of oxygen from an air bubble into solution.
(ii) The transfer of the dissolved oxygen through the fermentation medium to the microbial cell.
(iii) The uptake of the dissolved oxygen by the cell.
The individual steps involved in oxygen transfer from a gas bubble to the reaction site inside the
individual cell are given bellow: (Figure 33.2)
1. Diffusion of oxygen from the bulk gas to the gas liquid interface.
2. Transport across the gas liquid interface.
3. Diffusion of oxygen through a relatively stagnant liquid region adjacent to the gs bubble i.e.
from the gas-liquid interface to the well-mixed bulk liquid.
4. Transport of oxygen through the well-mixed liquid to a relatively unmixed liquid region
surrounding of the cells.
5. Diffusion through the stagnant region surrounding the cells.
6. Transport from the liquid to the pellet cell aggregate etc.
7. Diffusion transport of oxygen into the pellet etc.
8. Transport across the cell envelope.
9. Transport from the cell envelope to the intracellular reaction site, e.g., the mitochondria.
Figure 33.2 The individual steps involved in oxygen transfer from a gas bubble to the reaction site
inside the individual cell.
Pure Culture and Sterilization

Most of the products of bioprocesses are formed through the action of a single biocatalyst, either
a microorganism or an enzyme. If foreign organisms contaminate the process system, they may
disrupt its operation in a variety of ways. They can directly inhibit or interfere with the biocatalyst,
whether it is a single enzyme or a complete cell and they may even destroy the biocatalyst
completely. Alternatively, contaminating organisms may leave the catalyst unaffected but modify
or destroy the product. Also, there substances are difficult to separate from the primary product.
In the manufacture of pharmaceutical products, the risk of toxic impurities is of particular concern.
To avoid or minimize contamination, most current bioprocess technologies employ pure culture
techniques. The medium and its container are sterilized and a pure culture consisting of a population
of a particular species is introduced. In order to avoid subsequent contamination, all materials
entering the system, including the large amounts of air required for aerobic processes, are sterilized.
The apparatus must be designed and operated so that the opportunities for invasion by unwanted
organisms are minimized.
Bioprocessing Modes (Batch ver. Continuous Operation)

A bioprocess may, in principle, use any of the operating modes employed by conventional chemical
technology. These modes range from batch processing to continuous steady state processing
which include batch processing, semi-batch processing, fed-batch processing continuous
steady-state processing.
Batch Processing
In batch processing the reaction vessel (e.g., fermenter) is filled with the medium containing
substrate, sterilized, inoculated (or added biocatalyst) and the bioconversion is allowed to proceed.
It takes time ranging from a few hours to several days. During this time, nutrients as well as
substrates, agents for pH control and air are supplied and product gases are removed from the
reaction vessel. When conversion is complete, the reaction vessel is emptied and cleaned and
prepared for a fresh run. Simultaneously, the purification process begins. In some instances, this
turnover time between the batches can be prolonged.
A semi-batch processing allows either input or output of mass, but not both.
A fed-batch processing allows input of material to the system but not output.
Continuous Steady-state Processing

In this process raw materials are supplied to the vessel at a rate volumetrically equal to that at which
product and spent medium are withdrawn. The possibility of the long runs (i.e. several weeks
duration) is the potential advantage offered by continuous processing over batch processing which
leads to significantly higher productivity, greater ease of product recovery through the reuse of
contaminating biocatalyst and lower cost due to reuse of biocatalyst. The extent of increase in
productivity will depend on the ratio of processing time and turnover time: the smaller the ratio,
the more advantageous is continuous operation. A second advantage is that the growth rate of the
cells which is required for optimal product formation can be maintained.
Continuous culture is ideally suited for the production of biomass or simple metabolites or simple
metabolites such as ethanol where synthesis is proportional to cell density. It is not suitable for the
production of other metabolites such as amino acids and antibiotics where synthesis is not asso-
ciated with growth. The most widespread use of continuous culture is in the effluent disposal
industry where waste organic materials (substrate) are converted into microbial cells (biomass).
Batch operation currently dominates specially in chemical and pharmaceutical bioprocesses and is
likely to continue to do so in the near future. In addition to technical limitations on continuous
processing, other considerations have led manufacturers to choose the batch mode. Batch process-
ing is often used because it offers the operational flexibility needed when a large number of products
are manufactured, each at fairly low production levels. Each process unit, more or less standard
in design, can easily rapidly be switched from one product line to another. The simplest approach
to the implementation of a continuous processing system is to modify a batch reactor so that fresh
substrate and nutrients can continually be added while a product stream is removed. Furthermore,
a switch from batch to continuous processing is expensive. This arrangement has also one serious
drawback: the biocatalyst leaves the reactor continuously with the outlet stream and must be
separated from the product. Several techniques, all of which involve fixing the biocatalyst in some
manner, have been developed to avoid the biocatalysts escape with the reaction mixture and
allowed its repeated use. The development of techniques for the immobilization of continuous
bioprocesses, although still not widely employed for large scale bioprocess, the biocatalyst immo-
bilization techniques now available (discussed in Chapter 32) offer a diversity of new opportunities
for more effective bio-processing. Increased use of genetically manipulated biocatalysts could
affect the design and operation of bioconversion units. Harvey Blanch points out:
One of the difficulties which arise from the insertion of foreign DNA into the organism is
reversion. This can be minimized by placing the cell in an environment in which cellular replication
is minimized, while cellular activity such as the production of enzymes and products is maintained
at high levels.
Achieving the dual objective of minimal growth and maximum conversion activity requires
restrictive nutrient supplies and high cell densities. Immobilized biocatalysts could be used to
achieve these objectives.
Novel Reactor Designs in Bioprocess Monitoring

The main function of a bioreactor is to provide a controlled environment for the growth of
microorganisms or animal cells, to obtain a desired product. As a result of the growing need for
efficient and cost effective means of producing large quantities of important biological compounds
from living cells and enzymes, novel bioreactor types and the modes in which they are operated
have been developed. Currently bioreactors are used for small to large-scale production of many
different compounds from plant, microbial and animal cells as well as from isolated enzyme
systems.
In choosing a bioreactor type and mode of operation, a biotechnology company usually balances
medium costs, downstream processing costs and technological advantages.
During the designing and constructing a bioreactor following points must be considered (Figure
33.3):
1. The vessel should be capable of being operated aseptically for a number of days and should
be reliable in long-term operation and meet the requirements of containment regulations.
2. Adequate aeration and agitation should be provided to meet the metabolic requirements of
the microorganism. However, the mixing should not cause damage to the organism.
3. Power consumption should be as low as possible.
4. A system of temperature control should be provided.
5. A system of pH control should be provided.
6. Sampling facilities should be provided.
7. Evaporation losses from the fermenter should not be excessive.
8. The vessel should be designed to require the minimal use of labor in operation, harvesting,
cleaning and maintenance.
9. Ideally the vessel should be suitable for a range of processes, but this may be restricted
because of containment regulations.
10. The vessel should be constructed to ensure smooth internal surfaces, using welds instead
of flange joints whenever possible.
11. The vessel should be of similar geometry to both smaller and larger vessels in the pilot plant
or plant to facilitate scale-up.
12. The cheapest materials which enable satisfactory results to be achieved should be used.
13. There should be adequate service provisions for individual plants.
The first two points are probably the most critical. Its obvious from the above points that the
design of a bioreactor will involve co-operation between experts in microbiology, biochemistry,
chemical engineering, mechanical engineering and costing.
Figure 33.3 Diagram of a bioreactor.
Types of bioreactors: There are two types of bioreactors: 1) conventional bioreactors 2) and
membrane bioreactors (Figure 33.4).
Figure 33.4 Membrane bioreactor.

Conventional Bioreactors
Conventional bioreactors have been used almost universally for fermentation in food, beverage and
drug industries. Because of their widespread use and operator familiarity, they have also been
adapted for cell culture. Although most operations use batch mode, continuous operations are also
used. The continuous stirred tank reactor (CSTRs) is the work house of the fermentation industry
because of its reliability and versatility. Considerable power is needed to drive the agitators used
in large fermenters and to compress the air supplied. This leads to high utility costs, principally for
electricity.
Figure 33.5 Novel bioreactor.
One way of reducing the power requirements is to use the air supplied to agitate the culture.
This can be done by designing three novel reactors (Figure 33.5).
(i) Airlift fermenter.
(ii) Deep-shaft reactor.
(iii) Bubble column (tower) fermenter.
In an airlift fermenter, the air is introduced at high velocity at the bottom of the vessel. The use
of a draught tube enables the liquid to circulate with considerable turbulence. For efficient oxygen
transfer it is necessary for the fermenter to have a height to diameter ratio (10:1- 40:1) that is much
greater than with a conventional stirred tank reactor (1:1 4:1).
As a gas bubble ascends a column of liquid, the hydrostatic pressure on it decreases and the
volume of the bubble increases. Since there now is a decrease in the surface area to volume ratio
there is a decrease in the rate of oxygen transfer from the gas phase to the bulk liquid. Furthermore,
in a well agitated fermenter over 50% of the oxygen supplied is utilized. Thus, as the bubble ascends
the column, the partial pressure of oxygen within it will decrease and this too will decrease the rate
of oxygen transfer to the liquid.
A novel reactor design which overcomes these disadvantages is the deep-shaft reactor. Here the
air is introduced at high velocity at the top of the reactor and forces the liquid downwards. Although
oxygen is utilized during the downward passage the bubble size will diminish because of the
hydrostatic pressure and should give rise to a more uniform oxygen transfer rate.
Both the airlift fermenter and the deep-shaft reactor can be operated continuously. The entire
vessel contents are essentially homogeneous, both spatially and temporally. By contrast, in a bubble
column reactor, which is a variant of the airlift fomenter, the contents of the vessel are not
homogeneous. Since there is no mixing, the medium moves through the fermenter as a plug and
its composition varies spatially. By dividing the fermenter into sections it is possible to vary the
physiochemical environment at will e.g. pH 7.0 at temperature 30°C in one section and pH 40°C
in another section.
Membrane Bioreactors
Membrane bioreactors, which simulate the in vivo capillary tissue environment by retaining the
biological catalyst (cell or enzymes) behind a barrier, have proven to be quite successful in both
design goals (as discussed earlier). Membrane systems provide a very mild method of immobilizing
cells and enzymes composed with technique requiring chemical attachment. Other advantages
include their high surface to volume ratios (important for substrate delivery and waste removal)
and their ability to isolate the cells/enzymes from shearing and contamination. With recycle,
membrane systems can often achieve higher product concentrations, yields and productivities than
batch reactors. These reactors retain the cells in a stable environment with a continuous supply of
nutrients, substrates and cofactors while simultaneously removing metabolic wastes. In this man-
ner, cells can be maintained in a state of low or non-proliferating state for long periods of time while
still producing the desired products.
Typical system configurations are the hollow fiber and the plate and frame (flat sheet membrane
reactors have been used successfully for entrapped culture of microbial, plant mammalian cells.
Entrapment of selected cells/ enzymes in spiral wound modules is only beginning to be explored.
These membrane systems can also act as integrated production and separation devices.
Despite the advantages of using membrane bioreactors with or without perfusion for culturing
cells and producing large amount of products, these systems do have limitations. One of the
proclaimed disadvantages of membrane systems is the potential for concentration polarization and
fouling which can severely diminish the trans-membrane and nutrient transport.
Fouling can usually be avoided by using hydrophilic membranes with low surface charge density
which has the effect of minimizing adsorption on the pore walls and surfaces of the membrane.
Concentration polarization can be minimized by the use of high flow rates through the lumen space
which increases the wall shear rate and by using the highly porous membrane which lowers the
protein refraction coefficient. Another disadvantage is the existence of diffusional limitations. Since
in many systems transport of nutrients and by-products relies primarily on diffusion, cells further
from the membrane surface may be subjected to low substrate and high waste concentrations
leading to cell stagnation and a possible decrease in viability.
Other concerns with the use of hollow fiber bioreactors are the need to maintain aseptic
operation and the need to sterilize the bioreactor prior to each run. The use of steam and harsh
chemicals often degrades the membranes and the plotting materials such as polyurethane and epoxy
that hold and seal the fibers in the cartridge. Several approaches have been used to solve this
problem. Porous polypropylene and porous modified glass have been used as auto-cleavable
membrane materials. Cold sterilization techniques such as ethylene oxide and radiation have been
successfully used. Some companies offer pre-sterilized and disposable, semi- or fully autoclavable
hollow fiber cartridges.
The membranes used in reactors must be chosen carefully from the many available types. When
a membrane is used to act as a preamble barrier between cells and the cell-free culture medium
which carries both nutrients and products, it must be hydrophilic to allow for adequate transport
across the membrane. In other reactors where gases and /or organic solutions are used, a com-
bination of hydrophilic and hydrophobic membranes may be more appropriate. The pore size
(MWCO) must be chosen to allow the proper selectivity and mechanical strength should be
adequate to withstand operational pressures. Ultra filtration (MWCO form 103 to 106) or micro
porous (0.01- 10 µm pore size) membranes are typically used for immobilizing cells in reactors.
Micro porous membranes will retain cells yet allowed passage of product molecules while ultra
filtration membrane may retain some product molecule in the cell space, increasing its concentra-
tion with time. Table 33.1 lists some of the types of membrane and module as well as the catalysts
which have been used in bioreactors (Figure 33.6).
Table 33.1 Membrane materials used in bioreactors.

a
Membrane material Catalyst Module
Acrylic copolymer SV3T3 cells HF
Cellulose acetate Hepatome cells BHK and L929 cells hybridomas HF
Mixed cellulose esters B. Cells HF
Polyacrylonitrile SV 3T3 Cells HF
Polyamide Glucose oxidose HF
Polypropylene L. delpreuckii HF
S. cerevisiae HF
N. tabacum HS
S. aureofaciens HF
E. coli HF
Polysulfone SV3T3 cells HF
S. cerevisiae HF
D. corota HF
Hybridomas HF
Fibroblasts HF
H. henricii HF
Hydrophilic PVDF Hybridomas FS
Teflon Hybridomas HH
a HF. hollow fiber; FS, flat sheet
Figure 33.6 Various types of bioreactors.
Downstream Processing (Separation and Purification of Products)

The bioprocesses also release a variety of unwanted metabolites associated with useful products.
Separation and purification of useful products from these other associated unwanted metabolites
is also needed and is sometimes described as downstream processing. Separation and purification
techniques used in bioprocesses are the aspect of bioprocess engineering and needs of attention,
especially for the production of novel products such as proteins. Once fermentation is complete,
it is necessary to recover the desired and product. At a minimum this will involve separation of the
cells from the fermentation both but also may include purification of metabolites with or without
cell disruption. Such operations are referred to as downstream processing. Research is need to find
highly selective recovery techniques that leave as little residual product as possible in the medium
and thus lessen the labor intensity associated with downstream processing.
The choice of recovery process is based on the following criteria:

(i) The intracellular or extracellular location of the product.
(ii) The concentration of the product in the fermentation broth.
(iii) The physical and chemical properties of the desired product.
(iv) The intended use of the product.
(v) The minimal acceptable standard of purity.
(vi) The magnitude of bio-hazard of the product or broth.
(vii) The impurities in the bioreactor broth.
(viii) The marketable price for the product.
The recovery and purification of many compounds may be achieved by a number of alternative
routes. The decision to follow a particular route involves comparing the following factors to
determine the most appropriate under a given set of circumstances:
(i) Capital costs.
(ii) Processing costs.
(iii) Throughput requirements.
(iv) Yield potential.
(v) Product quality.
(vi) Technical expertise available.
(vii) Conformance to regulatory requirements.
(viii) Waste treatment needs.
(ix) Continuous or batch processing.
(x) Automation.
(xi) Personnel health and safety.
Some of the possibilities for improving recovery techniques now under consideration include the
following:
1. Some cells rapidly settle out of suspension once aeration and agitation of the fermented broth
cease and the setting may be assisted by the addition of flocculating agents. Many of these
flocculating agents are polyelectrolytes which neutralize the charge on the microbial cell
surface and keeps cells in suspension by electrostatic repulsion. Where cells settling occur,
or can be induced to occur, the cell removal becomes easy and inexpensive for the fermen-
tation broth and can be decanted or filtered off.
2. Where cell settling does not occur, cell removal can be effected by centrifugation.
3. An alternative to centrifugation is ultra filtration. Membranes and other filtration systems
such as porous metals offer many advantages, and considerable experience in other areas
of chemical technology is already available. Some U.S. companies such as Millipore, Amicon
and Needeopor are making advances in this area. The term ultra filtration describes pro-
cesses in which particles significantly greater in size than the solvent are retained when the
solution is forced through a membrane of very fine pore size, usually less than 0.5 µm.
4. The clarified fermentation liquor will contain microbial metabolites and extracellular en-
zymes and many methods are available for their recovery including continuous chromatog-
raphy, ion exchange chromatography, high performance liquid chromatography, precipita-
tion and solvent extraction. The use of ion exchange resins is particularly desirable since
they can be used for concentration as well as having capacity and being cheap and reusable.
5. Electrophoretic methods, especially continuous flow can separate proteins, peptides and
nucleic acids on the basis of their electrical charge. The advantage of this separation method
over some others is that it can run continuously and can effectively separate molecules in
large sample volumes.
6. Immobilized monoclonal antibodies are being used as purification agents for protein prod-
ucts (see in hybridoma technology). This technique best suits large molecular weight and
high value added products such as proteins.
If the desired product is retained within the cell then cell disruption will be necessary to affect
its release. Although many methods of cell disruption and used in the laboratory, most of them can-
not be used on a large scale. Two methods which are suitable for large scale use are high pressure
homogenization and the use of high speed bead mills. After cell disruption the cell debris needs to
be removed. For the removal of intact cells the most widely used methods are centrifugation and
ultra filtration.
Advantages of Bio-processing
Bioprocesses may offer the following advantages over conventional chemical processes:
(i) Milder reaction conditions (temperature, pressure and pH).
(ii) Use of renewable (biomass) resources as raw materials for organic chemical manufacture,
providing both carbon skeletons and the energy required for synthesis;
(iii) Less hazardous operation and reduced environment impart.
(iv) Greater specificity of catalytic reaction.
(v) Less expensive or more readily available raw materials
(vi) Requires less complex manufacturing facilities and smaller capital investments.
(vii) Improved process efficiencies (e.g., higher, yields, reduced energy consumption) and
(viii) The use of recombinant-DNA-technology to develop new processes.
Disadvantages of Bio-processing
Some of the conceivable disadvantages of bioprocesses on the other hand, the following:
(i) The generation of complex product mixtures requiring extensive separation and purification
especially when using complex substrates as raw materials (e.g., lignocelluloses)
(ii) Problems arising from the relatively dilute aqueous environments in which bioprocesses
function (e.g., the problem of low reactant concentrations and hence, low reaction rates.)
(iii) The susceptibility of most bioprocess system to contamination by foreign organisms, and
in some cases, the need to contain the primary organism so as not to contaminate the
surrounding.
(iv) An inherent variability of biological processes due to such factors as genetic instability and
raw material variability.
(v) For recombinant DNA systems, the need to contain the organisms and sterilize the waste
streams, an energy intensive process.
Solutions to some of these problems through the use of biotechnology may make
bioprocesses more competitive with conventional chemical synthesis.
APPLICATIONS OF BIOPROCESS TECHNOLOGY

Bioprocess technology has several implications in medical, veterinary, agricultural sciences and
industrial applications such as in production of cell matter (biomass itself) (e.g., banks yeast, single
cell protein); production of cell components (e.g., enzymes nucleic acids); production of metabo-
lites (chemical products of metabolic activity) including both primary metabolites (e.g., ethanol,
lactic acid) and secondary metabolites (e.g., antibiotics); catalysis of specific, single-substrate
conversion (e.g., glucose to fructose, penicillin to 6-aminopencillanic acid and; catalysis of mul-
tiple-substrate conversions e.g., biological waste treatment; production of microbial insecticide and
many more. Some of the important applications are discussed.
Medical Applications
In medical sciences bioprocess technology has several applications such as production of bacterial
vaccines (e.g., for cholera, whooping cough, plague, typhoid, paratyphoid fever and typhus fever);
production of the diversity of commercially useful low molecular weight compound produced by
microorganism such as antibacterial agent, antifungal agent, anti-heliminthic agent, pharmacologi-
cally active agents (e.g., ergot alkaloids (vasoconstrictor), cyclosporine (immuno-suppressor
drug), mevinolin (cholesterol reduction) vitamins, amino acids, therapeutically useful steroids,
etc.); Production of some human proteins with therapeutic potential using recombinant microor-
ganisms (e.g., the proteins include interferons, human growth hormone, insulin, etc.) and many
more.
Gene-Based Pharmaceuticals and Gene Therapy

New classes of products are being tested for use in humans and animals, all sharing genes as
common targets. Products based on antisense technology directed toward neutralizing messenger
RNA are probably being pursued most vigorously; gene therapy through permanent alteration of
chromosomes might hold the greatest potential for treatment of diseases like cancer and for
correction of genetic disease. The products depend either on classes of compounds that are related
to nucleic acids (oligonucleotides and oligonucleotide analogues), on cells that have been genetically
altered, of on viruses that bear appropriate nucleic acids. For the large-scale production of nucle-
otides and nucleotide analogues, new molecular techniques must be developed. There are now no
procedures for making substantial quantities of these types of materials in high purity and with
appropriate chiralitys. Basic chemical and biochemical techniques must be developed for their
preparation; new techniques (probably based on high-pressure chromatography) will be required
for large-scale purification, and biological methods might be required for preparation of precursors
and perhaps for formation of bonds. For genetically modified cells and viruses, the usual techniques
for mammalian-cell culture and molecular biology will be required, as will additional measures for
safety and for economical, patient-specific production.
Biopharmaceuticals and Biopesticides from Insect Cell-Baculovirus System

Insect cells from moths can be cultivated in a manner similar to (but not identical with) the manner
in which mammalian cells can. Unlike mammalian cells, insect cells are naturally continuous cell
lines. They can be adapted easily to growth in serum-free media. The unique biphasic life cycle
of the baculovirus, which readily infects cultured insect cells, makes it an ideal vector for expres-
sion of foreign genes. The baculovirus contains a late promoter that is very strong perhaps the
strongest eukaryotic promoter known. Consequently, the insect cell baculovirus expression can be
used to produce very high levels of protein (up to 40% of total protein) in a non-transformed host
cell (i.e., noncancerous) with a vector that is non-pathogenic to vertebrate animals. Those features
of high expression levels and increased safety distinguish this system from the commonly used
mammalian expression systems. However, the high expression levels are not typically obtained with
secreted, glycosylated proteins. Although insect cells have most of the post-translational machinery
of mammalian cells, proteins produced in the baculovirus system are not processed in precisely the
same way as in mammalian hosts. Currently, the insect cell-baculovirus system is widely used to
produce research quantities of proteins in many industrial laboratories. It is a convenient system
for gene expression, and most proteins are produced in the correctly folded conformation. One
product, a coat protein from HIV (the virus responsible for AIDS) produced from the baculovirus
system, is in Phase II clinical trials and could be produced commercially within 5 years. Other
commercial developments of the baculovirus system will depend on bioprocess research to in-
crease expression levels of secreted glycosylated proteins. The baculovirus itself has been approved
for use as a pesticide. Genetic modifications to the virus to increase its effectiveness (e.g., speed
of killing) are being tested. Large-scale production of such a biopesticide with cell culture will
present unprecedented challenges for large-scale, inexpensive animal-cell reactor designs.
Cells, Organs, and Biomaterials

Production of human skin is already a commercial business in the United States; clonal production
of lymphocytes is in an early stage of development. With the enormous advances in the biology
of differentiation and development, a clear target for the future is large-scale production of cells
(initially) and intact organs (later) for use in therapy and organ replacement. Bioprocess engineering
will be required to develop new types of reactors and to delineate biological mechanisms that affect
growth and maintenance of the cells and differentiated state of these cells. Although lymphocytes
in culture can be prepared now, economical production still requires substantial improvement of
existing techniques. The technology for production of organs will be much more complex. The
rapid increase in knowledge concerning the role of growth factors, cytokines, and other molecules
important in cellular communication, coupled with the realization that the three-dimensional matrix
in which tissue cells grow is crucial in maintaining cell phenotype, has opened up the possibility
of cultivation of viable functioning organ structures. The first applications, already well under way,
are in bone marrow cultures and tissues for cosmetic reconstruction. With the increasing concern
about the safety of blood products, the ability to culture specific types of blood cells might be
crucial for treatment of many diseases and wounds and for surgical procedures. There are serious
engineering challenges in developing a system for successful large-scale cultures. Important prob-
lems remain in understanding the role of mass transfer, protein matrix for cell attachment and
growth, and medium formulation, particularly the proper combination of growth factors required
to optimize production of specific cell types. Cosmetic applications include cartilage and artificial
skin. Great strides in the latter field in the last decade have resulted in many applications, from burn
treatment to reconstruction after surgical procedures, such as breast-cancer removal. But there is
need for much improvement even here. Longer-range opportunities include hepatic, pancreatic, and
kidney cell cultures. A complex three-dimensional substrate is required to maintain cell differentia-
tion and organ function. Again, many engineering problems must be solved. Providing new vas-
culature for substrate delivery and product removal is vital. Most organ cells have a polarity that
is required for proper function. The overall basic biology of the complex systems required to direct
and control differentiating cells is not now understood, and it is impractical to specify in any detail
the types of reactors that will be required in the future, other than to say that they will be much
more complex and interactive than those now used. With an aging population, there is increasing
interest in biologically compatible materials for replacement of organs, joints, and ligaments and for
related applications. Collagen, biologically derived polyesters, hyaluronic acid, and other materials
have all shown attractive properties in some applications. New biological materials (for example,
spider silk or protein adhesives from barnacle and mussel) might be usable for such applications
as sutures and bioadhesives. They are specialties, rather than true pharmaceuticals, even if they
are used in human health-care applications. Thus, the economics of production are more important
for them than for conventional drugs, and the process aspects of their production are critical.
Veterinary Applications
Transgenic animals
Transgenic animals are being developed for a wide variety of applications, and bioprocess engineer-
ing will play a role in the use of these animals in several ways. If transgenic animals are used as
factories for production of biological substances (e.g., as protein in milk), bioprocess engineering
will be required to develop appropriate techniques for isolation and purification of the desired
products. More important in the short term is that transgenic animals might provide test beds for
proving the safety of new pharmaceutical entities and for accelerating their passage through the
regulatory process. Bioprocess engineering can play an important role in controlling the testing
technologies, perhaps in maintaining transgenic tissues and organs, and in coupling the testing
available for use in transgenic animals with the development of processes that are acceptable and
robust from a regulatory viewpoint.
Agricultural Applications
Agricultural chemicals and food
Bioprocess engineering in agriculture sciences involves the application of biocatalysts (living cells
or their components) to produce useful and value-added products, and it offers opportunities to
design and produce new or improved agricultural and food products and their manufacturing
processes. This will likely have a great impact on the U.S. food-processing industry, which has
estimated annual sales of $255 billion. In our increasingly health-conscious society, genetically
engineered microorganisms and specialty enzymes will find increased use in improving the nutri-
tional, flavoring, and storage characteristics and safety of food products. Products under devel-
opment range from genetically improved strains of freeze-resistant yeast used in frozen bakery
products to phage-resistant dairy (yogurt) starter cultures. Chymosin, a product of recombinant
E. coli, is already used in the milk-clotting step of cheese manufacture, and a recombinant
maltogenic amylase is being used as an anti-staling agent. Enzyme-based immunoassays could
develop into a widely used method for detecting pesticides in foods at parts-per-billion concentra-
tions. Challenges that must be addressed include the economics of production and regulatory
issues. The most important applications of bioprocess-engineering research and development
related to agriculture and food involve production of agricultural chemicals for control of animal
and plant diseases, growth-stimulating agents for improved yield, and biological insecticides and
herbicides; increasing bioprocess efficiencies for fermented foods, natural food additives, food
enzymes as processing aids, and separation and purification of the products; use of plant-cell
culture systems to produce secondary metabolites or chemical substances of economic impor-
tance; and efficient use of renewable biomass resources for production of liquid fuel and chemical
feedstock and efficient treatment and management of agricultural wastes and wastes from food-
processing industries.
Plant-cell culture
The commercial potential of plant-cell tissue culture has not yet been fully recognized and is under-
exploited. Plant-cell tissue culture has two primary products: plant tissue for efficient
micropropagation of plants and the use of plant-tissue culture to produce specialty chemicals.
Plant-cell, -tissue, and-organ cultures can be used in processes analogous to traditional fermenta-
tion processes for producing chemicals. Although less than 5% of the worlds plants have even
been identified taxonomically, from among the known plants over 20,000 chemicals are pro-
ducedabout 4 times as many as from all microorganisms. Very few of the chemicals in pure or
semipure form have been tested for their pharmacological activity for other uses. The enzymatic
systems in plants can be used to generate completely new compounds when supplied with ana-
logues of natural substrates; thus, plants contain underused biochemical diversity. Even the limited
use of this vast biochemical potential has had important impacts on mankind; in western countries,
about one-fourth of all medicines are derived from compounds extracted from plants. Other plant
products are used as flavors, fragrances, or pesticides. Plant-cell tissue culture to produce chemi-
cals commercially has been exploited in Japan, although regulatory approval for medicinal uses has
proved difficult and commercial production is restricted to food uses and pigment production. In
Japan, a government-sponsored consortium of universities and corporations was recently devel-

oped to establish a foundation for plant-cell culture exploitation (i.e., a precompetitive research
thrust). In the United States, plant-cell tissue is not being exploited for chemical production,
although at least two companies are actively developing processes for the production of the
chemotherapeutic agent taxol. The major technical barriers to the commercial exploitation of plant-
cell tissue culture are low growth rates and relatively low product yields. To mitigate those
problems, research is needed in subjects as diverse as bioreactor strategies to maintain high-density
cultures and enable large-scale production of chemicals through organ cultures and a mechanistic
understanding of the role of elicitors in activating pathways for secondary metabolites that could
lead to higher productivities of compounds with therapeutic value.
Plants and seeds

Basic research in plant molecular biology has allowed the clonal production of plants through the
process of somatic embryogenesis, in which somatic cells develop through the stages of embryo-
genesis to yield whole plants without gamete fusion. Somatic embryos have been induced from a
variety of plant tissues, and this system is commercially attractive for the high-volume multiplica-
tion of genetically improved embryos in culture. The clonal embryos are synthetic seeds that can
be delivered to commercial growers. For many applications, somatic embryos have powerful
advantages over conventional clonal propagation methods and other in vitro regeneration systems
for mass propagation. One advantage is the very high multiplication rates. Depending on the plant
species, virtually unlimited numbers of embryos can be generated from a single explant. A second
advantage is that, for many species, growth and tissue development of somatic embryos can be
carried out in a liquid medium. That fact gives somatic embryogenesis the potential to be combined
with engineering technology to create large-scale culture systems. The development of such
engineering technology is the limiting step in commercialization. The use of submerged liquid
culture for the efficient mass production of embryos, artificial seeds, or plant propagates is a
promising industrial technique now used in Israel and other countries. For crop and forest plants,
micropropagation on solid medium is too expensive. The use of bioreactors, which reduces labor
costs greatly, will probably be necessary for mass production of crop and forest plants generated
by genetic engineering or nontraditional breeding methods. The key engineering problem in such
systems is the control of environmental conditions necessary for development of organized tissues
from unorganized, minimally differentiated tissues. Fundamental studies on the interaction of
concentration gradients with cellular developmental processes are required.
Transgenic plants
Transgenic plants are capable of generating specialty chemicals or other bioproducts. Special
bioprocessing capabilities will then also need to be developed for extracting, concentrating, and
purifying such products from plant tissue. This sector of bioprocess engineering might also be
important to the prospects of expanding crops or developing new varieties that are rich in ferment-
able carbohydrates, which are readily used as feedstocks for large-scale manufacturing of specialty
and industrial chemicals. Transgenic tobacco plants have been developed to produce monoclonal
antibodies identical in function with the original mouse antibody. Other proteins produced in plants
are human serum albumin and enkephalins. Processes to recover and purify proteins from plant-
cell extracts will be needed if such systems are commercialized.
Industrial applications
New catalysts
New types of catalysts based on biological systems are being developed. Among them are catalytic
antibodies (abzymes) and catalytic nucleic acids (ribozymes). Those types of materials can, in
principle, be used both in specialty-chemical production and in human therapy. Although the
techniques required for preparing abzymes will be the same as those used in other kinds of
monoclonal-antibody manufacture, production of ribozymes (initially for applications in human
health) will require an entire new array of manufacturing, purification, and production technologies;
there are no large-scale methods for preparation, isolation, and purification of high-molecular-
weight nucleic acids.
Microbial metabolites
The metabolites produced during the trophophase of culture are referred to as primary metabolites
and include amino acids, nucleotides, proteins, nucleic acids, lipids and carbohydrates, and the by-
products of energy-yielding metabolism are ethanol, acetone and butanol. Some of the commer-
cially important primary metabolites are given in Table 33.2.
Table 33.2 Some commercially important microbial primary metabolites.
Primary metabolite Producing microbe Commercial signification/application

Ethanol Saccharomyces cerevisiae Production of alcohol
Acetone, butanol Clostridium acetobutyricum Solvents
Lysine Corynebacterium glutamicum Feed supplement
Glutamic acid Corynebacterium glutamicum Flavour enhancement
Polysaccharides Xanthomonas spp. Food industry, enhanced oil recover
The metabolites produced during the idiophase are referred to as the secondary metabolites.
Secondary metabolites are synthesized from the intermediates and end-products of primary me-
tabolism. Few of the secondary metabolites include antibiotics (penicillin. cephalosporin, tetracy-
clines, streptomycin and griseofulvin), anti-tumour agents (actinomycin), anticancer agents
(krestin and bestatin) and immunosuppresant (cyclosporin A). The production of microbial metabo-
lites may be achieved in continuous, as well as in batch systems. The secondary metabolites are
produced by slow-growing organisms in continuous cultures.
Microbial enzymes
Microbial enzymes are most widely used in the food and beverage industries and to a lesser extent
in clinical and analytical laboratories and as protease detergents in washing powders. The most
economical and convenient method of producing these enzymes is by microbial fermentation.

Bacillus stearothermophilus produces amylases as secondary metabolites, but most other microbes
produce enzymes as primary metabolites, during exponential growth. The major commercial
utilization of microbial enzymes is in the food and beverage industries, in pharmaceutical textile and
leather industries, in analytical processes as well as in detergent and dairy industry. Most enzymes
are synthesized in the logarithmic phase of the batch culture and may, therefore, be considered
primary metabolites. However, some, for example, amylases of Bacillus stearothemophilus are
produced by idiophase, type cultures and may be considered equivalent to secondary metabolites.
Enzymes may be produced from animals and plants as well as microbial sources, but the production
by microbial fermentation is the most economical and convenient method. For example, microbial
rennin is being used instead of calf rennin and proteases used in detergents are produced by
microbes. It is now possible to engineer microbial cells to produce animal or plant enzymes. Most
of the enzymes in industrial use are extracellular proteins produced by Aspergillus sp. or Bacillus
sp. and include a-amylase, b-glucanase, cellulase, dextranase, lactase, lipase, pectinase, proteases
and others. The extent of purification required for most of these enzymes is minimal, and they can
be produced in tonnes without serious problems. Some of the important microbial enzymes, their
source and applications are described in Table 33.3.
Table 33.3 Some important microbial enzymes and their applications.
Source (genus) Enzymes Reaction Application

Bacillus a-Amylase Starch hydrolysis Converts starch to glucose
or dextran in food industry
Proteases Protein digestion Help laundering
Escherichia Penicillin Acylase Benzoyl cleavage Production of 6-APA
L-Asparaginase Removal of L-asparagine Leukaemia/cancer treatment
involved in tumour growth
Aspergillus Amyloglucosidase Dextrin hydrolysis Glucose production
b-Galactosidase Lactose hydrolysis Lactose hydrolysis in milk
or whey
Aminoacylase Hydrolysis of acylated Resolution of racemic mixtures
L-amino acids
Glucose oxidase Oxidation of glucose Glucose detection in blood
Streptomyces Glucose isomerase Conversion of glucose Production of high fructose
of fructose syrups
Several marine Luciferase Bioluminescence Assay for ATP
bacteria
Several bacteria Nucleases (restriction Hydrolysis of phospho-
and cyanobacteria endonucleases) diester bonds in nucleic Genetic engineering
acids
Fermented Foods
Thousands of kinds fermented foods are being produced industrially in Japan, Korea, China and
other oriental countries. Miso, shoyu, ontjam, khimchi, beet, tempeh and fermented fish and meat
are good examples. Fermentation often makes the food more nutritious, more digestible, safer, or
having better flavour. It also tends to preserve the food, increasing its shelf life and lowering the
need for refrigeration. Fermentation can be applied to all kinds of foods. Eight classes of fermented
food may be recognized, viz., beverages, cereal products, diary products, fish products, fruit and
vegetable products, legumes, meat products and starchy products. Of these, dairy products, cereal
products and beverages are the most common. Beer is produced from cereal grains which have
been malted, dried and ground into fine powder. The powder is washed in warm water. Fermen-
tation of the washed powder is mediated either by bottom yeast (e.g., Saccharomyces uvarum) or
by a top-yeast (S. cerevisiae). The final product (beer) has up to about 8% alcohol. Grapes can
be directly fermented by yeasts to wine. Wine is made by distillation of the alcoholic broth formed
by fermentation of grape juice (Table 33.4).
Table 33.4 Industrial chemicals produced by fermentation.
Chemicals Microbial source Some uses

Formic acid Aspergillus Textile dyeing, leather treatment, electroplating, rubber
manufacture
Ethanol Saccharomyces Industrial solvent, intermediate for vinegar, esters and
ethers; beverages
Oxalic acid Aspergillus Printing, dying, bleaching; cleaner; reducing agent
Glycerol Saccharomyces, Solvent; Plasticizer, sweetener, manufactures of
Dunaliella explosives; printing, cosmetics; soaps
Propylene glycol Bacillus Antifreeze solvents; synthetic resin manufacture; mould
inhibitor
Acetone Clostridium Industrial solvent; intermediate for several organic
chemicals
Malonic acid penicillium Manufacture of barbiturates
Lactic acid Lactobacillus, Food acidulant; dyeing; intermediate for lactates; leather
Streptococcus treatment
Acrylic acid Bacillus Industrial intermediate for plastics
Butanol Clostridium Industrial solvents; intermediate for several organic
chemicals
Methylethyketone Chlamydomonas Industrial solvent; intermediate for explosives and
synthetic resins
Succinic acid Rhizopus Manufacture of lacquers
Tartaric acid Acetobacter Acidulant, tanning, printing, esters for lacquers
Itaconic acid Aspergillus Textile and paper manufacture
Other Biotechnological Applications

Nontraditional organisms
Many unusual chemicals or enzymes with unique properties come from organisms that are difficult
to culture effectively. In particular, marine microbes (especially algae) and extremophiles (primarily
the archaebacteria) present important, but long-range opportunities. Bioprocess engineers have
already demonstrated the ability to design devices and protocols to culture microbes from deep-
sea vents and undoubtedly have the skills necessary to develop techniques for other difficult-to-
culture organisms.
Energy and renewable resources

A broad range of technical opportunities might require large-scale engineering. For example, if
biologically produced materials (surfactants and viscosifiers) are used in enhanced oil recovery and
in transportation of heavy crudes and coal slurries; appropriate biological manufacturing facilities
must be developed. Particularly for those purposes, the ability to produce materials economically
on-site from local raw materials might be important and might require development of completely
new technology. Although ideas for desulfurization of coal and liquid hydrocarbons seem, at
present, to be improbable, the development of materials from biological processes to aid in trans-
portation and burning of coal, for example, seems plausible. The largest-volume process that
bioprocess engineering might be called on to address would be consumption of carbon dioxide
(CO2) from combustion. If it is necessary to reduce CO2 emissions from stationary sources
substantially to ameliorate the greenhouse effect, a plausible approach would be to couple CO2
release with growth of a CO2-requiring organism (either photosynthetic or non-photosynthetic).
The scale of any facility that would be used in this type of application would be enormous and
would require innovative engineering to minimize costs. In addition to those large-scale processes,
there are plausible uses for biological catalysts on a smaller scale in a number of fields related to
energy production and bioprocessing. For example, fuel cells based on enzymatic catalysis would
provide an attractive method of using ethanol and perhaps other biologically derived fuels with high
thermodynamic efficiency in an environmentally acceptable way. A range of chemicals can, of
course, in principle be produced from renewable resources. The process for production of
acrylamide from acrylonitrile has re-emphasized the practicality of large-volume production of
some types of commodity chemicals with enzymatic catalysis. If energy costs continue to go up,
if petroleum feedstocks become more expensive or more erratic in supply, and if processes based
on conventional non-biological catalytic systems become unacceptable from an environmental point
of view, biological processing might be able to overcome what is usually an intrinsic economic
disadvantage. In that event, bioprocess engineering will be called on to provide reactors and control
systems appropriate for the production of commodity products (chemicals and fuels) with tight
environmental and economic constraints. Bioprocess engineering is an essential component for
addressing those challenges. Fundamental understanding of the changes that occur in pre-treated
cellulose to make it more reactive and identification of inexpensive approaches to the engineering
of large-scale processes are first steps in producing a reactive substrate. Improvement of both the
rate and the extent of xylose fermentation to ethanol require an understanding of microbial physi-
ology and of the optimal control of fermentation conditions to maximize productivity and yield. The
fractionation of solutes from water is a challenge common to most fermentation, where the product
is present at less than 10% concentration and is less volatile than water. Identification of sorbents,
for example, that remove the solutes from the water could be an important first step in improving
the process.
The environment
Many environmental problems will require bioprocess engineering. Some are discussed elsewhere
in this report. Here we mention that, in addition to the types of problems commonly considered
(minimization of manufacturing waste, treatment of municipal waste, environmentally friendly

manufacturing), new classes of problems might arise from the concept of life-cycle environmental
responsibility. That idea is being actively considered in various European countries for such
products as automobiles. The manufacturer would be responsible for its product throughout its life
cycle, including its ultimate disposal. If that responsibility becomes a reality in any of the major
markets, it will be necessary to develop new classes of processes for final disposal of components.
The need for biodegradable polymers, for example, coupled with efficient biodegradation systems
for disposing of manufactured components, would become a reality. Large volume and low cost
in operation would be essential. Because there are virtually no counterparts for them at present,
there is no experience to guide future development; these types of engineering and process prob-
lems would be truly novel. The removal and oxidation of organic gases from contaminated air by
microorganisms fixed in beds of soil, compost, or other solid materials might gain expanded
acceptance as a process by which air is cleaned through biological means. Bio-filtration has already
enjoyed industrial success in Europe and Japan. Nonetheless, significant bioprocess engineering
challenges remain in the use of organisms to remove gas-phase organic chemicals. These include
gas-solid contacting, maintaining stable microbial populations, and predicting performance for
scale-up purposes.
Space
The unique problems presented by the manned exploration and colonization of space pose major
and important challenges for bioprocess engineering. Space biologythe behavior of living sys-
tems in a zero-gravity (and perhaps high radiation) environment is just beginning to be investigated.
The long-term influence of the space environment on cellular development remains to be deter-
mined. In principle, however, a plausible (from an economic point of view) application of process-
ing in space would be to produce new cell lines. If it is possible to carry out manipulations of genetic
materials or cell types in space that cannot be conducted on earth and if the modifications of cell
behavior or germ-line composition that result from the manipulations can be preserved on return
to earth, very high value could, in principle, be achieved. Because cells can be propagated and
relatively small volumes of starting material (genetically altered cells) can be converted into large
numbers of product cells after return to earth, the very high cost of manipulations in space would
have a smaller effect on the overall cost of genetic manipulation of cell lines than of products that
are sold by weight. The development of reactor systems and assay systems that are appropriate
for use in space thus represents an important investment in this speculative field. Reduced gravity
(Moon and Mars) and microgravity (space station) would present some unique opportunities for
the study of complex biological systems. One new interest is in the possible use of a microgravity
environment to produce three-dimensional differentiated tissue structures in the fluid phase without
a solid support. That could lead to a new tool for tissue engineering and the study of cell differ-
entiation and developmental biology. Limitations in the knowledge of how cell suspensions and cell-
to-cell communication affect the growth of such biomaterials might be quickly overcome once
experiments are carried out on a space platform in low earth orbit. The objective would be research
on manufacturing technologies, rather than manufacturing itself. Specific objectives include devel-
oping and testing experimental models for testing mammalian cell and tissue properties and for
providing the necessary nutrients, bio-modifiers, gases, and control. Important studies in develop-
mental biology should be possible, including the generation of high-order tissue morphology of
primary cells and ultimately perhaps complete organ generation. Once developed, the necessary
techniques could be brought back to earth for manufacture. Bioprocess engineering is an element
of all phases of such a project, from the design and implementation of orbiting bioprocess labo-
ratory experiments to the implementation of the results for manufacturing processes on earth. The
problem of development of life-support systems for humans and other organisms in space is
separate and highly important (if specialized). The engineering of even conventional equipment for
the space environment presents a unique set of problems. If the National Aeronautics and Space
Administration (NASA) continues with its long-term goal of manned exploration of space, a major
component of the reduction of space biology to engineering systems appropriate for life support
of astronauts on long voyages will require the development of a specialized, light-weight, gravity-
insensitive set of operations for handling and maintaining the appropriate environments for biologi-
cal systems in space. The number of appropriately trained bioprocess engineers required for that
effort would be substantial. NASA supports relatively small programs in space biotechnology
through the Microgravity Science and Applications Division (MSAD) and the Life Sciences Divi-
sion. One field in which the lack of density gradient-driven convection could be important is protein
nucleation and crystallization. Several active research projects are supported by MSAD (NASA,
1991). Another potential use of microgravity is in separation of large biological molecules (such as
chromosomes) or cells (such as lymphocyte subpopulations), in which again gravity-induced
sedimentation and density-gradient-driven convection could destroy separation ability. This com-
mittee feels that space manufacturing of biological products for use on earth will not be fiscally
feasible in the near future. For example, improvements in earth-based crystallization and separation
systems and rapid developments in three-dimensional matrices for tissue engineering will make
space-manufactured biomaterials too expensive. The space environment does provide a laboratory
for interesting experiments in developmental biology and physiology, which should be pursued.
Integration of those experiments into the space stations or the Moon-and Mars-based modules will
require substantial collaboration between bioprocess engineers and basic biological scientists. That
is particularly true because changes in local mass transport and fluid mechanics can often have
important biological consequences on cell growth and structure formation, which must be sepa-
rated from any effects of microgravity alone.
FUTURE PROSPECTS
Bioprocess engineering is concerned with translating biological science into biologically based
manufacturing. To be prepared for the biological manufacturing systems of the future, it is
important to identify the fields of science and technology that have reached or will soon reach early
prototypes and to begin to develop engineering systems to deal with them. The lead time in
development of any new technology is long. Biological science is so prolific that any present list
of future developments of technology must be incomplete. It is, however, straightforward to
identify subjects in which new engineering techniques must be developed now, if the technologies
are to be available when they are needed in large-scale manufacturing 5-15 years from now.
CONCLUSION
The challenge of bioprocess engineering lies in identifying the needs of the industry and promoting
technology transfer and training of engineers who will fit the wide range of markets, activities, and
products that are being encompassed by biotechnology and bioprocess-engineering developments.
The synthesis and innovation to develop the enabling technologies for industry to exploit the
potential of modern biology and chemistry fully is a significant part of this challenge. The vignettes
in this chapter illustrate how bioprocessing technology facilitates amplification of existing microbial
products, application of new manufacturing techniques for existing products, and the production
of proteins derived from mammalian systems that would otherwise not be available in sufficient
quantities for practical use as therapeutic agents. Challenges that bioprocess engineering faces
include specialty-equipment design that meets regulatory, biological, and economic constraints;
integration of manufacturing processes into environmentally acceptable and economically feasible
process concepts; and rapid purification and monitoring of purification processes to obtain high
quality, high purity, and consistent output. Improvements in the design of bioreactors and in
conditions for cultivating microorganisms and obtaining products are also needed. Both solid-
substrate and submerged fermentations present major opportunities for bio-manufacturing and
improvement through bioprocess engineering. Novel bioreactor designs, membrane-separation
technologies, and processing aids could all improve the economics of producing specialty
bioproducts. The challenges and opportunities of bioprocessing technology for the manufacture of
specialty bioproducts are similar to those related to biopharmaceuticals, except for the special
hazards of pathogens and the specialty-product emphasis on economics of productionthe lower
the value of the product, the more intense the economic focus.
FURTHER READINGS
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Goldstein, eds. Academic. Press, New York, pp. 97-156.
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Kumar A, Ray DK & Gupta SM (2012) “Bioprocess Technology”. In:

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Requirements for Bioprocessing

Pure Culture and Sterilization

Bioprocessing Modes (Batch ver. Continuous Operation)

Continuous Steady-state Processing

Novel Reactor Designs in Bioprocess Monitoring

Figure 33.3 Diagram of a bioreactor.

Figure 33.4 Membrane bioreactor.

Figure 33.5 Novel bioreactor.

Table 33.1 Membrane materials used in bioreactors.

Figure 33.6 Various types of bioreactors.

Downstream Processing (Separation and Purification of Products)

The choice of recovery process is based on the following criteria:

APPLICATIONS OF BIOPROCESS TECHNOLOGY

Gene-Based Pharmaceuticals and Gene Therapy

Biopharmaceuticals and Biopesticides from Insect Cell-Baculovirus System

Cells, Organs, and Biomaterials

Japan, a government-sponsored consortium of universities and corporations was recently devel-

Plants and seeds

Table 33.2 Some commercially important microbial primary metabolites.

Primary metabolite Producing microbe Commercial signification/application

economical and convenient method of producing these enzymes is by microbial fermentation.

Table 33.3 Some important microbial enzymes and their applications.

Source (genus) Enzymes Reaction Application

Table 33.4 Industrial chemicals produced by fermentation.

Chemicals Microbial source Some uses

Other Biotechnological Applications

Energy and renewable resources

(minimization of manufacturing waste, treatment of municipal waste, environmentally friendly

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