Topic 1 INTRODUCTION TO BIOPROCESSES

Bioprocess operations make use of microbial, animal and plant cells and components of cells such as enzymes to manufacture new products and destroy harmful wastes
biological systems are very complex and beautifully constructed, but they obey the rules of chemistry and physics and they are susceptible to engineering analysis

Bioprocess Engineering
is the study of engineering principles applied to processes involving cells or enzyme catalysts

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Objective of this subject:

focus primarily on the application of some engineering principles to systems containing biological materials, and with emphasis on those system making use of biotechnology

FIVE groups of commercially important product from bioprocess operations:

1. Production of microbial cell (biomass) microbial inoculum for biofertilizer, probiotics, starter culture, single cell protein (SCP) .. 2. Production of microbial enzymes protease, amylase, glucoamylase .. 3. Production of microbial metabolites organic acid, ethanol, solvent 4. Production of recombinant products 5. Bio-transformation processes to modify a compound which is added to the fermentation broth, for example, ethanol to acetic acid
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1) Production of microbial cell (biomass)

Two main applications:a) b)

as a source of protein Example: SCP as a commercial inoculum (starter culture)

a) SCP production

refer to microbial biomass used as food and feed additives either the isolated cell protein or the total cell material may be called SCP great nutritional value high protein, vitamin, and lipid content and the almost complete range of all essential amino acids
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Single Cell Protein Microbial biomass or protein extracted there from processes in which bacteria, yeast, fungi or algae are cultivated in large quantities As human or animal protein supplement in animal feed or in human nutrition As protein supplement (for infant as nutritional food protein)

Table 1: Composition (%) of SCP compared with soya meal and milk powder Component Alkane Methanol Alga Soya yeast bacterium meal 60 9 80 9.5 9.5 2.8 72.6 7.3 4.7 3.6 42 4 6.5 10 Milk powde r 34 1 8 5

Raw protein Fat

Mineral salts 6 moisture 4.5

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Sample of the single cell protein 10 biosolids after the drum dryer

Protein content in bacteria 60 to 65% Selected fungi UEMK3233 and Topicyeast 1 33 to 45%

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 Some psychological barriers:

microorganism involved must be safe & acceptable; non-pathogenic; non-toxic forming (for example, aflatoxins are produced by some fungi); genetically stable Toxic or carcinogenic substances used during the production of microbial e.g., hydrocarbon, polycyclic aromatic compounds . may cause indigestion and allergic reactions

b) as a commercial inoculants (or known as starter cultures)

Major applications (as food starter cultures), such as, in baking and dairy industries Functions of food starter cultures:

Texture modifications Preservation Flavor development Nutritional improvement

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Examples:i. Bakers yeast bread making ii. Cheese-starter cultures Streptococcus cremoris, Streptococcus lactis iii. Yoghurt manufacturing Stretococcus thermophilus, Lactobacillus bulgaricus, L. acidophilus

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1 m (a) Spherical (cocci)

Topic 1 - Prokaryotes

2 m

5 m

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(b) Rod-shaped (bacilli)

(c) Spiral

Table 2: Some applications of microbial inoculants

Applications Probiotics Waste treatment** Microbial species Lactobacillus acidophilus, Streptococcus faecium Methanogens Pseudomonas spp., Acinetobacter spp., extracellular enzyme-producing bacteria Function Increase digestibility of animal feeds Anaerobic digestion, degrade alkanes and waste compounds, hydrolyse waste proteins, carbohydrates and fats Increase nitrogen fixation in plants Increase root nutrient and water uptake, protection against disease

Trickling filter

Nitrogen fixation Plant growth acceleration using mycorrhizal fungi

Free-living and symbiotic nitrogenfixing prokaryotes Endomycorrhizae of the family Endogonaceae, phylum Zygomycota .

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Activated sludge plant

b) Production of microbial enzymes

First enzyme (1894)
Fungal amylase takadiastase employed as a pharmaceutical agent (for digestive disorder) in US produce in large quantities by defined and established fermentation techniques easier to control the microbial system Improve the productivity of final enzyme yield

Advantages

(Returned Activated Sludge)

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Examples of commercial applications of microbial enzymes

c) Production of microbial metabolites Growth of microbial divided into few stages:Lag phase ii. Log or exponential phase iii. Stationary phase iv. Death phase
i.

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Growth curve of a bacterial culture in batch conditions 21

Phases Lag

Processes

Product formation during microbial growth:a) Primary metabolites

referred as primary products of metabolism are essential for life and reproduction of cells produced during log phase (trophophase) e.g., amino acids, proteins, carbohydrates, lipids. examples (refer Table 3 and 4) also known as ???? produced by wild type OR genetically modified microorganism ???
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Growth does not occur; adaptation period Log/ Cells grow gradually at constant exponential rate until reach maximum growth rate stationary death Growth ceases and growth rate reduced Viable cell number declines
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 some may be produced in concentrations far higher than those required by the producing strains
Table 3: Excessive production of certain primary metabolites

Table 4: Examples of primary product from microbial metabolism and their commercial application Primary metabolite Ethanol Citric acid Acetone & butanol Glutamic acid Vitamins Polysaccharides Commercial applications Active ingredient in alcoholic beverages; Food industry Solvents Flavour enhancer Feed supplements Food industry; oil industry
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b) Secondary metabolites
secondary products of metabolism produced during stationary phase (idiophase) (Figure A) are seemingly not essential for growth and reproduction produced from the intermediates and products of primary metabolism (refer Figure B) and their formation is extremely dependent on environmental conditions every secondary metabolite is formed by only a few organisms Importance of secondary metabolites in fermentation industries
a) Antimicrobial activity b) Growth promoters c) Pharmacological properties

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Wild-type microbial ??? Genetically modified microbial?? also known as ????? UEMK3233 Topic 1

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d) Recombinant products
advent of recombinant DNA technology extend the range of potential fermentation products genes from higher organisms may be introduce into microbial cells recipients are capable of synthesizing foreign proteins hosts E. coli, S. cerevisiae and filamentous fungi products from GMO interferon, insulin, epidermal growth factor, chymosin etc important factors to consider
i. ii. iii.
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Figure A: Production of antibiotics (e.g. Penicillium spp. & Streptomycetes)
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secretion of the product minimization of the degradation of the product maximizing the expression of the foreign gene
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e) Microbial transformation / Bioconversions

to chemically modify a wide variety of organic compound to convert a compound into a structurally related, financially more valuable compound examples:
1. 2. production of vinegar conversion of ethanol to acetic acid production of steroids, antibiotics and prostaglandins

advantageous over the use of chemical reagents: operate at low temperature without requirement of any potential heavy metal pollution

disadvantages: require large amount of biomass

reaction involved:- oxidation, dehydrogenation, dehydration, condensation, amination, isomerization,

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Oxidation of ethanol to acetic acid

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Many words have been used to describe engineering working with biotechnology:-

Biochemical engineering
extension of chemical engineering principles to systems using a biological catalyst to bring about desired chemical transformations often subdivided into bioreaction engineering and bioseparation engineering

Bioengineering is a broad title include work on medical and agricultural systems its practitioners include agricultural, electrical, mechanical, industrial, environmental Biological engineering is similar but emphasizes applications to plants and animals

Biomedical engineering
the application of engineering techniques to the understanding of biological systems and to the development of therapeutic technologies and devices. kidney dialysis, synthetic skin, artificial joints.. etc are some products of biomedical engineering.
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Approach to research biologists vs engineers

fundamental training distinctly different For biologist,
mathematical theories and quantitative methods play 2nd role very strong with respect to laboratory tools (interpretation of laboratory data from complex systems) BUT they are often have incomplete backgrounds in mathematics usually better at the formation of testable hypotheses, experimental design, and data interpretation from complex systems results are qualitative and descriptive
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Engineer
usually possess a very good background in the physical and mathematical sciences theory formulate mathematical equation (predictive model) and then the validity of the theory is tested by comparing predicted responses to those in experiments typically unfamiliar with the experimental techniques and strategies used by life sciences

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The skills of engineer and life sciences are complementary integration of skills To become a bioprocess engineer:
needs a solid understanding of biology (need to take further courses in microbiology, biochemistry, cell biology.) need to have improvements in experimental tools learn more advanced work in biochemical engineering

How biologists and engineers work together

Biochemical engineering extension of chemical engineering principles to systems using a biological catalyst to bring about desired chemical transformations often subdivided into bioreaction engineering and bioseparation engineering
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Figure 1: Steps in development of a complete bioprocess for commercial manufacture of a new recombinant-DNA-derived product

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4 Gene cut from chromosome 1 biochemicals 3 Part of animal chromosome

9 Insertion into microorganism

Steps in bioprocess development

The interdisciplinary nature of bioprocessing is evident if we look at the stages of development required for a complete industrial process as shown in Figure 1, several steps are required to convert the idea of the product into commercially reality these stages involve different types of scientific expertise
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2 Animal tissue

10 Plasmid multiplication and gene expression 8 Recombinant plasmid

5 Microoganism such as E. coli

6 Plasmids

7 Cut plasmid

11 Cell division

12 Small scale culture 13 Bench-top bioreactor 14 Pilot scale bioreactor

UEMK3233 Topic 1 15 Industrial scale bioreactor 16 Product recovery

17 Packaging and marketing

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4 Gene cut from chromosome 1 biochemicals 3 Part of animal chromosome

9 Insertion into microorganism

Step 1 to 11
concerned with genetic manipulation of host organism a gene from animal DNA is cloned into Escherichia coli genetic engineering done in laboratories on a small scale by scientists trained in molecular biology and biochemistry experiment tools: petri dishes, micropipettes, microcentrifuges, nano- or microgram quantities of restriction enzymes, electrophoresis gels for DNA, protein fractionation Parameters of major importance stability of the constructed strains and level of expression of the desired product bioprocess development
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2 Animal tissue

10 Plasmid multiplication and gene expression 8 Recombinant plasmid

5 Microoganism such as E. coli

6 Plasmids

7 Cut plasmid

11 Cell division

12 Small scale culture 13 Bench-top bioreactor 14 Pilot scale bioreactor

UEMK3233 Topic 1 15 Industrial scale bioreactor 16 Product recovery

17 Packaging and marketing

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Step 12
Small-scale cultivation (lab-scale) is mostly carried out using shake flasks (250-mL to 1-L) to study the growth and production characteristics of cells culture environment (medium composition, pH, temperature..and other environmental condition) allow optimal growth of microorganism and productivity of product practical skills microbiology, fermentation calculated parameters cell growth rate, specific productivity and product yield used to describe performance of the organism
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Step 13 to 14 (scaling up)

once the culture conditions for production are known, scaleup of the process starts Bench-top bioreactor (cultures can be more closely monitored in bioreactor than in shake flasks better control over the process)
Lab scale 1- or 2-litre Equipped with instruments for measuring and adjusting temperature, pH, DO, stirrer speed, and many other process variables Information collected: oxygen requirements of the cells, shear sensitivity, foaming characteristics . Identify the limitation imposed by the reactor on the activity of the organism whether or not the reactor can provide conditions for optimal activity of the cells is of prime concern!!
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Step 14 Pilot-scale bioreactor Engineers trained in bioprocessing are normally involved in pilot-scale operations a vessel of capacity 100 to 1000 L is built according to specifications determined from the bench-scale prototype Aim of pilot-scale studies to examine the response of cells to scale-up
Changing the size of the equipment seems relatively trivial; however, loss or variation of performance often occurs Even though the geometry of the reactor, method of aeration and mixing, impeller design and other features may be similar in small and large fermenters, the effect on activity of cells can be great! Loss of productivity following scale-up may or may not be recovered Economic projections often need to be re-assessed as a result of pilot-scale findings
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Step 15 (Industrial-scale operation)

This part of process development is clearly in the territory of bioprocess engineering As well as the reactor itself, all of the auxiliary service facilities (air supply and sterilisation equipment; steam generator; supply lines; medium preparation; sterilisation facilities; cooling-water supply. etc) must be designed and tested Need to ensure the fermentation to be carried out aseptically

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Step 16 (Product recovery or downstream

processing) After the production process, raw broth is treated in a series of steps to obtain pure product Product recovery is often difficult and expensive accounts for 60% or 80 90% of the total processing cost Actual procedures used depend on the nature of the product and the broth physical, chemical or biological methods Techniques applied industrially for downstream processing are first developed and tested using small-scale apparatus many operations which are standard in the laboratory become uneconomic or impractical on an industrial scale Scientists trained in chemistry, biochemistry, chemical engineering and industrial chemistry play important roles in designing product recovery and purification systems
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Step 17 (Packaging and marketing)

After the product has been purified in sufficient purity, it can be packaged and marketed New pharmaceuticals products require medical and clinical trials to test the efficacy of the product Bioprocess engineers with a detailed knowledge of the production process are often involved in documenting manufacturing procedure for submission to regulatory authorities manufacturing standards must be met, particularly in the case for recombinant products where a greater number of safety and precautionary measures is required!
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As shown in this example, a broad range of disciplines is involved in bioprocessing Scientists working in this area are constantly confronted with biological, chemical, physical, engineering and sometimes medical questions!!

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