Processes and devices in biotechnology: educational manual 9786010432468

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Processes and devices in biotechnology: educational manual
 9786010432468

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Introduction

AL-FARABI KAZAKH NATIONAL UNIVERSITY

N. Sh. Akimbekov A. S. Kistaubayeva I. S. Savitskaya

PROCESSES AND DEVICES IN BIOTECHNOLOGY Educational manual

Almaty «Qazaq University» 2018

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Processes and devices in biotechnology

UDC 60 (075.8) LBC 30.16 я 73 А 29 Recommended for publication by the decision of the Academic Council of the Faculty of Biology and Biotechnology, Editorial and Publishing Council of Al-Farabi Kazakh National University (Protocol №4 dated 29.12.2017)

Reviewers: Doctor of Biological Sciences, Associate Professor A.K. Bisenbaev Candidate of Biological Sciences, Associate Professor S.Sh. Asrandina

А 29

Akimbekov N.Sh. Processes and devices in biotechnology: educational manual / N.Sh. Akimbekov, A.S. Kistaubayeva, I.S. Savitskaya. – Almaty: Qazaq University, 2018. – 140 p. ISBN 978-601-04-3246-8 The book contains the scientific aspects for the engineering design of upstream processing, fermentation, separation and purification of the target products with industrial stages, the principles for assessing the effectiveness and safety of biotechnological productions. The application of biotechnology in various branches of national economy and typical schemes of industrial processes are provided. Each chapter of the book is accompanied by control questions. In terms of volume and awareness of the material, such book in English is published in our country for the first time. It has been prepared taking into account the world practice in biotechnology as a production sector and represents one of the most important elements of the general education for a biotechnologist. The book is intended for students, undergraduates and PhD students, who are trained on the basis of credit technology of education in the specialty of «Biotechnology». Published in authorial release.

UDC 60 (075.8) LBC 30.16 я 73 ISBN 978-601-04-3246-8

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© Akimbekov N.Sh., Kistaubayeva A.S., Savitskaya I.S., 2018 © Al-Farabi KazNU, 2018

Introduction

FOREWORD

T

he biotechnological industry is one of the priority areas for the welfare of mankind in the foreseeable future. When the entire technological chain is conducted by bio-objects in artificial conditions, they must have maximum comfort, which in turn provides the necessary nutrition sources and protection from external adverse effects. The engineering, technical base, processes and devices of biotechnological productions play an important role in regulating proper function of any given bio-object. The book contains the main concepts of macro-kinetics of cultivation, isolation, purification and drying of final products. The regulation of such processes as sterilization of liquid and air, the stage of fermentation are described. The main features of hardware design of biotechnological productions are considered as well. Particular attention is paid to the safety of these industries. The book mainly contains the scientific aspects for the engineering design of upstream processing, fermentation, separation and purification of the target products with industrial stages, the principles for assessing the effectiveness and safety of biotechnological productions. The application of biotechnology in various branches of national economy and typical schemes of industrial processes are provided. Each chapter of the book is accompanied by control questions. At the end of the book, there is a list of recommended references. In terms of volume and awareness of the material, such book in English is published in our country for the first time. It has been prepared taking into account the world practice in biotechnology as a production sector and represents one of the most important elements of the general education for a biotechnologist. The book is intended for students, undergraduates and PhD students, who are trained on the basis of credit technology of education in the specialty of «Biotechnology». 3

Processes and devices in biotechnology

INTRODUCTION

B

iotechnological industry is the sphere of production with its specific hardware design. Bio-industry produces the following types of products: – Proteins (yeast, protein products, amino acids); – Physiologically active substances (antibiotics, vitamins, enzymes, hormones, growth promoters); – Organic acids (citric, lactic, itaconic, acetic); – Bacterial products for the control of pests in agriculture, as well as for intensification of farming (entobacterin, boverin, dendrobacillin, nitragin, azotobacterin). The production of these substances through biological synthesis is more beneficial than their production by chemical ways. It should be noted that some of these products (bacterial, enzyme products, etc.) can only be obtained by microbiological means. The possibilities of microbiological synthesis, first of all, related to high intensity and great variability of microbial metabolism. In some cases the whole biochemical activity of the cell is directed not at growth and reproduction, but at the synthesis of any desirable substance, and the ability of microorganisms to synthesize a particular product can be significantly enhanced. Biosynthesis is characterized by a specific action of enzymes and involving reactions take place at ordinary temperature and pressure. An important advantage of biological synthesis is that when it is used in industrial conditions, in most cases it is possible to use nondeficient raw materials. Typically, these waste products are processed through agricultural products, waste hydrolysis industry and widely distributed products such as oil and natural gas used as well. Ultimately, the technical and economic efficiency of biotechnological productions in many cases is higher than using methods of 4

Introduction

chemical technology. For instance, microbiological synthesis is the only possible way for production of enzymes, bacterial products, protein, some antibiotics, etc. A number of industries belong to the processes related to industrial biotechnology. They can be divided into 2 large groups. The first should include some food production, engaged in the processing of agricultural raw materials (flax), and, finally, fermentation (brewing, winemaking). For these industries (except fermentation), the cultivation of large masses of microorganisms or the accumulation and recovery of useful products of their metabolism are not characterized. The application of a biological object here is limited to any one stage of the technological process or they are used in small quantities. The specific technological equipment associated with the use of microorganisms, in these industries has a small specific gravity. The second group of industries is the biotechnology industry, which includes production, where the main stage of the technology is cultivation (growing microorganisms or accumulating target products of microorganisms). The main production of the microbial industry on the basis of technology can be divided into two subgroups. 1. Large-tonnage production associated with the production of vast quantities of biomass of microorganisms (yeast), organic acids (citric, lactic, acetic) or alcohols. A characteristic feature of production, which determines the type of basic technological equipment is the use of submerged (suspension) growth of microorganisms. The cultivation conditions are, as a rule, it does not require a high degree of asepsis. Due to the relatively low requirements for protecting the process from extraneous microflora in the production of this group, it is not necessary to solve complex technical problems of reliable sterilization of large quantities of liquid, fine air purification, sealing and reliable sterilization of equipment. A feature of the technology of large-tonnage production is also that the isolation stage of the finished product is usually simple (separation of yeast, rectification of solvents), the final products are available in liquid form or are subjected to heat drying in atmospheric spray dryers, since they are sufficiently thermostable. 2. Small-tonnage production of fine microbiological synthesis associated with obtaining bacterial products and substances of com5

Processes and devices in biotechnology

plex organic structure, most of which have physiological activity (medical and fodder antibiotics, enzyme preparations, bacterial fertilizers, starter cultures, amino acids, vitamins, blood substitutes, some organic acids, growth stimulants, vaccines, hormonal drugs, etc.). The main technological stages of fine microbial synthesis is also submerged cultivation of microorganisms. However, there are a number of features, the most important of which is the increased requirement to protect the working environment at the stage of cultivation, and sometimes in the subsequent stages from contamination by extraneous microflora. This is due to the fact that the cultivation of bioproducers used in these industries takes place in conditions close to optimal for most representatives of wild microflora (pH 6.2-7.2, temperature 25-35 °C, the medium contains carbohydrates, plant protein, phosphorus and nitrogenous salts). As a producer, usually specially selected strain is used. Under these conditions, extraneous microorganisms that have introduced from the environment into the medium can completely suppress the growth of a useful bio-producer or drastically reduce the yield of the desired metabolic product. In this regard, an important role is played by equipment for fine microbiological synthesis, including purification of aerating air, reliable sterilization of nutrient media and various additives. Chemical purification or isolation of the target product from culture liquids for most of the productions of this group occurs in a more complicated way than in large-tonnage yeast industries. At this stage, apart from separation and filtration, processes such as precipitation, extraction, evaporation, ion exchange are carried out. A number of products are thermal unstable, which requires the use of low temperatures and rapid separation processes after cultivation stage. Most products of fine microbiological synthesis are produced in dry form. The most common types of drying are spray, thermal vacuum, fluidized bed and sublimation. Processes in microbiological production can be divided, as in chemical technology, into mass exchange, heat exchange, hydrodynamic and mechanical processes, but, as a rule, they are accompanied by biological processes. Therefore, growth, exchange and death of microbial population in a cultivator are impossible without mass-heat exchange, hydrodynamic and mechanical processes. 6

Introduction

The need to implement specific technological processes entailed the development and creation of special equipment used only in microbiological industries. At the same time, standard equipment of chemical technology is widely used here. Regardless of its field of activity (science, design, production), a biotechnologist must know well the regularities and kinetics of the main processes, the methods for calculating, and the main principles of hardware design. It should be able to solve problems of scaling and optimization of technological processes. Knowledge of the basic standard processes and unification of their hardware design become particularly important for further production with a flexible universal technological scheme allowing the rapid development of new products.

The Tree of Biotechnology (adapted from Rolf D. Schmid)

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Processes and devices in biotechnology

The Foundations of Biotechnology (adapted from Rolf D. Schmid)

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Chapter 1. Biological systems are indispensable agents of industrial ...

CHAPTER 1

BIOLOGICAL SYSTEMS ARE INDISPENSABLE AGENTS OF INDUSTRIAL BIOTECHNOLOGY

1.1. Biological agents and highlights Industrial biotechnology is a set of operations that use living systems or their derivatives to generate target products or processes valuable for the national economy promoting sustainable development. On a commercial scale, biotechnology represents a robust bio-industry. It includes, on the one hand, industries in which biotechnological methods can replace commonly used in conventional chemical technology, and on the other hand, industries in which biotechnology plays a leading role (Fig. 1.1).

Figure 1.1. Biotechnologies: the range of application in various industries

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Processes and devices in biotechnology

These technologies are based on the application of the catalytic potential of various organisms and bio-systems, such as microorganisms, viruses, plant and animal cells and tissues, as well as extracellular substances and cell components (Fig. 1.2). Biological agents and non-biological components employed in industrial biotechnology are called product systems or biotechnological processes. The main elements composing biotechnological processes are biological agents, substrates, equipment and products (Table 1.1).

Figure 1.2. Levels of organization in biotechnologically valuable living systems

In this way, industrial biotechnology is the research, development and production of valuable bio-based material from biological systems. A biological agent is a vigorous active principle in biotechnological processes and one of its most important elements (Table 1.2). A bio-agent can be: ‒ Integrated holistic multicellular or unicellular organism; ‒ Subcellular structures, like viruses, plasmids, DNA, RNA, proteins; 10

Chapter 1. Biological systems are indispensable agents of industrial ...

‒ Cell cultures of plants and animals, protoplasts and any biological systems capable of biosynthesis and conversion; ‒ Multi-enzyme complexes isolated from cells; ‒ Individual isolated enzyme. Table 1.1 Examples of substrates, biological agents and products generated in biotechnological processes Substrates Molasses, sugar cane juice, hydrolysates of plant polymers; Sugars, alcohols, organic acids; Oil paraffin; Products, precursors of biotransformation; Natural gas, hydrogen; Waste from agriculture and forestry, including processing of fruits and vegetables; Household waste, waste water; Milk serum; Potatoes, grain; Green biomass of plants.

Biological agents Microorganisms, plant and animal cells; Viruses; Cell components: membranes, protoplasts, mitochondria, enzymes; Extracellular products: enzymes, coenzymes; Immobilized cells of microorganisms, plants and animals, their components and extracellular products, etc.

Products Biogas; Clean products, medicines, diagnostics; Hormones and other biotransformation products; Organic acids; Polysaccharides; Amino acids, proteins; Food products; Extracts, hydrolysates; Alcohols, organic solvents; Antibiotics; Enzymes, vitamins; Metals, nonmetals; Monoclonal antibodies; Biofertilizers and bioinsecticides, microbial biomass, vaccines, etc.

The functions of any bio-agent are the capability to complete biosynthesis of the desired product. A bio-system responsible for carrying out the biosynthesis of the target product is called «bio-producer» or «bio-factory». «Bio-agent», which is an individual enzyme that fulfills the function of a single reaction is called «biocatalyst». The nomenclature of biological organisms is expanding rapidly, but in the industrial biotechnology the most important place among these bio-agents is occupied by the indigenous and geneticallyengineered microbial cell (Fig. 1.2). 11

Processes and devices in biotechnology Table 1.2 Examples of microorganisms used to produce desired compounds Biological agent

Product

Bacteria Acetobacter aceti, Gluconobacterium Vinegar suboxidans Actinoplanes missouriensis Glucose isomerase Azotobacter vinelandii Alginates Bacillus subtilis Proteases, Antibiotics Bacillus amyloliquefaciens α-Amylase Bacillus licheniformis Proteases, α-Amylase Bacillus thuringiensis, Bacillus popilliae Bioinsecticides Brevibacterium flavum Glutamate, lysine and other amino acids Clostridium acetobutylicum п-Butanol, acetone Corynebacterium glutamicum Glutamate, L-lysine, inosinic acid, ribonucleotides Escherichia coli (recombinant strain) Insulin, growth hormone, interferon Lactobacillus acidophilus, Lactococcus Yoghurt and sour-milk products lactis Lactic acid Leuconostoc mesenteroides Dextran Propionibacterium freudenreichii Vitamin BRR12 Propionibacterium shermanii, Yogurt, Swiss cheese Streptococcus thermophilus Xanthomonas campestris Polysaccharides, Xanthan Zymomonas mobilis Ethanol Yeasts Candida utilis Microbial protein Kluyveromyces fragilis Lactase Phaffia rhodozyma Astaxanthin Saccharomyces cerevisiae Ethanol, baker's yeast, wine, ale, sake Saccharomycopsis lipolytica Lipase Fungi Aspergillus oryzae Amilase, Sake Blakeslea trispora β-carotene Chehalosporium acremonium Cephalosporins Endothia parasitica Rennet extract Penicillium chrysogenum Penicillins Penicillium roqueforti Roquefort cheese Rhizopus nigricans Transformation of steroids

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Chapter 1. Biological systems are indispensable agents of industrial ...

Cellular forms, including prokaryotic and eukaryotic organisms differ in many principal features. However, there are a number of common properties for microorganisms in terms of technological aspects: ‒ The high rate of reproduction and metabolism processes. This is due to the large ratio of the exchange surface to the cell volume. For microorganisms, the entire surface of the cell is capable to metabolize and exchange. Since bacterial cells are the smallest, they grow and reproduce faster than all other microorganisms, followed by yeasts and fungi. In contrast, the rate of metabolic processes in microorganisms is tens and hundreds of thousands of times higher than in animals, e.g. in the bovine body weighing 500 kg approximately 0,5 kg of protein is formed in 24 hours; during the same time, 500 kg of yeast can synthesize more than 50,000 kg of protein. Therefore, the biosynthesis of complex substances (proteins, antibiotics, antibodies, etc.) is more economical and technologically more accessible than chemical synthesis. ‒ Metabolic flexibility. Great ability to adaptation, i.e. adjustment to new conditions of existence. The incomparably vast versatility of metabolic processes of microorganisms in comparison with plants and animals is because of their ability to synthesize inducible (adaptive) enzymes, which are generated in the cell if there are appropriate substances in the medium; ‒ The high level of variability. Wider degrees of variability of microorganisms in comparison with macroorganisms is due to the fact that most microbes are single-celled organisms. It is easier to manipulate an individual cell than a whole organism composed of multiple cells. High level of variability, rapid growth and development, metabolic activity, fast reproduction, all these properties make microorganisms extremely convenient agents for genetic analysis, since experiments can be conducted in a short time on a large number of individuals. ‒ The possibility of carrying out biotechnological processes in industrial scales at the presence of the corresponding technological equipment, raw materials and processing technology. While choosing a biological agent and settling it in manufacturing process, first it should be complied the principle of technological 13

Processes and devices in biotechnology

effectiveness of strains. This means that the microbial cell, population or community of an individual organism should maintain their basic physiological and biochemical properties during long-term fermentation. Bio-producers must be resistant to mutational influences, phages, contamination with extraneous microflora, be safe, absent of toxic products and wastes during cultivation, have high yields of product and acceptable technical and economic indicators, etc. (Fig. 1.3).

Figure 1.3. Criteria for the selection of a biotechnological agent, particularly bacteria

Nowadays, many industrial microbial technologies are based on the use of heterotrophic organisms, and in the future, autotrophic microorganisms will occupy the decisive place among producers, not requiring growth in scarce organic media, and extremophiles, organisms that flourish in extreme conditions (thermophilic, alkalophilic and acidophilic microbes). The use of mixed microbial cultures and their natural associations are gaining more attention around the world. In comparison with monocultures, microbial consortiums are able to assimilate complex, heterogeneous substrates, mineralize organic composition, having 14

Chapter 1. Biological systems are indispensable agents of industrial ...

high ability to biotransformation and increased resistance to the impact of adverse environmental factors and toxic substances, as well as increased productivity and the possibility of sharing genetic information between certain types of community. The main applications of mixed cultures are environmental protection, biodegradation and assimilation of complex substrates. A special group of biological agents in biotechnology are enzymes, so-called catalysts of biological origin. Enzymes are commonly used in different biotechnological processes and industries; however, this area is limited by difficulties in obtaining them, instability and high cost. As a specific sector in establishment and application of new biological agents the immobilized enzymes should be emphasized, which are a harmoniously functioning system, implication of which is determined by the right choice of enzyme, carrier and technique of immobilization. The advantage of immobilized enzymes in comparison with free ones is the stability and increased activity, retention in the volume of the reactor, the possibility of complete and rapid separation of the target products and the organization of continuous fermentation processes with reuse of a biological agent. Immobilized enzymes offer new opportunities and challenges for constructing biological micro-devices in analytics, energy conversion and bio-electrocatalysis. Non-traditional biological agents of biotechnology include plant and animal tissues, involving hybridomas and transplants. A great deal of attention is currently being paid to obtaining the novel biological agents, such as transgenic cells of microorganisms, plants, animals by genetic engineering methods. Modern methods have also been advanced that make it possible to obtain artificial cells using miscellaneous synthetic and biological materials (membranes with specified properties, isotopes, magnetic materials, antibodies, etc.). Approaches are being developed for the construction of enzymes with particular properties that have increased reactivity and stability. At present, the synthesis of polypeptides with desired configuration is also implemented. Some proteins and secondary metabolites can only be attained by culturing eukaryotic cells. Plant cells can serve as a source of a number of compounds, e.g. atropine, nicotine, alkaloids, saponins, etc. Cell 15

Processes and devices in biotechnology

cultures of animals and human also produce multiple biologically active compounds, in particular, pituitary cell lines – lipotropin (fatsplitting stimulant) and somatotropin (growth regulating hormone). Cultured cell lines of animals that secrete monoclonal antibodies are widely used for the disease diagnosis. Veterinary science broadly applies contemporary biotechnological methods, as the culture of cells and embryo, ovogenesis in vitro, artificial insemination and etc. Hence, a variety of biological agents with different levels of organization from molecular to the cell can serve as the source of biotechnological products and processes. The biological agents during the life course synthesize new products and metabolites possessing different physicochemical properties and biological activity, and are divided into four categories: 1. The cells themselves as a source of the desired product. For example, bacteria or viruses are used to produce live or subunit vaccines, yeast is a feed protein and source for obtaining hydrolysates for culture media, etc. 2. The macromolecules, synthesized by cells in the process of growing. These metabolites include enzymes, toxins, antigens, antibodies, polysaccharides and others. 3. Primary metabolites, low molecular weight substances necessary for cell growth, such as amino acids, vitamins, nucleotides, organic acids, etc. 4. Secondary metabolites, low molecular weight compounds, which are not required for cell growth, considering antibiotics, alkaloids, hormones and toxins (Fig. 1.4). There are several ways of using bio-agents in biotechnological production: ‒ The most common one is based on obtaining biomass followed by its use as a semi-product or the desired product. These are single cell proteins (SCP) for fodder and food, bakery and brewer's yeast, normal-flora, bio-pesticides, bio-fertilizers, some vaccines, diagnostic bacteriophages. ‒ Other option is the application of waste products from bioagents that accumulate in their environment. Amino acids, vitamins, enzymes, antibiotics are harnessed in such way. A variation of this 16

Chapter 1. Biological systems are indispensable agents of industrial ...

option can be considered biotransformation, when a biological object is used for a specific biochemical reaction at some stage of drug production. Application of acetic acid bacteria in the production of vitamin C (translation stage of sorbitol to sorbose), mycobacteria in the production of steroids (at the stage of conversion of sitosterol to 17-ketoandrostan) are some examples. ‒ The third way is based on immobilization, which provides the following advantages: increasing the stability and sustainability of the bio-producer, process automation, reduction of costs for the isolation and purification of the resulting reaction products.

Figure 1.4. The various groups of biological compounds synthesized by microorganisms

Gels, membranes, fibers, inert micro-particles are comprehensible for immobilization. This technology includes methods of mechanical, physical or/and chemical processing. Viable biological agents (whole cells of microorganisms, plants and animals), as well as individual enzymes or their complexes are usually immobilized. Questions for thought and review 1. List the main elements of biotechnological process. 2. What is the role of the biological agents in biotechnological production? 3. What can be used as biotechnological agents in industrial biotechnology?

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Processes and devices in biotechnology 4. What are the classifications of biological agents? 5. What are the options for using biological agents in biotechnological production? 6. What factors determine the choice of microorganism-producer in industrial production of targeted products? 7. List the advantages of agent cultivation of microbial origin in comparison with plant and animal biological agents. 8. Describe the requirements for industrial microorganisms-producers. 9. What is meant by the term «Technological strain»?

1.2. Improvements of biological agents. Selection and mutagenesis Biotechnology production is aimed either at obtaining the maximum possible amount of biomass, or at achieving the maximum yield of products generated by cell activity. The main strategy is the extension of product yield per unit of producer biomass. If the organism isolated from the environment is not improved, then the production process is either economically impractical or technically unfeasible. The goals that should be attained while improving the bio-producer: 1. Productivity enchantment in order to achieve the large yield of useful substances per unit of biomass. 2. Formalization of the ability for bio-producers to demand less scarce and cheaper media. 3. The bio-producer should not be a subject to feedback inhibition the biosynthesis of the final product. 4. Stability of the bio-producer to viral infections (bacteriophages). 5. Low requirements for equipment, i.e. biosynthesis should not decline by using old-fashioned devices (e.g., achieving less foaming of the culture liquid). 6. Optimization of the hygienic properties of the bio-producer (e.g., lack of unpleasant smell, etc.). The biotech industry currently uses thousands of strains of various organisms. Mainly three types of microbial strains are employed in industry which is shown in Fig. 1.5. 18

Chapter 1. Biological systems are indispensable agents of industrial ...

Figure 1.5. Industrially important types of microbial strains

In most cases, they are improved by induced mutagenesis and subsequent selection, which allows for large-scale synthesis of various substances. Most frequently, this trend apparent in the case of biological agents belonging to microorganisms (Fig. 1.6. and 1.7.).

Figure 1.6. General stages of microbial selection

Selection is artificial screening of organisms with the best properties of its generation. Selection procedures include searching natural forms of microorganisms with a beneficial interest, further improving it to create on its base the industrial strains. Traditionally, for increasing the productivity of microbial strains the screening and selection of suitable options are preferable. Normal population of microorganisms is heterogeneous: «+» – version carries a desirable trait, while «-» – version does not. Variations are due to spontaneous 19

Processes and devices in biotechnology

mutations (uncontrolled, random, the causes of which are not identified).

Figure 1.7. Types of mutagenesis

During selection process at each stage the most high-performance clones are selected from a microbial population. However, the method of selection based on spontaneous mutations is limited by their low frequency, making it difficult to process intensification. Changes in the structure of DNA are rare (10PP-10PP per gene). Despite this, in a very large population (10PP15PP…. 10PP17PP cells) even in one generation 10PP5PP ... 10PP7PP cells appear per each mutant gene. Spontaneous mutations assist the microbial populations to adapt to new conditions of existence (Fig. 1.8). In this way, strains of beer, wine, baker's yeast, acetic acid, propionic acid bacteria, etc. were selected for a long time. An example of the most productive mutants during cultivation in a continuous mode is the selection of Saccharomyces uvarum yeasts on the basis of resistance to ethanol, a product of the vital activity of yeast. There a feedback formed between the parameter characterizing the vital activity of the culture (its CORR2RR release) and the intake of ethanol as an inhibitory factor into the bioreactor. Mutant yeasts resistant to inhibitory effect of ethanol in concentrations up to 10% are obtained with prolonged cultivation in such way. This approach is used to increase the stability of biological agents to various factors, such as acids, alkalis, metabolic products, heavy metal ions, etc. The main drawback of this method is its extreme duration. Induced mutagenesis is a way of improving biological agents by radical means. Induced mutagenesis contributes to a dramatic increase 20

Chapter 1. Biological systems are indispensable agents of industrial ...

of the frequency of bio-agent mutations during artificial genome damage. Ultraviolet, X-ray or gamma-radiation, corpuscular radiation of the type of fast electrons, positrons, protons, neutrons, and some of the chemical compounds have mutagenic effects that cause changes in the primary structure of DNA. Nitrous acid, alkylating agents, acridine dyes, bromouracil and others belong to the most proven mutagens. Phages and plasmids are biological mutagens (Table 1.3).

Figure 1.8. Typical procedure in mutation and spontaneous strain selection process

The main phenotypic feature of microbial improvement is the ability to form a large amount of product. Selection system is developed for screening mutants fulfilling this requirement. Resistance to antibiotics, poisonous substances or phage infection can serve as an indication of selection. The cells obtained after the mutagenesis are plated on solid selective media, where only those colonies that are resistant can multiply. The replicas of colonies are inoculated on minimal medium with metabolites for the selection of auxotrophic mutants. 21

Processes and devices in biotechnology

The main advantage of this technique is the ability to conduct simultaneous analysis of a large number of mutants obtained (e.g. several hundred on a single agar plate). It is feasible to receive various mutants using different selection media (replica plating, penicillin technique, etc.) (Fig. 1.9). After treatment, the number of mutants as «positive» and «negative» suddenly increases. Thus, in some mutants the traits dramatically alter and the greater the dose of the mutagen, the more lethal process happens. It is necessary to maintain a balance between lethal mutations and the number of surviving mutants. Selection of mutants can be carried out in a continuous-culture chemostat; microbial cells are incubated in the medium with a mutagenic agent and subjected to selective pressure, e.g., by gradually replacing a good carbon source with a poor one. Under such conditions, because of mutagenesis, only those microorganisms survive which have acquired the ability to utilize the new source of carbon. However, this method cannot be used for selecting mutants that form a desired metabolite in higher concentrations. Table 1.3 Mutagens used for strain improvement Mutagens Physical agents Iodizing radiations (x-rays, γrays) Short wavelength (UV light) Chemical agents Nitrate

Mechanism

Leads to ss and dsDNA Major genetic breakage alterations Thymidine and cytidine form Point mutations dimers

Deaminates adenine to hypoxanthine, cytidine to uridine Alkylating agents (NTG, EMS) Alkylate purines Base analogs (5-Chlorouracil, 5- Incorporated into replicated bromouracil) DNA Intercalating agents (Acridine Intercalates into DNA orange, ethidium bromide) Biological agents Phage, plasmid, DNA Transfer DNA elements transposons within a chromosome

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Applications

Point mutations

Point mutations Major genetic alterations Major genetic alterations Gene markers

Chapter 1. Biological systems are indispensable agents of industrial ...

Therefore, it is necessary to examine not only the ability of the mutants to form the target product, but also maintaining the traits that possess a wild strain. Usually the selected cells are grown in shaking flasks under conditions similar to those in the production process and cells with improved properties are preferred. The best candidates may then be crossbreed with wild type or less mutated strains to reduce the negative effects arising from many passages of random mutation. Under natural conditions, cell metabolism is administered according to the principles of the strictest conservation, which is provided by a complex system of its regulation. Therefore, creation the mutant forms of microorganisms, i.e. super-producers of the corresponding products is very crucial. The over-synthesis of the required metabolic product can be achieved both by changing the organism’s genetic program and by altering its regulatory systems. Mutagens cause DNA changes leading to a shift in metabolic reactions, as a result some of the typical cells turn into overproducers.

Figure 1.9. The selective media technique

Increased ability to form the target products using mutants provided by: 23

Processes and devices in biotechnology

– Changes in structural and regulatory genes (deletion, inversion, transposition and point mutation) (Fig. 1.10). In deletion, transposition or inversion, the genome of a mutant organism undergoes changes that result either in the loss of a certain trait by the mutant or in the appearance of a new trait. Genes in new places turned to be under control of other regulatory systems. In addition, hybrid proteins that are unusual for the original organism may appear in the mutant cells, due to the fact that polynucleotide chains of two (or more) structural genes previously remote from each other emerged in one controlled promoter. Repressor genes that regulate the synthesis of the desired product can be suppressed. Disorder of the retro-inhibition system is an effective way to increase the formation of the desired product. Enhancing activity of the producer can also cause by mutations that change the transport system of predecessors of the target product. Another mechanism is a promotion of the producer’s resistance to the substance formed by it and the lack of a suicidal effect, which is especially necessary for producers of antibiotics.

Figure 1.10. Types of mutations

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Chapter 1. Biological systems are indispensable agents of industrial ...

Substitutions in one pair of nucleotides (type of point mutations) during the transfer of the genetic code at the translation stage lead to the appearance in the encoded protein instead of one amino acid another. This can dramatically change the conformation of the protein and accordingly, its activity, especially if the replacement occurred in the active or allosteric center. – Increase of the gene dose (duplication, amplification). Amplification is a significant increase of the number of copies in a specific gene of the microbial cell, leading to a dramatic extension during production of the substance encoded by this gene. This technique is associated with increasing the number of plasmids per cell. Normally they are present in 1-30 copies per cell and contain 2-250 genes. Meanwhile, it is possible to achieve up to 3,000 copies of plasmid genes per cell. The amplification technique has been widely used for Escherichia coli, and now it is possible to transfer any chromosomal gene or gene group to the plasmid. Then plasmid is transferred to the bacterium, where it can be amplified. Moreover, it became possible to transfer from one cell to another the plasmid of Bacillus after transformation in the presence of polyethylene glycol. Plasmids from Pseudomonas can be transferred to other gram-negative bacteria, like Acinetobacter, Agrobacterium, Klebsiella, Proteus, Rhizobium and Salmonella. Plasmids of Staphyloccus aureus can also be replicated and expressed in Bacillus subtilis. Virtually all microbial-producing antibiotics contain plasmids that carry either the structural genes for the biosynthesis of antibiotics or the genes that regulate their expression. Therefore, amplification of these genes can significantly extend the production of antibiotics. The main successes of mutational-selection steps were achieved with microorganisms, as they easily multiply, have a large number of mutants and easier to select with properties of interest. The sequential impact of one or more mutagenic factors in mutational-selection step allows one to gradually increase the productivity of the strain. For example, by selection and mutagenesis, the activity of the penicillin producer was increased 100,000 times by amplifying the genes encoding the formation of the LLD tripeptide, disturbing the retroinhibition system and increasing the permeability of the wall. 25

Processes and devices in biotechnology

The yield of the producer of streptomycin is increased 20 000 times by repeated mutational-selection step treatment with UV light and X-ray. Microorganisms capable of synthesizing chlortetracycline and oxytetracycline provide the mutants that generate other antibiotics (6-dimethyltetracycline, 7-chloro-6-dimethyltetracycline, 7-chloro-5a (11a) -dehydrocetracycline, 2-acetyl-2 decarboxyamidooxytetracycline) in addition to the main ones. The same results were found with bio-producers of vitamins and amino acids. For example, highly productive strains of Bacillus subtilis is received, capable of secreting up to 75 kg of vitamin B2 from a ton of the nutrient mixture. As a result, the improvement of the biological agent is the obtaining of bio-producers with mutations in the genome that differ from the original bio-agent towards improving biotechnological traits, in particular, increasing the formation of the desired product. Advantages of the mutational-selection step method: ‒ There is no need for preliminary hypotheses and knowledge about the role of a protein in the emergence of this phenotype; ‒ Allows the selection of productive strains that are not genetically studied, and produce metabolites which the pathway of biosynthesis has not been set; ‒ Induced mutagenesis provides new findings and information for biochemistry and molecular biology. Disadvantages of the induced mutagenesis and subsequent mutational-selection step methods: ‒ Labor intensity, i.e. extensive screening for productivity; ‒ Lack of information about the nature of mutations; ‒ Necessity to map mutations and evidence of the absence of other mutations that affect the phenotype; ‒ Decrease in the rate of mutant selection. These disadvantages of mutational-selection step can be overcome by combining it with genetic exchange methods. Therefore, the general way of selection represents the process from the blind selection of the right producers to the conscious construction of their genome. One of them is cell engineering in vivo. 26

Chapter 1. Biological systems are indispensable agents of industrial ... Questions for thought and review 1. The purposes of bio-producer improvement. 2. What properties of the biological agents can be used to improve in order to create efficient and safe production of the targeted compounds? 3. Describe the directions of bio-producer selection. 4. Describe the methods of bio-producer selection. 5. Enumerate the general stages of microbial selection. 6. What methods of mutagenesis are used to improve producers? 7. What mechanisms provide an opportunity to increase the yield of the target product in mutants? 8. What are structural and functional genes? 9. What are the functions of regulatory genes? 10. What mutations can lead to the change in the biosynthetic activity of microorganisms? 11. Describe the conventional procedure in mutation and spontaneous strain selection process. 12. What is the selective media technique? 13. What are the advantages and disadvantages of spontaneous and induced mutagenesis in bio-producer selection?

1.3. Cell engineering In the case of cell engineering, the whole cell is considered for obtaining producers with desired properties based on DNA recombination. Recombination is an important tool for the genetic programming; it arranges genes or parts of genes and combines genetic information from two or more organisms in a single organism. Different processes of genetic information exchange of living cells lead to recombination; the sexual and parasexual cycle of eukaryotic cells, transformation, conjugation and transduction in prokaryotes, as well as a universal method of protoplast fusion. Homologous recombinetion occurs if bacterial or eukaryotic chromosomes (having similar base sequence in DNA) get together because of coupling and exchange their DNA parts by processes of rupture and reunion. The easiest way to create organisms with the desired complex of genetically determined traits is a crossing the organisms belonging to the opposite sex. Genetically marked strains are taken for hybridization (auxotroph, resistant to growth inhibitors). As a result of cell fusion (coupling) the hybrids in yeast, fungi and algae are formed. If the 27

Processes and devices in biotechnology

parent cells are haploid due to the nuclear fusion diploid cell (zygote) arises which carries a double set of chromosomes in a nucleus. The nucleus of the individual representatives immediately falls down in meiosis, during which each of the chromosomes splits. Homologous chromosomes pair up and share some of their chromatids resulting crossover. Further haploid sex spores are formed, each contains a set of genes that differ from parent cells. Hybridization allows using heterosis, combination of various economically important traits of their parent cells and the high degree of hybrid variability. However, until recently, the hybridization as the selection method in microbiology and biotechnology was used extremely rarely, because crossing is unusual for microbial world and is found in nature as an exception. However, natural hybridization, giving basis for recombination occurs in both eukaryotic and prokaryotic microorganisms. Sexual and parasexual processes are widely used in genetic practice of industrially important fungi-producers. Hybridization has been successfully applied in yeasts that have a sexual mode of reproduction. This technique created many valuable cultures for bakery industry. By crossing spores and vegetative cells of the original strains a group of large-cell yeasts, which are resistant to molasses was obtained. A hybrid yeast adapted to the wort capable for fermenting not only glucose, but also raffinose which is one more example of hybridization. Hybrids can also be acquired from other microorganisms that lack of sexual process. Vegetative hybridization is preferable for fungi; the genetic material of two vegetative cells is exchanged because of cell fusion, i.e. the parasexual cycle. The operation consists of several consecutive steps. At the first step, two adjacent hyphae of mycelia (possibly belonging to different strains of fungus) are fused via cytoplasmic bridge. Through the bridge the cytoplasmic material of both cells, including the exchange of nuclei occurs. The nuclei in the heterocaryon merged forming a diploid nucleus. Diploidization sometimes leads to an increase in cell productivity, for example, the biosynthesis of organic acids or antibiotics. The mitotic division of the nucleus occurs very often in diploid cells; as a result, both haploid and diploid recombinants can arise. Consequently, during parasexual hybridization the main goal is achieved, i.e. the formation of a recombinant 28

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hybrid, in the genome of which is combined with genetic information from different individuals. Vegetative hybridization of fungi is a natural process and occurs both in nature and in microbiological practice. This method is quite suitable with fungi of such industrial genera as Aspergillus, Penicillium, Cephalosporium, and Fusarium. Most successfully, it was used during selection of Penicillium, which secretes penicillin. The hybrid-producer of oxytetracycline with new useful properties has been obtained; the ability to grow on a more productive concentrated medium and weakened ability to medium foaming. Hybridization provided excellent results in the creation of more active strains of fungi, the producers of organic acids and enzymes. Unlike eukaryote no true sexual reproduction is found in bacteria because they lack sexual structures, no gamete fusion takes place, karyogamy and meiosis is also absent, and by all means bacteria are haploid (Fig. 1.11).

Figure 1.11. The process of merozyogote formation

Although prokaryotes do not undergo sexual reproduction, they possess the ability to exchange genes and undergo genetic recombination. Except homologous recombination, which results in the exchange of analogous genes in chromosomes, there are other forms of gene recombination in which new genes are induced to the genome of the cell. The features of the bacterial recombination: gene transfer in bac29

Processes and devices in biotechnology

terial are not able to produce zygotes but partial diploid called «merozygotes»; the original genome of recipient is named as «endogenote»; while the portion of DNA introduced from donor cell into recipient cell is called exogenote. Bacteria are known to exchange genes in nature by three fundamental processes (Fig. 1.12): 1. Transformation 2. Conjugation 3. Transduction In transformation, DNA is acquired directly from the environment, having been released from another cell. High molecular weight DNA must bind to the cell surface. The bound DNA is taken up through the cell membrane. The donor DNA fragment is then integrated into the host chromosome or replicates autonomously as a plasmid. Transformation occurs in nature and it can lead to increased virulence. In addition, transformation is used extensively in recombinant DNA technology. Conjugation requires cell-to-cell contact for DNA to be transferred from a donor to a recipient. Conjugation is the most prevalent process of sexual reproduction in bacteria. In conjugation, two parental cells physically contact between two genetically different cells of the same or closely related species and transfer their genetic material through a small tube, called «conjugation tube». The genetic material from one cell (donor) is transferred to other (recipient). This type of gene recombination is carried out through plasmids. In many cases, plasmids include genes that determine the resistance of the cell to a particular antibiotic, the ability of microorganisms to form toxins, and additional hereditary information. The plasmid is able to integrate with a specific chromosome of the host. Among Gram-negative bacteria, this is the major way to transfer bacterial genes. The process can occur between different species of bacteria. Multiple antibiotic resistance due to conjugation has become a major problem in the treatment of certain bacterial diseases. Since the recipient cell becomes a donor after transfer of a plasmid it is easy to see why an antibiotic resistance gene carried on a plasmid can quickly convert a sensitive population of cells to a resistant one. 30

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Figure 1.12. Gene transfer types in bacteria

Gram-positive bacteria also have plasmids that carry multiple antibiotic resistance genes, in some cases these plasmids are transferred by conjugation while in others they are transferred by transduction. Transduction is the bacteriophage-mediated transfer of genetic material from donor bacterial cell to the recipient cell. There are generally two types of transduction: 1. Generalized transduction. This type of transduction starts with the infection of bacteria by bacteriophages. This process is controlled by the DNA segments called as «prophage», a particle present in cytoplasm of the bacterial cell. During the infection of lysogenic bacterial cell by phages, the DNA of the host breaks down into small fragments and the nucleic acid of the bacteriophage utilizes the bacterial enzymes and synthesize new phage components. At the same time when these progeny phage particles are assembled the DNA fragments of the bacteria incorporate into the DNA particles of the phage. The genetic material or DNA fragments of the previous bacterial cell is transferred to the new bacterial cell infected by progeny phage particles. In generalized transduction, all genes have an equal probability of being transduced. Any bacterial gene from the donor 31

Processes and devices in biotechnology

can be transferred to the recipient. Thus, generalized transduction is the process where bacteriophage plays an active role in the transfer of DNA fragments of the bacterial cell. 2. Specialized transduction. Andre Lwoff reported that certain bacterial strains are able to survive for a long time even after infected by the bacteriophage and the case nothing like lysis of the bacterial cell. There is a fusion of bacterial DNA with the phage DNA and both DNA, i.e., bacterial DNA and phage DNA replicate commonly. These bacteria are known as lysogenic bacteria and the phage is called as prophage. This bacterial cell can survive in lysogenic stage for many generations, which is due to the synthesis of a special repressor protein. This protein inhibits the synthesis of phage particle inside the bacterial cell. As the synthesis of this protein ceases the bacterial cell starts the synthesis of phage components. The DNA of both i.e., phage DNA and bacterial DNA break down before the synthesis of the phage particles start. At the same time, some bacterial genes are carried out by phage DNA and replicate with the phage DNA. These resultant progeny phage particles are entirely different from the parent one when these particles infect a new bacterial cell; some of the gene (from previous bacterial cell) are also transmitted to the newly infected bacterial cell. In this type of transduction only those special genes are transmitted which are attached very closely to the phage DNA. Hybridization can be achieved by means of laboratory techniques. At present, methods of artificial vegetative hybridization so called cellular engineering in vivo have been developed. Cellular engineering is one of the most important areas in biotechnology. The basis of it is the hybridization of somatic cells i.e., the fusion of non-sex cells with the formation of a single whole cell. Somatic hybridization has broader possibilities for crossing phylogenetically distant organisms than sexual interbreeding. The fusion of cells can be complete or the recipient cell can acquire separate parts of the donor cell, like cytoplasm, mitochondria, chloroplasts, nuclear genome or its large parts. Cellular engineering is a technique for exchanging fragments of DNA, sections of chromosomes in prokaryotes and any chromo-somes in eukaryotes, regardless of the remoteness of organisms from an 32

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evolutionary point of view. In the case of cellular engineering, there are no species and patrimonial barriers, i.e. genetic material is exchanged between organisms, which under normal conditions the sexual process do not occurs, which opens up great opportunities in creating biological agents. The set of methods of cellular engineering used to construct new cells include the cultivation and cloning of cells in specially selected media, cell hybridization, cell nucleus transplantation and other microsurgical operations to «disassemble» and «assemble» (reconstruct) viable cells from individual fragments. These methods are based on compulsory fusion of protoplasts, as well as on a number of cellular engineering techniques that allow the protoplasts of even genetically separated cells to be deliberately fused and thereby expand the possibilities of hybridization. Protoplast technique leads to a change in microorganisms, which makes possible to achieve the improvement of bio-agents.

Figure 1.13. Protoplast fusion technique

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For the exchange of chromosome fragments, firstly protoplasts must be obtained. Protoplast is a cell without cell walls, surrounded by a cytoplasmic membrane. Then fusion of protoplasts is carried out to form diploids, which are incubated for several hours to «break» and reunite chromosomal strands in different variants. The suspensions are then plated onto a solid nutrient medium, and the cells are formed capable for reproduction, which form corresponding colonies. They are closely examined and clones with new traits of interest are selected (Fig. 1.13). The protoplast technique consists of several stages: 1. Removal of the cell wall. Protoplast is obtained by treating the cell with enzymes that hydrolyse the polymers in the cell wall. Lysozyme (for bacterial cells), snail zymolyase (for fungal cells) and a complex of cellulases, hemicellulases and pectinases (for plant cells) are used for this purpose. 2. Stabilization of protoplasts. Removal of the cell wall and at the same time preserve the integrity of the protoplast membrane can be done only by equalizing the osmotic pressure inside the cell and in the medium. An osmotic stabilizer is used to isolate viable protoplasts. Typically, this is a hypertonic solution composing 20% mannitol or sucrose, 10% sodium chloride. The ionic strength of the solution is such that the cell is in the state of turgor, but does not burst, and the solution is also washed with the enzyme. In addition to the osmotic properties of the medium, pH, illumination, temperature and other factors must be selected. Conversion of the cell suspension into the protoplast suspension is usually controlled by phase contrast microscopy. 3. Fusion of protoplasts. The close contact must be established to carry out the fusion between plasma membranes of cells. It is hampered by the presence of a surface charge on natural membranes caused by negatively charged groups of proteins and lipids. To eliminate this obstacle, an alternating electric or magnetic field, Ca PP2+PP ions, polyethylene glycol detergent are used, which breaks the cellular cytoplasm and the DNA of the two protoplasts are combined. When animal cells are fused, the Sendai virus is also used, which partially hydrolyzes the proteins of the cytoplasmic membrane. 34

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The process of fusion of protoplasts occurs gradually. The cell must be competent to facilitate the cell fusion process. For this, the cell is treated with heavy metals, enzymes, or quickly frozen and thawed. A protoplast with two sets of chromosomes (diploid set) is obtained while fusing, i.e., DNA recombination takes place when a hybrid is formed. 4. Regeneration is the restoration of the protoplast cell wall. In this case, the cell wall is regenerated. Protoplast behaves like an isolated cell and is able to divide and form a clone of cells. Some of these cells have a hybrid chromosome. The culture of such cells possess new properties. Screening of the resulting hybrid cells is carried out as follows: ‒ Consideration of phenotypic signs; ‒ Creation of selective conditions in which only hybrids survive that have integrated genomes of the parent cells. An example is the preparation of «hybrid antibiotics». Among the streptomycetes there are various types of antibiotics belonging to different types of glycosidic structure with varying aglycons and sugars. For example, erythromycin has a 14-limb macrocyclic aglycon and two sugars. Anthracycline aglikon consists of four condensed carbonic six-membered rings, linked to an amino-sugar. With the help of cellular engineering, bio-producers of antibiotics were acquired in which the macrolide aglycone of erythromycin was bound to the carbohydrate part of anthracycline. Conversely, anthracycline aglikon with sugars inherent in erythromycin. The fusion of two species of Streptomyces resulted in a recombinant that produced a new anthraxcycline, and Nocardia mediterranei mutants that did not produce rifamycin, after fusion, were given strains synthesizing three new rifamycins. Similar work is being done to obtain hybrid beta-lactam antibiotics. Protoplast fusion may contribute to the improvement of some industrial strains that have accumulated many irregularities in the selection program. They may be subjected to interspecies recombination producing less but robust strains. So two strains of Nocardia lactamdurans, producing cephamycin have been improved. The subsequent fusion led to the emergence of recombinants, which separated cultures produced 10-15% antibiotic than the best of the parent strains. 35

Processes and devices in biotechnology

During protoplast fusion amplification phenomenon in the number of genes can be occurred. Biosynthetic genes of expected product will be in one haploid in a doubled state and its chromosome will have two biosynthetic gene cluster. Of course, this feature can have only a small part of the cultures obtained after regeneration. Therefore, after the protoplast fusion «+» – and «-» – producing variants are detected, compared to the original culture performance, wherein purpose products (vitamins, hormones, antibiotics) increase. Cells with the gene amplification is «+» option. However, in the cell, the repair (recovery) systems of the DNA molecule exist, and gradually these recombinants are returned to the original (wild) type. Reparations overcome in two ways: «+» options are treated with mutagens for overcoming the repair system action or «+» options cells are immobilized. The significance of the hybridization method: ‒ The possibility of combining in one organism (cell) the desired properties of two or more strains or species; ‒ Use of recombinants, selected from the second generation of hybrids, with original properties, uncharacteristic to parent cells; ‒ Enrichment of the genome of the grown microorganism by mutations obtained independently from different strains; ‒ The possibility of transferring into the cell of the microorganism genes, unusual for this species, as well as the possibility of increasing the number of already existing genes, thereby strengthening those properties of the microorganism for which this gene is responsible. Methods of cell engineering in vivo are universal way of introducing genetic information into cells of various origins. The simplicity of the method makes it available for selection of industrially important producers. The technique opens up new opportunities for obtaining not only interspecific and intergeneric hybrids, but also for interbreeding phylogenetically distant forms of the living organisms. At present, interspecific hybrids of tobacco, potatoes, and petunias; sterile intergeneric hybrids of potato and tomato; interspecific and intergeneric yeast hybrids; hybrids of fungi and bacteria have been successfully obtained. 36

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It is also possible to obtain asymmetric hybrids carrying a complete set of genes of one parent and a partial set of another parent. As a rule, such hybrids arise during the cell fusion of organisms, phylogenetically distant from each other. Asymmetric hybrids are more stable, prolific and viable than symmetrical ones. Very promising trend is the hybridization of cells, which carry various improvement data: the fusion of cells of various tissues or organs, the fusion of normal cells with cells that have neoplastic transformation. As a result, hybridoma cells – hybridomas are obtained, inheriting from the normal parent cell the ability to synthesize a useful compound, and from malignant – the ability for rapid growth. Thus, currently, the selection of microorganisms has achieved great success, and improved by the achievements of molecular biology. There are many highly productive microorganisms that synthetize many valuable substances under industrial conditions. However, it significantly extends the possibilities of selection using the methods of cell engineering in vitro i.e., genetic engineering. Questions for thought and review 1. Give the definition for cellular engineering. 2. What is genetic recombination? 3. List the types of recombination patterns. 4. What is the significance of hybridization in the selection of industrial bioproducers? 5. What ways of sharing genetic information exist in prokaryotes? 6. What is protoplast? 7. What techniques of protoplast separation are used in biotechnology? 8. How are protoplasts cultured? 9. Describe the process of obtaining protoplasts from prokaryotes and its stages. 10. Discuss the technique of protoplasts fusion in the selection of highproductive valuable strains of microorganisms. 11. What are the advantages of protoplasts fusion technique over other breeding methods? 12. What are the promising areas of development of cellular engineering in biotechnology. 13. What are the areas of practical application of cellular engineering achievements?

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1.4. Genetic engineering Advances in the field of genetics and molecular biology have enabled biotechnologists, since the 1970s of the last century, move from blind selection of mutant strains to conscious genome construction; the basis of modern genetic engineering using recombinant DNA technology. By recombinant means, a DNA formed by combining in vitro two or more DNA fragments isolated from various biological sources. Recombinant DNA is constructed in two ways. The first way is the preliminary isolation of the necessary gene from the donor organism or its synthesis with the subsequent integration of the gene into the vector molecule. The second way is to create collections of recombinant DNA and search in a collection of recombinants with the necessary built-in gene. In genetic engineering, an isolated DNA serves as vital purpose in vitro. The essence of the technology is the combination of DNA fragments in vitro, followed by the introduction of isolated DNA into a living cell. The scope of mere genetic engineering is a technique of combining isolated DNA fragments of natural or synthetic origin or a combination of them in vitro. Further, it is necessary to introduce the resulting recombinant structures into a living cell. The inserted DNA fragment is either incorporated into the chromosome and replicated, or autonomously expressed. Consequently, the introduced genetic material becomes part of the cell's genome. Hence, the necessary conditions for the implementation of genetic engineering are following: 1. It has to be a biological agent able to synthesize a heterogenous protein, would perceive and transmit genetic information. 2. The host organism should not reject the product synthesized by the producer. 3. The cell must be divided, it is necessary that the genes producing the desired product in the cells, formed after division, be expressed. 4. It is necessary to have a transport tool for introducing DNA into the producer's cell; a vector in the form of plasmids, cosmids and phages. 38

Chapter 1. Biological systems are indispensable agents of industrial ...

Natural methods of transferring genetic information are usually used to introduce a vector into a prokaryotic cell: 1. Conjugation. The genetic material of the cells upon approaching passes from one cell to another in the form of a plasmid. 2. Transduction. Transfer of a genetic material through a virus or phage to a cell. 3. Transformation. The transfer of the genetic material to a cell of isolated DNA, as a result of which genome is altered. In conclusion, the cells containing the recombinant DNA are identified and selected. It is mainly conducted on a selective medium by marker genes. Most widely used as a marker are antibiotic resistance genes. If the vector molecule contains a gene for resistance to an antibiotic, the correspondingly transformed cells will be resistant only to this antibiotic. On this basis, they are selected. The introduction of recombinant DNA (vector) into a host-eukaryote cell is implemented by various methods with different efficacy. All of them are based on the formation of pore in the cytoplasmic membrane, through which DNA can penetrate inside the cell (Fig. 1.14). 1. Microinjection. By means of the finest glass tube and micromanipulator, a vector DNA can be inserted into the nucleus of the cell with the transgene included in it. The number of DNA molecules inserted per injection can range from 100 to 300000. 2. Electroporation. The permeability of membranes is reversibly increased under the influence of high voltage electric pulses. As a result, through the micro-pores which are formed for a short time in the membrane, DNA from the surrounding medium penetrates into the cell. 3. Transfection. The vector is treated with calcium ion. The resulting nano-complexes of ions and vectors penetrate into the cell by pinocytosis. The method is used to introduce transgenes into eukaryotic cells. 4. Packing in liposomes (lipofection). Liposomes are spherical formations coated with phospholipids and containing a vector inside that can penetrate the cell due to their dissolution in the lipids of the plasma membrane. 39

Processes and devices in biotechnology

Figure 1.14. Methods of the vector delivery into eukaryotic cells

5. Microparticle bombardment (gene gun, ballistic transformation, agrolistic method). This is one of the most effective methods of plant transformation. For the implementation, immature embryos of seeds are used to exposure the bombarding by particles of gold or tungsten, which are coated with vectors. «Gene guns» are charged with these particles, after the shots of which the particles penetrate into the cells. The cells in the position of the shot are often killed, while in the center zone around 0,6 -1 cm are most successfully transformed cells remain. The particles can penetrate a depth of 2-3 cell layers. At an enough rate, these particles can penetrate directly into the nucleus, which greatly improves the efficiency of transformation. Using this same method, it is possible, to transform DNA containing cellular organelles, such as chloroplasts and mitochondria (Fig. 1.15). 6. For dicotyledons there is a natural vector for horizontal transfer of genes, so called agrobacterial plasmids. In the process of transformation, the cell wall of a plant is damaged under the action of bacterial pectolytic enzymes and a close contact of bacteria with the plant cell 40

Chapter 1. Biological systems are indispensable agents of industrial ...

plasma membrane is ensured. Because of this contact, bacterial DNA is transferred to the plant cell. The most studied and widely used are 2 types of agrobacteria, first one is Agrobacterium tumefacienes (has Ti-plasmid (tumor inducing), part of which is embedded in the chromosomes of plant cells) and next one is Agrobacterium rhizogenes (in the damaged root zone many new roots are generated due to the phenomenon of Ri – plasmid (root inducing).

Figure 1.15. Agrobacterium and microparticle bombardment methods

7. Infection with virus. The most promising is white cabbage mosaic virus, which affects mainly the plants of the cabbage family. The small genome size (circular DNA of 8000 nm length) allows to manipulate in vitro a viral DNA as bacterial plasmid, and then introduce it into the plants by rubbing the leaves. In this case, all cells become infected, because the virus multiplies rapidly and is transmitted from cell to cell. 41

Processes and devices in biotechnology

The advantage of virus-based vector systems is the small size of the genome, the high concentration of viral DNA in plant cells (up to 50000 per cell), the presence of robust promoters that ensure the effective expression of foreign genes. 8. In recent years, scientists have proposed a new approach to produce transgenic plants with «antisense RNA» (inverted or antisense RNA), which allows to control the action of the gene of interest. In this case, when constructing a vector, a copy of the DNA (c-DNA) of the inserted gene is inverted by 180°. As a result, besides normal mRNA molecule, an inverted mRNA molecule is formed, which due to the complementarity of normal mRNA with it composes a complex and the encoded protein is not synthesized. The strategy of antisense constructs is widely applicable to the modification of gene expression. This strategy is used not only to obtain plants with new qualities, but also for fundamental research in plant genetics (Fig. 1.16).

Figure 1.16. Antisense RNA strategy

After carrying out in one or another transformation method of plant tissue, it is placed in vitro on a special medium with phytohormones, which promotes the multiplication of cells. The medium typically contains a selective agent for which transgenic but not control cells become resistant. Regeneration often passes through the stage of 42

Chapter 1. Biological systems are indispensable agents of industrial ...

callus, after which, with proper selection of the media, organogenesis (shoot formation) begins. The formed shoots are transferred to the rooting medium, also containing a selective agent for a more rigorous selection of transgenic individuals. In addition, the main object of cellular engineering – protoplasts are used extensively in genetic engineering, what is called cell genetic engineering. The exchange of molecules and DNA fragments in protoplasts is achieved more easily than in cells with their complex cell wall. There are three ways of cell genetic engineering (Fig. 1.17).

Figure 1.17. Techniques of cellular genetic engineering

1. The method of gene rearrangement. There is a certain set and sequence of genes in the cell. The genes are rearranged during protoplast fusion. There is also the activation of silent genes. 2. Fusion of genes. Prokaryotes are fused with eukaryote; a recombinant is formed, containing original and foreign DNA. 43

Processes and devices in biotechnology

3. Transformation. a) a cell with a certain set of genes in DNA → living protoplast without rearranging the genes → protoplast hybrid-recombinant → recombinant regenerating cell. b) isolating of DNA, i.e. vector (a piece of isolated DNA from another microorganism. If there is a cell and an isolated DNA with the desired gene, the vector can be in the form of a phage, plasmid, virus, cosmid (plasmid + phage), then it is possible to integrate the vector in an isolated protoplast. For the transformation it is necessary to make the cell competent, i.e. increase its permeability. This is achieved as follows: 1. To affect the cell using heavy metal ions (zinc, cobalt, lithium, magnesium). 2. To influence the cell with enzymes (lysozyme, complex enzyme of grape snail). 3. Quick freezing and thawing. Competent cells more easily absorb the vector. They have a cytoplasmic membrane, which in some places forms a pore to the surface, where the vector as isolated DNA easily penetrates, as well as in the form of phage, virus, cosmid. The necessary gene construction by gene and cell engineering the methods allows to manage the heredity and vital activity of animals, plants and microorganisms and to create organisms with desired traits useful that were not previously observed in nature. Principles and methods of genetic engineering are established, first, on microorganisms, particularly bacteria and actinomycetes (prokaryotes), and yeast and mold fungi (eukaryotes). With the help of genetic engineering techniques, according to a certain plan it can be designed new forms of microorganisms capable of synthesizing a variety of products (including plant and animal origin). Microorganisms have a high growth rate and productivity, the ability to recycle a variety of raw materials. However, during constructing new microbial producers, certain problems arise: 1. Gene products of plant, animal and human origin secrete into the intracellular environment that is foreign to them, where they are subjected to destruction by proteases. For example, peptides like 44

Chapter 1. Biological systems are indispensable agents of industrial ...

somatostatin are hydrolyzed in a few minutes. To protect genetically engineered proteins in a microbial cell, the following approaches used: ‒ Applying protease inhibitors; ‒ The peptide of interest is obtained as a part of the hybrid protein molecule, for this purpose the peptide gene is cross-linked to the natural gene of the recipient organism; ‒ Amplification (increase the number of copies) of genes. 2. In most cases, the product of the transplanted gene is not released into the culture medium and accumulates inside the cell, which makes it difficult to isolate. For example, the production of insulin using E. coli involves the destruction of cells and subsequent purification of insulin. It should be noted that E.coli excretes relatively few proteins. In addition, its cell wall contains a toxic substance, which must be carefully separated from products, used for pharmacological purposes. Currently, more promising objects of genetic engineering are gram-positive bacteria of the genera Bacillus, Staphylococcus, Zymomonas, Streptomyces, as well as eukaryotic microorganisms, like yeasts Saccharomyces (wine, baker's, brewer's yeast), or Candida, Pichia (single cell protein). 3. Most of the hereditary characteristics are encoded by several genes, thus genetic engineering should include the stages of consecutive transplantation of each gene. In some cases, a simultaneous transplantation of whole blocks of genes with a single plasmid is possible. The microbial recombinant strains during cultivation synthesize the product that are not characteristic of them, encoded by an embedded foreign gene. At the moment, hundreds of recombinant strains of bacteria, yeasts, viruses have been obtained, which are capable of producing a variety of biologically active substances, such as antigens, antibodies, enzymes, hormones, immunomodulators, etc. Technologies have been developed for obtaining hundreds of medicines based on genetic engineering. Many of them are introduced into practice and applied in medicine. These are hormones (insulin and human growth hormone), anticoagulants and thrombolytics (tissue activator of plasminogen, factors VIII and IX), vaccines (yeast vaccine against hepatitis B), immunomodulators (interferons and interleukins) enzymes (urease), diagnostic drugs (HIV infection, viral hepatitis, etc.), 45

Processes and devices in biotechnology

monoclonal antibodies, colony-stimulating factors (macrophage, granulocyte, etc.), and many biologically active peptides. The application of genetic engineering in biotechnology is always effective when the right substance cannot be obtained in any other way and when it is safe for human and the environment. For example, antigens for making vaccines against non-cultivable microorganisms (plasmodium malaria, a causative agent of syphilis) can only be attained by genetic engineering. Genetically manipulated interferon is superior in activity to interferon, obtained from blood leukocytes, and much cheaper in comparison. Preparation of drugs from pathogen antigens of especially hazardous infections (plague, cholera) can be replaced by biosynthesis with their recombinant strains of nonpathogenic bacteria. The method of genetic engineering is progressively used in biology and medicine. This method will allow to realize new effective drugs, principally new polyvalent live (vector) vaccines, regulatory proteins, implemented in gene diagnostics and gene therapy. Genetic engineering offers the possibility to enhance the effectiveness of microorganisms used in production. A common method of increasing the yield of a useful product is amplification, i.e. an expansion in the number of genes copies. By amplification of genes in the composition of vectors highly efficient producers of threonine and proline are produced. Biotechnologically valuable products, including antibiotics, amino acids and vitamins are characterized in most cases by long and complex biosynthetic pathways, which is provided by dozens of different genes. The isolation of these genes and their amplification are often difficult to achieve. If the synthesis of antibiotics occurs in multi-enzyme complexes encoded by a single operon, the latter can be easily embedded in a suitable vector and cloned. If genes are scattered throughout the genome, an increase in the yield of the product is accomplished by cloning the genes, corresponding to complex biosynthetic sites. Strains can be constructed in the way of their adaptation to special conditions of technological process called technological engineering. Antibiotic producing operons can be transferred from slowly growing 46

Chapter 1. Biological systems are indispensable agents of industrial ...

streptomycetes to fast-growing bacteria, such as E.coli or B. subtilis. This would allow a much higher yield to be achieved through better control of cultivation conditions. The structural genes for the biosynthesis of certain antibiotics are usually concentrated in chromosomes, that they can be incorporated into plasmids and transferred to other actinomycetes or E.coli. Such plasmids were constructed on the basis of plasmids SLP1.2 S.lividans and SCP2 of S.coelicolor. Conventional types of fermentation can be improved by transferring the genes, encoding amylase of Aspergillus or Rhizopus, the glucose isomerase Streptomyces or the rennin Mucor into bacterial cells. The production of enzymes would become more economical, and the processes of fermentation more effective. The introduction amylase and glucoamylase genes into Saccharomyces cerevisiae cells ensures the production of alcohol from starch. A number of bacteria have a problem of not the synthesis of the product, but its excretion from the cell. Increasing the efficiency of transporting the product from the cell to the culture liquid accelerates the synthesis of new portions of the product, for example, amino acids. Intensifying the effectiveness of traditional methods is achieved with the help of localized (site-specific) mutagenesis in vitro, for example, not all genomes in the cells, but their fragments with the gene of interest, are treated with chemical mutagens. A biotechnologist, skilled with genetic engineering techniques, moves from gene to product; shifts nucleotide sequence of DNA, thereby controls changes in the corresponding protein, which is the fundamental of protein engineering. In this way, optimization of the structure of enzymes, hormones, vaccines-antigens can be realized. The inserting of heterologous genes and regulatory elements allows to construct new metabolic pathways with production-friendly characteristics. This approach is aimed to optimize cellular activity, and the change in the enzymatic, transport, regulatory properties of bacteria using recombinant DNA technology is called metabolic engineering. One of the first successes in this field was the production of a recombinant strain with altered nitrogen metabolism and used as fodder microbial protein. Assimilation of ammonia in methylotrophic bacteria Methylophilus methylotrofus occurs with the participation of 47

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aminotransferase and glutamine synthetase. The process runs with the expenditure of 1 mole of ATP per 1 mole of assimilated ammonia. Simultaneously, in E.coli only glutamate dehydrogenase (GDH) participates in ammonia assimilation and the process does not require ATP. In this regard E.coli GDH gene was cloned and introduced into glutamine synthetase-free mutant Methylophilus methylotrofus cells. When this gene was expressed the yield was 5% higher than in the case of the parent strain. Cloning with subsequent introduction into cells and expression of heterogeneous genes can be used to enhance the catabolic capabilities of the microorganism and, accordingly, the spectrum of the substrates used. For example, the lac operon of E.coli was chosen to create lactose-utilizing strains of Alcaligenes eutrophus, Corynebacterium glutamicus and Xanthomonas compestris. Optimization of strains can reduce the cost of production by acquiring the ability to grow on cheap substrates or more specific properties. This can be a change in the nutritional needs of the producer in order to expand the raw material base of the industry and increase the efficiency of conversion of the substrate. For example, after the introduction of appropriate genes to E.coli cells-producers of threonine, it started to utilize sucrose. Thus, genetic engineering opens great prospects for biotechnologists, connected both with the creation of fundamentally new producers of valuable compounds for humans, and with the increase in the efficiency of producers already used in production. At the moment, genetic engineering has mastered all the realms of the living. The main directions of the development of plant genetic engineering include: ‒ Enrichment of cultivated plants with additional reserve substances by means of genes taken from other plants; ‒ Increasing the efficiency of plant photosynthesis based on ribulose-1,5-bisphosphate carboxylase genes, chlorophyll a/bbinding proteins, etc. ‒ Changing nitrogen metabolism; ‒ Providing resistance to herbicides, salinization of soils, high and low temperatures, and other unfavorable factors of the environment. 48

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The current stage in the development of genetic engineering of plants is called metabolic engineering. Mainly, this is due to the fact that the goal is not only to improve certain existing qualities of the plant like in traditional breeding, but also how to manipulate the plant to produce completely new compounds really important in medicine, chemical production and other fields. These compounds can be, for example, special fatty acids, useful proteins with a high content of essential amino acids, modified polysaccharides, edible vaccines, antibodies, interferons and other «medicinal» proteins, as well as new polymers that are safe for the environment. Genetic engineering methods allow solving a number of critical tasks to advance the resistance of new forms, lines, varieties and hybrids of agricultural plants to various pathogens. However, genetic engineering manipulations with plants can lead not only to the expected results. For example, resistance to herbicides, caused by the transplantation of one gene, can cause serious problems in crop rotations; the herbicide-tolerant plant, cultivated on a given crop area will act next year to its successor crop as a weed against which herbicides are helpless. Another threat is biochemical changes caused by genetic modifications can lead to loss of food or fodder value by plants and even their acquisition of toxicity. This problem is inherent not only in genetic engineering, but also in traditional methods of selection. With the help of genetic engineering manipulations with animals, the genes of ß-globin mice, tyrosine tRNA of E.coli, thymidine kinase, and human ß-interferon in clones of insects and mammals have been cloned. The application of methods of genetic engineering in livestock farming is promising for changing a number of properties of the organism, like increasing productivity, resistance to diseases, expanding the growth rate, improving product quality. Thus, the creation and development of genetic and cellular engineering methods can artificially create new highly productive forms of organisms suitable for utilization in industrial scale. The introduction of genetic engineering into microbiological production led biotechnology to a new level of its development, 49

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allowing conscious and purposeful management of complex cellular processes. First, the productivity of industrial microorganism-producers of classical products has been significantly increased by initiating additional genes, raising their number or activity. Secondly, introducing new genes into the microbial cell made possible to change the nutritional demands of the microorganism. Further, microorganisms «taught» to synthesize substances that were not natural to them and thus increased the diversity of biotechnological products. Finally, the whole logic of selection of microorganisms-producers was reviewed. So, if previously an active strain of a microorganism was searched for and then a specific biotechnology, taking into account the physiological properties and nutritional needs of the producer, was created but now one can take the strain adapted to the production conditions and introduce a gene structure into it that will ensure efficient synthesis of the target product. The microbial producer was replenished with new sources (the culture of isolated cells and tissues of plants and animals) for obtaining useful substances. On this basis, new methods of biotechnology were created, and fundamentally new techniques for selecting eukaryotes were developed. Especially huge success was achieved in the field of microclonal reproduction of plants, as well as the production and use of transgenic plants and animals. Questions for thought and review 1. What is the fundamental difference between the methods of cellular and genetic engineering? 2. What are the necessary conditions for the implementation of genetic engineering? 3. What is recombinant DNA? 4. What are the main ways of creating recombinant DNA? 5. List the main types of enzymes used in the technology of genetic engineering, give a brief description of their purpose. 6. What is a vector? What can be used as vectors? 7. List the main stages of genetic engineering technique. 8. What natural ways of transferring genetic information are usually used to introduce the vector into prokaryotic cell? 9. What problems arise when designing new microbial producer using methods of genetic engineering? 10. How are genetically transformed microorganisms selected?

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Chapter 1. Biological systems are indispensable agents of industrial ... 11. By what methods are recombinant DNA inserted into the recipient cells of eukaryotes? 12. What are the main directions in the development of genetic engineering of plants. 13. What is the purpose of genetic engineering methods used in animal husbandry? 14. What is cellular engineering? What are the ways of its implementation? 15. What is «metabolic engineering»? 16. List the prospects for applying the methodology of genetic engineering in science and practice.

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CHAPTER 2

BIOTECHNOLOGICAL PRODUCTION IN INDUSTRY

2.1. Main stages of biotechnological manufacturing Biotechnology is the goal-directed production of valuable compounds and relevant services using biochemical activity of biological agents or their components, the system generally considered as a green technology. Advantages of biotechnological productions: ‒ The possibility of obtaining specific and unique natural substances, some of which still cannot be obtained using conventional way, like chemical synthesis or extraction; ‒ The biotechnological processes can be conducted at relatively low temperatures and pressures ‒ High rates of bio-agent growth and accumulation of biomass; ‒ One-step synthesis; complex compounds to be synthesized in one or very few processing stages; ‒ Use of cheap agricultural and industrial waste as raw materials; ‒ Usually environmentally friendly, produce less hazardous waste and are close to natural processes taking place in nature; ‒ Biotechnological products are biodegradable; ‒ The technology and equipment of biotechnological productions are simple, and also inexpensive. Biotechnological green processes, unlike chemical synthesis processes, are realized under mild conditions, normal pressure, active reaction and low ambient temperatures. Moreover, they are less likely to pollute environment with waste and by-products, less depend on weather conditions; do not require large land areas and xenobiotics, etc. Therefore, biotechnology is among the most priority areas of 52

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scientific and technological progress and is a prime example of sustainable technologies, which is connected with the prospects for the development of many industries. Biological technologies are currently in a phase of rapid development, but the level of their development is largely determined by scientific and technical potential of nation. The mission of any biotechnological process is the development and optimization of scientifically based technology and equipment. This process has a significant difference from chemical due to the fact that biotechnology uses more complex organization substances – biological systems. Every single biological object is an autonomous self-regulating system. The process of typical biotechnological production consists of a certain number of constituents and has different levels of complexity. These challenges are determined by the terms of a particular biotechnological process, which vary depending on the biological agents and final product. If the target product is biomass, such as SCP, then accordingly the technological line is shorter; if it is a substrate for the production of highly purified pharmaceuticals, then the production scheme is more complicated and the processing line is quite longer. If the source of target product is any microorganism, then aseptic conditions, appropriate equipment and right processing scheme for the process are required for its proper cultivation. In general, any biotechnological process involves three main stages: upstream processing (pre-fermentation), midstream processing (fermentation) and downstream processing (post-fermentation). The basic scheme for the realization of biotechnological processes in general form can be represented by flowchart (Fig. 2.1). At upstream processing, the inoculum (producer culture) is stored and activated, nutrient substrates or media, fermentation equipment, technological and recycled water and sterile air are prepared. Maintaining a pure culture of biological agents is very important point of upstream processing stage, since the producer, its physiological and biochemical characteristics and properties determine the effectiveness of the whole biotechnological process. The cultivation of inoculum involves scaling principles, that is, the producer's biomass is gradually increased in section containers, then in a series of fermenters. The resulting inoculum is sent further to the bioreactor in which the 53

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fermentation stage is realized. Preparation of nutrient media is carried out in special reactors equipped with agitators. Depending on the solubility and compatibility of media components, individual reactors can be used. The technology of media preparation is considerably complicated if they contain insoluble components. Different substrates are applied in various biotechnological processes, so the process of their preparation varies greatly. Dosing of nutritional components is selected and carried out individually at each production facility, weight and volume devices used as a dosing equipment in industrial scale. Transport of substances is carried out by pumps and conveyors. Bulk components are fed to the fermenters using vacuum pumps. Due to the exceptional diversity of biotechnological processes, media, methods and equipment employed for their implementation, these elements will be further related to specific biotechnological production.

Figure 2.1. The general process scheme for biotechnological production

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Chapter 2. Biotechnological production in industry

The fermentation stage is the key step in biotechnological process, as the bio-producer interacts with the substrate and the formation of the target products, including biomass itself, intra and extracellular products. This stage is conducted in a biochemical reactor and can be organized depending on the characteristics of producer used and the requirements for the type and quality of the end product (Fig. 2.2). Fermentation can take place in strictly aseptic conditions.

Figure 2.2. Flow diagram of a typical industrial biotechnological process

The downstream processing stage ensures the production of finished products and not least, the disposal of waste and by-products. Depending on the final product location in a cell and its nature, different equipment and techniques of isolation, purification can be applied. The first step of post-fermentation stage is the fractionation of culture liquid and separation of suspended biomass phase. The most common technique for this purpose is the separation, which is carried out in separators. In order to improve the efficiency of the separation process, preliminary special treatment (pH change, heating, addition of chemical agents, etc.) of culture is used. Depending on the properties of final product, various formulation methods are operated. For the stabilization the properties of biotechnological products, various substances are added as fillers, lubricants, dying ingredients, etc. 55

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The main elements that make up biotechnological processes are shown in Fig. 2.3:

Figure 2.3. The core elements of biotechnological processes

Biotechnological production based on the use of recombinant bioproducers has its own characteristics, which involves enhanced control over the stability of the product, and, in addition, precise and constant compliance with measures to prevent the microorganism from contaminating the environment. Such arrangements provide for the use of special equipment and the observance of certain rules relating directly to the technological regime. In modern biotechnological production, the most frequent biological agents are the strains of microorganisms grown in apparatusesfermenters (bioreactors) in various types. 56

Chapter 2. Biotechnological production in industry

The most important condition for successful bioconversion or fermentation is the sterility of the process. In real conditions to achieve, sterility is not easy. This is hindered by the large volume of fermenter and the complexity of the medium composition, as well as the large volume of air passing through it and the sophistication of fermenter design. Microbial contamination of culture medium during fermentation lead to a change in its composition, pH and rheological properties, which ultimately decreases the yield of desired product. The contamination can also cause by different micro-impurities, the compounds formed as a result of vital activity of extraneous microbial associations. Questions for thought and review 1. Describe the modern concepts of organization of industrial biotechnological productions. 2. What is the structural organization of biotechnological production. 3. What are the distinctive features of biotechnological production from traditional types of technology. 4. Name the advantages and disadvantages of biotechnological production in comparison with traditional technologies. 5. Give a typical scheme of biotechnological production. 6. Describe the main elements that make up biotechnological processes.

2.2. Sterilization in biotechnological industry The term «sterilization» is to be understood as the complete destruction of all viable organisms. Terminology is essential when the control of microorganisms is considered because some terms often are used mistakenly. ‒ Sterilization (Latin sterilis, unable to produce offspring) is the entire inactivation (distraction or removal) of all forms of microbial life (viable cell, spores, infectious agents) and acellular entities (viruses, viroids, and prions) in relation to the ability to reproduce. ‒ The chemical compound that used for sterilization is referred as sterilant. This concept should be distinguished from the concept of «disinfection». 57

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‒ Disinfection means only reducing the number of contaminating microorganisms to level of «safe», not causing infection, but not necessarily their complete destruction. ‒ Disinfectants are chemical compounds, used to conduct disinfection and normally applied only on not alive objects. Some spores and viable microorganisms remain indestructible after treatment of disinfectants. ‒ Sanitization is closely associated with disinfection. Here, the viable microorganisms are decreased to levels that are believed safe by public health standards. ‒ 6T6TSanitizer is an6T6T agent designed to 6T6Tkill germs, usually applied on6T6T 6T6Tfood-processing machinery and6T6T 6T6Tequipment.6T6T It also is often essential to control foreign cells on or in living tissue with chemical agents. ‒ Antisepsis (Greek anti, against, and sepsis, decay) is the prevention of infection, putrefaction or sepsis and is achieved by antiseptics, which are chemicals agents used to tissue to avoid infection by inhibiting germ growth. As the host tissue should stay protected, antiseptics are in most cases not as harmful as disinfectants. ‒ Chemotherapy is the application of chemical compounds to kill or inactivate the microbial growth of within host tissue. Various agents that inactivate organisms often have the suffix – cide (Latin cida, to destroy); thus: ‒ Germicide deprive of pathogen or non-pathogen existence (and many nonpathogens) but not certainly endospores. According to the particularly group of microorganisms, they are categorized as bactericide, fungicide, viricide and algaecide. ‒ Some chemicals agents do not totally inactivate, but rather inhibit growth. If these chemicals are removed, growth will continue and have the suffix –static (Greek statikos, causing to stop), examples; bacteriostatic, fungistatic and algistatic. Aseptic requirements in biotechnological processes Aseptic or sterilization is a network of efforts aimed at preventing contamination. It must be taken into account that any input or material flow is a potential source of microorganisms. 58

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Air dust or droplets of moisture in the air contain on its surface a layer of attached microorganisms or spores. The dust particles have different sizes and they are divided into three fractions: larger particles (>100 μm diameter), smaller particles (