Food and Industrial Microbiology

Module 5. Bioprocessing

Lesson 19
OVERVIEW OF BIOPROCESSING

19.1 Introduction

Bioprocessing is the use of biological materials (organisms, cells, organelles, enzymes) to carry out a process for commercial, medical or scientific reasons.

Bioprocess operations should ideally manufacture new products and destroy harmful wastes. Use of microorganisms to transfer biological material for production of fermented foods has been an essential part of many foods, chemical and pharmaceutical industries. Since then, bioprocesses have been developed for an enormous range of commercial products, from relatively cheap materials such as industrial alcohol and organic solvents to expensive speciality chemicals such as antibiotics, therapeutic proteins and vaccines.

Advantages of bioprocessing:

· They are specific in their action

· They are extremely efficient

· They are biodegradable

· Safer

· They work in mild conditions and thus energy saving.

Tools of modern biotechnology such as recombinant DNA, gene probes, cell fusion and tissue culture offer ways to produce new products or improve bioprocessing methods. Modern Biotechnology has allowed us to envision sophisticated medicines, cultured human tissues and organs, biochips for new-age computers, environmentally-compatible pesticides and powerful pollution-degrading microbes.

19.2 A Brief on Historical Developments in Bioprocessing

Modern bio-process technology is an extension of ancient techniques for developing useful products by taking advantage of natural biological activities.

Earliest example of bioprocess is alcoholic beverages that are a combination of yeast cells and nutrients (cereal grains) to form a fermentation system in which the organisms consumed the nutrients for their own growth and produced by-products (alcohol and carbon dioxide gas) that helped to make the beverage.

Modern biotechnology was started in the 19th century when knowledge about biological system, their components, and interaction between them grew. In the first half of the 20th century the first large scale fermentation processes, namely citric acid and penicillin, were realized. The process of recombinant gene technology then led to a substantial increase in the number of bioprocesses and their production volume starting with insulin, the first product manufactured with recombinant technology, in the early 1980s.

The development of genetic engineering and monoclonal antibody technology, which started in the 1970s, has led to the introduction of a large number of new products with application in many different areas. The most highly visible applications have been in the area of human health care, with product such as human insulin, interferon’s, tissue plasminogen activator, erythropoietin, colony-stimulating factors, and monoclonal antibody-based products.

New products for agriculture, food industry, fine chemicals and the environmental protection are also under intense development.

Today, the bio-industries have reached a critical size and are additionally based on a broad understanding of genomics, proteomics, bioinformatics, genetic transformation and molecular breeding. However, this knowledge waits to be transfer to technology and market products. The knowledge of molecular breeding, stem cell technology and pharmacogenomics might lead to strongly personalized therapies and therapeutics.

19.3 Industrial Fermentation Products & Producer Organisms

The word fermentation comes from the Latin verb fevere, which means to boil. It originated from the fact that early at the start of wine fermentation gas bubbles are released continuously to the surface giving the impression of boiling.

It has three different connotations when used in industrial microbiology

a) The first meaning relates to microbial physiology. In strict physiological terms, fermentation is defined in microbiology as the type of metabolism of a carbon source in which energy is generated by substrate level phosphorylation and in which organic molecules function as the final electron acceptor (or as acceptors of the reducing equivalents) generated during the break-down of carbon-containing compounds or catabolism.

b) The second usage of the word is in industrial microbiology, where the term ‘fermentation’ is any process in which micro-organisms are grown on a large scale, even if the final electron acceptor is not an organic. Thus, the production of penicillin, and the growth of yeast cells which are both highly aerobic, and the production of ethanol or alcoholic beverages which are fermentations in the physiological sense, are all referred to as fermentations.

c) The third usage concerns food. A fermented food is one, the processing of which microorganisms play a major part. Microorganisms determine the nature of the food through producing the flavor components as well deciding the general character of the food, but microorganisms form only a small portion of the finished product by weight.

19.4 Types of En Products of Fermentation

Types of end products of fermentation include:

• Microbial cells (e.g. bacteria, yeast, fungal spores)

• Microbial enzymes (e.g. milk clotting enzymes or rennets, recombinant fungal and bacterial rennets for cheese manufacture)

• Microbial metabolites (e.g. alcohols– ethanol, butanol, 2, 3-butanediol, isopropanol; chemicals– lactate, propionate, proteins, vitamins, antibiotics; and fuels– methane)

• Recombinant products (e.g. hormones)

Fermentation products can be broadly divided into two categories: high volume, low value products or low volume, high value products. Examples of the first category include most food and beverage fermentation products, whereas many fine chemicals and pharmaceuticals are in the latter category.

Products of industrial microorganisms may also be divided into two broad groups, those which result from primary metabolism and others which derive from secondary metabolism.

19.4.1 Products of primary metabolism

Primary metabolism is the inter-related group of reactions within a microorganism which are associated with growth and the maintenance of life. Primary metabolism is essentially the same in all living things and is concerned with the release of energy, and the synthesis of important macromolecules such as proteins, nucleic acids and other cell constituents. When primary metabolism is stopped the organism dies. Products of primary metabolism are associated with growth and their maximum production occurs in the logarithmic phase of growth in a batch culture. Primary catabolic products include ethanol, lactic acid, and butanol while anabolic products include amino-acids, enzymes and nucleic acids. Single-cell proteins and yeasts would also be regarded as primary products (Table 19.1)

Table 19.1 Examples of industrial products resulting from primary metabolism

19.4.2 Products of secondary metabolism

In contrast to primary metabolism which is associated with the growth of the cell and the continued existence of the organism, secondary metabolism (Table 19.2), which was first observed in higher plants, has the following characteristics

• Secondary metabolism has no apparent function in the organism.

• Secondary metabolites are produced in response to a restriction in nutrients.

• Secondary metabolism appears to be restricted to some species of microorganisms.

• Secondary metabolites usually have ‘unusual chemical structures and several closely related metabolites may be produced by the same organism in wild-type strains.

• The ability to produce a particular secondary metabolite is easily lost.

• Owing to the ease of the loss of the ability to synthesize secondary metabolites, secondary metabolite production is believed to be controlled by plasmids rather than by the organism’s chromosomes.

The factors which trigger secondary metabolism, the inducers, also trigger morphogenesis.

Table 19.2 Examples of industrial products of microbial secondary metabolism

19.5 Criteria for Selection of Industrially Important Microorganisms

Since prehistoric times, by means of trial and error, people developed strains of microbes that were used in the production of 'beverages, food, textiles, and antibiotics without knowing that microbes were the responsible agents. With the discovery that microorganisms existed, and the subsequent development of culture methods, came the birth of modem biological technology or biotechnology. Expanded development of microbial screening and cultural techniques has brought us to a point where microbially produced products are a major part of our life. While production of food, drink, and textiles remains a large part of the biotechnology industry, the discovery of penicillin in the first half of this century revolutionized microbial screening for other useful products such as antibiotics, enzymes, and speciality chemicals.

During the past 40-50 years, screening of industrially important microorganisms has evolved steadily, although somewhat haphazardly, while still relying on the original underlying techniques of enrichment, pure culture, mutagenesis, and sheer labor. Classical methods are still used extensively with modifications resulting from the use of chemical analogs, coupled

Colorimetric reactions, membrane technology, immunological techniques, and advances in instrumentation.

Although the well-known ubiquity of microorganism implies that almost any natural ecological entity–water, air, leaves, tree trunks – may provide microorganisms, the soil is the preferred source for isolating organisms, because it is a vast reservoir of diverse organisms. In recent times, other ‘new’ habitats, especially the marine environment, have been included in habitats to be studied in searches for bioactive microbial metabolites or ‘bio-mining’.

Identification and isolation of required micro-organisms is very critical for any microbiological process, since some micro-organisms may be toxic to the useful microbes and may use up the nutrients all by themselves, producing metabolites that are different from the desired ones. Isolation of micro-organisms also helps to screen them to determine, if they can be used for any industrial process. Such microorganisms should satisfy some specified criteria.

19.5.1 Important criteria in the choice of organism

• The nutritional characteristics of the organism

• The optimum temperature of the organism

• The reaction of the organism with the equipment to be employed and the suitability of the organism to the type of process to be used

• The stability of the organism and its amenability to genetic manipulation

• The productivity of the organism, measured in its ability to convert substrate into product and to give a high yield of product per unit time

• The ease of product recovery from the culture

• Irrespective of the origins of an industrial microorganism,

Other features that may be exploited are thermophilic or halophilic properties, which may be useful in a fermentation environment. Also, particularly for cells grown in suspension, they should grow well in conventional bioreactors to avoid the necessity to develop alternative systems. Consequently, they should not be shear sensitive, or generate excessive foam, nor be prone to attachment to surfaces.

19.5.2 Some general screening methods are described below

1. Isolation de novo of Organisms Producing Metabolites of Economic Importance

1.1 Enrichment with the substrate utilized by the organism being sought

1.1.1 Enrichment with toxic analogues of the substrate utilized by the organism being sought

1.2 Testing microbial metabolites for bioactive activity

1.2.1 Testing for anti-microbial activity

1.2.2 Testing for enzyme inhibition

1.2.3 Testing for morphological changes in fungal test organisms

1.2.4 Conducting animal tests on the microbial metabolites

19.5.3 Strain improvement

Several options are open to an industrial microbiology organization seeking to maximize its profits in the face of its competitors’ race for the same market. The operations in the fermentor may also be improved by its use of a more productive medium, better environmental conditions, better engineering control of the fermentor processes, or it may genetically improve the productivity of the microbial strain it is using. Of all the above options, strain improvement appears to be the one single factor with the greatest potential for

contributing to greater profitability.

To appreciate the basis of strain improvement it is important to remember that the ability of any organism to make any particular product is predicated on its capability for the secretion of a particular set of enzymes. The production of the enzymes, themselves depends ultimately on the genetic make-up of the organisms. Improvement of strains can therefore be put down in simple term as follows

1. Regulating the activity of the enzymes secreted by the organisms

2. Increasing the permeability of the organism so that the microbial products can find their way more easily outside the cell.

3. Selecting suitable producing strains from a natural population

4. Manipulation of the existing genetic apparatus in a producing organism

5. Introducing new genetic properties into the organism by recombinant DNA

6. Technology or genetic engineering

19.5.3.1 Examples of targets for strain improvement

• Rapid growth
• Genetic stability
• Non-toxicity to humans
• Large cell size, for easy removal from the culture fluid
• Ability to use cheaper substrates
• Modification of submerged morphology
• Elimination of the production of compounds that may interfere with downstream processing
• Catabolite derepression
• Phosphate deregulation
• Permeability alterations to improve product export rates
• Metabolite resistance
• Production of additional enzymes and compounds to inhibit contaminant microorganisms

19.5.3.2 Manipulation of the genome of industrial organisms in strain improvement can be done by mainly two ways

1. Methods not involving foreign DNA

1.1 Conventional mutation

2. Methods involving DNA foreign to the organism (i.e. recombination)

2.1 Transduction

2.2 Conjugation

2.3 Transformation

2.4 Heterokaryosis

2.5 Protoplast fusion

2.6 Genetic engineering

2.7 Metabolic engineering

2.8 Site-directed mutation

19.6 Media for Industrial Inoculums Development

The use of a good, adequate, and industrially usable medium is as important as the deployment of a suitable microorganism in industrial microbiology. Unless the medium is adequate, no matter how innately productive the organism is, it will not be possible to harness the organism’s full industrial potentials. Indeed not only may the production of the desired product be reduced but toxic materials may be produced.

Fermentation media must satisfy all the nutritional requirements of the microorganism and fulfill the technical objectives of the process. The nutrients should be formulated to promote the synthesis of the target product, either cell biomass or a specific metabolite.

19.6.1 The main factors that affect the final choice of individual raw materials are as follows

1. Cost of the material

The cheaper the raw materials the more competitive the selling price of the final product will be. Due to these economic considerations the raw materials used in many industrial media are usually waste products from other processes. Corn steep liquor and molasses are, for example, waste products from the starch and sugar industries, respectively.

2. Ready availability of the raw material

The raw material must be readily available in order not to halt production. If it is seasonal or imported, then it must be possible to store it for a reasonable period.

3. Transportation costs

Proximity of the user-industry to the site of production of the raw materials is a factor of great importance, because the cost of the finished material and its competitiveness can all be affected by the transportation costs.

4. Ease of disposal of wastes resulting from the raw materials

The disposal of industrial waste is rigidly controlled in many countries. When choosing a raw material therefore the cost, if any, of treating its waste must be considered.

5. Uniformity in the quality of the raw material and ease of standardization

The quality of the raw material in terms of its composition must be reasonably constant in order to ensure uniformity of quality in the final product and the satisfaction of the customer and his/her expectations.

6. Adequate chemical composition of medium

The medium must have adequate amounts of carbon, nitrogen, minerals and vitamins in the appropriate quantities and proportions necessary for the optimum production of the commodity in question.

7. Presence of relevant precursors

The raw material must contain the precursors necessary for the synthesis of the finished product. Precursors often stimulate production of secondary metabolites either by increasing the amount of a limiting metabolite, by inducing a biosynthetic enzyme or both.

8. Satisfaction of growth and production requirements of the microorganisms

Many industrial organisms have two phases of growth in batch cultivation: the phase of growth, or the trophophase , and the phase of production, or the idiophase . Often these two phases require different nutrients or different proportions of the same nutrients.

19.6.2 Components of media for industrial inoculums development

The media should support the metabolic process of the microorganisms and allow bio-synthesis of the desired products.

Carbon & Energy source + Nitrogen source + Nutrients Product(s) + Carbon Dioxide + Water + Heat + Biomass

Media are designed based on the above equation using minimum components required to produce maximum product yield. Important components of the medium are carbon sources, nitrogen sources, minerals, growth factors, chelating agents, buffers, antifoaming agents, air, steam, and fermentations vessels.

1. Carbon sources

A carbon source is required for all biosynthesis leading to reproduction, product formation and cell maintenance. In most fermentations it also serves as the energy source. Carbohydrates are traditional carbon and energy sources for microbial fermentations, although other sources may be used, such as alcohols, alkanes and organic acids. Animal fats and plant oils may also be incorporated into some media, often as supplements to the main carbon source.

a. Molasses

Pure glucose and sucrose are rarely used for industrial scale fermentations, primarily due to cost. Molasses, a byproduct of cane and beet sugar production, is a cheaper and more usual source of sucrose. This material is the residue remaining after most of the sucrose has been crystallized from the plant extract. It is a dark coloured viscous syrup containing 50–60% (w/v) carbohydrates, primarily sucrose, with 2% (w/v) nitrogenous substances, along with some vitamins and minerals.

b. Malt Extract

Aqueous extracts of malted barley can be concentrated to form syrups that are particularly useful carbon sources for the cultivation of filamentous fungi, yeasts and actinomycetes. The composition of malt extracts varies to some extent, but they usually contain approximately 90% carbohydrate, on a dry weight basis. This comprises 20% hexoses (glucose and small amounts of fructose), 55% disaccharides (mainly maltose and traces of sucrose), along with 10% maltotriose, a trisaccharide. Malt extracts also contain some vitamins and approximately 5% nitrogenous substances, proteins, peptides and amino acids.

c. Starch and Dextrins

These polysaccharides are not as readily utilized as monosaccharides and disaccharides, but can be directly metabolized by amylase-producing microorganisms, particularly filamentous fungi.

d. Sulphite Waste Liquor

Sugar containing wastes derived from the paper pulping industry are primarily used for the cultivation of yeasts. Waste liquors from coniferous trees contain 2–3% (w/v) sugar, which is a mixture of hexoses (80%) and pentoses (20%). Hexoses include glucose, mannose and galactose, whereas the pentose sugars are mostly xylose and arabinose. Usually the liquor requires processing before use as it contains sulphur dioxide.

e. Cellulose

Cellulose is predominantly found as lignocellulose in plant cell walls, which is composed of three polymers: cellulose, hemicellulose and lignin. Lignocellulose is available from agricultural, forestry, industrial and domestic wastes. Relatively few microorganisms can utilize it directly, as it is difficult to hydrolyse. It is potentially a very valuable renewable source of fermentable sugars once hydrolysed, particularly in the bioconversion to ethanol for fuel use.

f. Whey

Whey is an aqueous byproduct of the dairy industry. This material is expensive to store and transport. Therefore, lactose concentrates are often prepared for later fermentation by evaporation of the whey, following removal of milk proteins for use as food supplements. Lactose is generally less useful as a fermentation feedstock than sucrose, as it is metabolized by fewer organisms. S. cerevisiae, for example, does not ferment lactose.

g. Alkanes and Alcohols

n -Alkanes of chain length C₁₀–C₂₀ are readily metabolized by certain microorganisms. Mixtures, rather than a single compound, are usually most suitable for microbial fermentations. However, their industrial use is dependent upon the prevailing price of petroleum. Methane is utilized as a carbon source by a few microorganisms, but its conversion product methanol is often preferred for industrial fermentations as it presents fewer technical problems. Ethanol is less toxic than methanol and is used as a sole or co substrate by many microorganisms, but it is too expensive for general use as a carbon source.

2. Fats and Oils

Hard animal fats that are mostly composed of glycerides of palmitic and stearic acids are rarely used in fermentations. However, plant oils (primarily from cotton seed, linseed, maize, olive, palm, rape seed and soya) and occasionally fish oil, may be used as the primary or supplementary carbon source, especially in antibiotic production.

3. Nitrogen sources

Most industrial microbes can utilize both inorganic and organic nitrogen sources. Inorganic nitrogen may be supplied as ammonium salts, often ammonium sulphate and di ammonium hydrogen phosphate, or ammonia. Ammonia can also be used to adjust the pH of the fermentation. Organic nitrogen sources include amino acids, proteins and urea. Nitrogen is often supplied in crude forms that are essentially byproducts of other industries, such as corn steep liquor, yeast extracts, peptones and soya meal.

a. Corn Steep Liquor

Corn steep liquor is a byproduct of starch extraction from maize. The exact composition of the liquor varies depending on the quality of the maize and the processing conditions. Concentrated extracts generally contain about 4% (w/v) nitrogen, including a wide range of amino acids, along with vitamins and minerals.

b. Yeast Extracts

Yeast extracts may be produced from waste baker’s and brewer’s yeast, or other strains of S. cerevisiae. Alternate sources are Kluyveromyces marxianus grown on whey and Candida utilis cultivated using ethanol, or wastes from wood and paper processing.

c. Peptones

Peptones are usually too expensive for large-scale industrial fermentations. They are prepared by acid or enzyme hydrolysis of high protein materials: meat, casein, gelatin, keratin, peanuts, soy meal, cotton seeds, etc..

4. Water

All fermentation processes, except solid-substrate fermentations, require vast quantities of water. In many cases it also provides trace mineral elements and is important for ancillary equipment and cleaning. Before use, removal of suspended solids, colloids and microorganisms is usually required. When the water supply is ‘hard’, it is treated to remove salts such as calcium carbonate.

5. Minerals

Normally, sufficient quantities of cobalt, copper, iron, manganese, molybdenum, and zinc are present in the water supplies, and as impurities in other media ingredients. Occasionally, levels of calcium, magnesium, phosphorus, potassium, sulphur and chloride ions are too low to fulfil requirements and these may be added as specific salts.

6. Vitamins and growth factors

Many bacteria can synthesize all necessary vitamins from basic elements. For other bacteria, filamentous fungi and yeasts, they must be added as supplements to the fermentation medium. Most natural carbon and nitrogen sources also contain at least some of the required vitamins as minor contaminants. Other necessary growth factors, amino acids, nucleotides, fatty acids and sterols, are added either in pure form or, for economic reasons, as less expensive plant and animal extracts. Pharmamedia, cornsteep powder, distillers solubles and malt sprouts are some examples of media ingredients.

7. Precursors

Some fermentations must be supplemented with specific precursors, notably for secondary metabolite production. Examples include phenylacetic acid or phenylacetamide added as side-chain precursors in penicillin production. Threonine is used as a precursor in isoleucine production by Serratia marsescens, and anthranillic acid additions are made to fermentations of the yeast Hansenula anomala during tryptophan production.

8. Inducers and elicitors

If product formation is dependent upon the presence of a specific inducer compound or a structural analogue, it must be incorporated into the culture medium or added at a specific point during the fermentation. Inducers are often necessary in fermentations of genetically modified microorganisms (GMMs).

9. Inhibitors

Inhibitors are used to redirect metabolism towards the target product and reduce formation of other metabolic intermediates; others halt a pathway at a certain point to prevent further metabolism of the target product. An example of an inhibitor specifically employed to redirect metabolism is sodium bisulphite, which is used in the production of glycerol by S. cerevisiae.

10. Cell permeability modifiers

These compounds increase cell permeability by modifying cell walls and/or membranes, promoting the release of intracellular products into the fermentation medium. Compounds used for this purpose include penicillins and surfactants.

11. Oxygen

Depending on the amount of oxygen required by the organism, it may be supplied in the form of air containing about 21% (v/v) oxygen, or occasionally as pure oxygen when requirements are particularly high. For most fermentations the air or oxygen supply is filter sterilized prior to being injected into the fermenter.

12. Antifoams

Antifoams are necessary to reduce foam formation during fermentation. Foaming is largely due to media proteins that become attached to the air–broth interface where they denature to form astable foam. If uncontrolled the foam may block air filters, resulting in the loss of aseptic conditions; the fermenter becomes contaminated and microorganisms are released into the environment. Natural antifoams include plant oils (e.g. from soya, sunflower and rapeseed), deodorized fish oil, mineral oils and tallow.