Project of glucoamylase production by submerged cultivation of Aspergillus awamori

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НАЦІОНАЛЬНИЙ АВІАЦІЙНИЙ УНІВЕРСИТЕТ

Інститут екологічної безпеки

Кафедра біотехнології

ЗАВДАННЯ

на виконання курсового проекту

Тема курсової роботи:

Проект виробництва глюкоамілази шляхом глибинного культивування Aspergillus awamori. Відділення біосинтезу.

студентки Суслової Віолетти

2011

Тема курсової роботи: Проект виробництва глюкоамілази шляхом глибинного культивування Aspergillus awamori. Відділення біосинтезу

1. Термін виконання роботи: з 7.10.2011р. до 16.12.2011р.

2. Вихідні дані до роботи:

- фермент глюкоамілаза

- мікроорганізм продуцент виду Aspergillus awamori

- підігрівач для поживного середовища, що надходить у ферментер:

- тиск гострої пари - 4 кг/см²

- температура початкова - 30 ^оС

- температура кінцева - 130 ^оС

- кількість поживного середовища - 27 м³

- тривалість процесу стерилізації - 3 год

3. Етапи виконання курсової роботи

- опрацювання літературних даних 7.10.11 - 21.10.11

- розробка методики визначення 22.10.11. - 4.11.11

- написання основної частини роботи 5.11.11. - 17.11.11

- виконання креслень 18.11.11. - 2.12.11

- оформлення роботи та її захист 2.12.11. - 16.12.11

4. Завдання видав (доцент, к.т.н. Карпенко О.П.)

5. Завдання прийняв до виконання___

Курсова робота захищена з оцінкою

Голова комісії: д.б.н. Гаркава К.Г. 16.12.2011 р.

Члени комісії: доцент, к.т.н. Карпенко О.П. ___.

NATIONAL AVIATION UNIVERSITY

Institute Ecological Safety

Department of Biotechnology

TASK

on the execution of yearly project

student Suslova Violetta

The theme of course work: Project of glucoamylase production by submerged cultivation of Aspergillus awamori. Department of biosynthesis.

1. The term of work execution: from 7.10.2011- 16.12.2011

2. Initial data to the work:

- enzyme glucoamylase

- producer microorganism Aspergillus awamori

- Heater for nutrient medium supplied to fermenter:

- sharp steam pressure - 4 kg/cm²

- temperature initial - 30 ^оС

- temperature final - 130 ^оС

- quantity of nutrient medium - 27 m³

- duration of sterilization process - 3 h

3. Stages of yearly project creation

- processing handling of literature data 7.10.11 - 21.10.11

- elaboration of determination method 22.10.11. - 4.11.11

- writing of principal part of the work 5.11.11. - 17.11.11

- creation of drawings 18.11.11. - 2.12.11

- issuance of the work and its defense 2.12.11. - 16.12.11

4. Task was given by (associate professor Karpenko V.I..).

(sign of supervisor) (full name of supervisor)

5. Took task for execution_____.

The yearly project is defended with a mark__.

Chief of commission: Doctor Garkava K.G. 6.12.2011.

Members of commission: associate professor, Karpenko V.I..

ABSTRACT

Explanatory note for the yearly project “Project of glucoamylase production by submerged cultivation of Aspergillus awamori. Department of biosynthesis” contains 54 pages, 23 references, 2 drawings, 6 figures, 5 tables, 3 appendixes.

The purpose of this work is to investigate general method of producing enzyme glucoamylase by the most suitable method and conditions of cultivation that is submerged cultivation.

Investigation method - literature data processing, description of apparatus and of technological flowsheets of glucoamylase production in the department of biosynthesis, drawing up the equipment scheme of glucoamylase biosynthesis by Asp. awamori culture, calculation of heater for medium supplied to fermenter.

РЕФЕРАТ

Пояснювальна записка до курсового проекту на тему «Проект виробництва глюкоамілази шляхом глибинного культивування Aspergillus awamori. Відділення біосинтезу» містить 54 сторінки, 23 літературних джерела, 2 креслення, 6 рисунків, 5 таблиць, 3 додатки.

Мета роботи полягає в дослідженні загального методу виробництва ферменту глюкоамілази найбільш придатним способом та умовами культивування , а саме глибинним культивуванням.

Метод дослідження: обробка літературних даних, опис технологічної схеми виробництва глюкоамілази у відділенні біосинтезу, креслення апаратурної схеми біосинтезу глюкоамілази продуцентом Asp. awamori, розрахунок нагрівача для поживного середовища, що постачається у ферментер.

CONTENT

INTRODUCTION

1. LITERATURE REVIEW

1.1 Characteristics of final product

1.1.1 General notion about enzymes

1.1.2 Classification of enzymes

1.1.3 Characteristics of glucoamylase

`1.2 Characteristics of microorganisms producers of glucoamylase. Aspergillus awamori

2. TECHNOLOGICAL PROCESS

2.1 Grounds of choosing technological scheme

2.2 Description of technological scheme

2.2.1 Additional works

2.2.2 Technological processes

2.2.3 Micribiological and chemical control

2.2.4 Processing of waste water and air

2.3 Description of equipment scheme. Specification of equipment

3. DESCRIPTION AND CALCULATION OF HEATER FOR NUTRIENT MEDIUM

3.1 Description of heater

3.2 Calculation of heater

CONCLUSIONS

REFERENCES

Appendix

INTRODUCTION

Enzymes are biological catalysts that bring about chemical changes in substances. With the development of the science of biochemistry has come a fuller understanding of the wide range of enzymes present in living cells and of their modes of action.

Without enzymes, there can be no life. Although enzymes are only formed in living cells, many can be separated from the cells and can continue to function in vitro. This unique ability of enzymes to perform their specific chemical transformations in isolation has led to an ever-increasing use of enzymes in industrial and food processes, in bioremediation, and in medicine, and their production is collectively termed `enzyme technology'. Commercially produced enzymes will undoubtedly contribute to the solution of some of the most vital problems with which modern society is confronted, e.g. food production, energy shortage and preservation, and improvement of the environment, together with numerous medical applications.

The activity of an enzyme is due to its catalytic nature. An enzyme carries out its activity without being consumed in the reaction, and the reaction occurs at a very much higher rate when the enzyme is present. Enzymes are highly specific and function only on designated types of compounds - the substrates.

Glucoamylase is exoenzyme that attacks starch from the nonreducing end of polysacharide chain and fully convert starch into glucose. Some general properties at majority of glucoamylases of microbal origin are distinguished. Glucoamylase is widely widespread in the nature. It is synthesized by many microorganisms and forms in animal tissues, especially in a liver, kidney etc. Glucoamylase is used preliminary for starch hydrolyses in beverages production.

The use of microorganisms as a source material for enzyme production has developed because of different reasons such as there is normally a high specific activity per unit dry weight of product, seasonal fluctuations of raw materials and possible shortages due to climatic change or political upheavals do not occur, in microbes, a wide spectrum of enzyme characteristics, such as pH range and high temperature resistance, is available for selection, industrial genetics has greatly increased the possibilities for optimizing enzyme yield and type through strain selection, mutation, induction and selection of growth conditions and, more recently, by using the innovative powers of gene transfer technology and protein engineering.

The task is to make the best choice of microorganism for production of certain enzyme that gives the highest yield and requires the cheapest raw materials.

As producers of amylolytic enzymes most often use the molds of genera Aspergillus. Presently at the industrial receipt of foods of hydrolysis of starch - decstrose, glucose and fructose syrups, on the stage of saccharification mainly use glucoamylases of producers, related to the species Asp. awamori, optimal conditions of action of which рН 5.0 and temperature 55^oС. Aspergillus are typical obligate aerobs, therefore they can develop only on the surface of solid or liquid medium or in a liquid, aerated enough medium.

The production of glucoamylase is actual problem nowadays because of its ability to hydrolase the starch which then can be applied as low-price glucose source for lots of industries. The purpose of this work is to investigate general method of producing glucoamylase enzyme and to choose the most optimal way of its production.

1. Literature review

1.1 Characteristics of final product

1.1.1 General notion about enzymes

Enzymes are biocatalysts produced by living cells to bring about specific biochemical reactions generally forming parts of the metabolic processes of the cells, they act as catalysts in bringing about chemical changes in substances.

Enzymes are highly specific in their action on substrates and often many different enzymes are required to bring about, by concerted action, the sequence of metabolic reactions performed by the living cell. All enzymes which have been purified are protein in nature, and may or may not possess a nonprotein prosthetic group.

Enzymes occur in every living cell, hence in all microorganisms. Each single strain of organism produces a large number of enzymes, hydrolyzing, oxidizing or reducing, and metabolic in nature. But the absolute and relative amounts of the various individual enzymes produced vary markedly between species and even between strains of the same species. Hence, it is customary to select strains for the commercial production of specific enzymes which have the capacity for producing highest amounts of the particular enzymes desired. Commercial enzymes are produced from strains of molds, bacteria, and yeasts. [1]

With the development of the science of biochemistry has come a fuller understanding of the wide range of enzymes present in living cells and of their modes of action. Without enzymes, there can be no life. Although enzymes are only formed in living cells, many can be separated from the cells and can continue to function in vitro. This unique ability of enzymes to perform their specific chemical transformations in isolation has led to an ever-increasing use of enzymes in industrial and food processes, in bioremediation, and in medicine, and their production is collectively termed “enzyme technology”.

The activity of an enzyme is due to its catalytic nature. An enzyme carries out its activity without being consumed in the reaction, and the reaction occurs at a very much higher rate when the enzyme is present. Enzymes are highly specific and function only on designated types of compounds - the substrates.

Table 1. Application of enzymes in different industries

Industry segment	Enzymes	Chemical(s) replaced	Process(es)
Detergents	Lipases, proteases, cellulases, amylases	Phosphates, silicates, surfactants	High temperature, energy
Textile	Amylases, cellulases, catalases	Acids, alkali, oxidizing agents, reducing agents	Energy, reduced machine wear
Starch (i.e. high fructose, corn syrup, fuel ethanol, etc.)	Amylases, pullulanases, glucose isomerases	Acids	High temperatures
Leather	Proteases, lipases	Sulfides, surfactants	High temperatures
Feed	Xylanases, lipases	Phosphorus	Lower environmental phosphate and waste (manure) levels
Film silver recovery	Proteases		Recovery of silver from used film

The catalytic function of the enzyme is due not only to its primary molecular structure but also to the intricate folding configuration of the whole enzyme molecule. It is this configuration which endows the protein with its specific catalytic function; disturb the configuration by, for example, a change in pH or temperature, and the activity can be lost.

Because of their specificity, enzymes can differentiate between chemicals with closely related structures and can catalyse reactions over a wide range of temperatures (0-110^oC) and in the pH range 2-14. In industrial applications this can result in high-quality products, fewer by-products and simpler purification procedures. Furthermore, enzymes are non-toxic and biodegradable (an attractive `green' issue) and can be produced especially from microorganisms in large amounts without the need for special chemical-resistant equipment.

Enzyme technology embraces production, isolation, purification and use in soluble or immobilised form. [2]

Use of microorganisms as a source material for enzyme production has developed for several important reasons:

(1) There is normally a high specific activity per unit dry weight of product.

(2) Seasonal fluctuations of raw materials and possible shortages due to climatic change or political upheavals do not occur.

(3) In microbes, a wide spectrum of enzyme characteristics, such as pH range and high temperature resistance, is available for selection.

(4) Industrial genetics has greatly increased the possibilities for optimizing enzyme yield and type through strain selection, mutation, induction and selection of growth conditions and, more recently, by using the innovative powers of gene transfer technology and protein engineering.

Commercially produced enzymes will undoubtedly contribute to the solution of some of the most vital problems with which modern society is confronted, e.i. food production, energy shortage and preservation, and improvement of the environment, together with numerous medical applications.[3]

1.1.2 Classification of enzymes

Enzymes are divided into six main classes according to the type of reaction catalyzed. They are assigned code numbers which contain four elements separated by points and have the following meaning:

1. the number first indicates to which of the six classes the enzyme belongs,

2. the second indicates the subclass,

3. the third number indicates the sub-subclass, and

4. the fourth is the serial number of the enzyme in its sub-subclass.

The six classes are distinguished in the following manner:

1. Oxidoreductases

This class encompasses all enzymes that catalyze redox reactions. The recommended name is dehydrogenase whenever possible, but reductase can also be used. Oxidase is used only when O₂ is the acceptor for reduction. The systematic name is formed according to donor: acceptor oxidoreductase.

2. Transferases

Transferases catalyze the transfer of a specific group, such as methyl, acyl, amino, glycosyl, or phosphate, from one substance to another. The recommended name is normally acceptor group transferase or donor group transferase. The systematic name is formed according to donor: acceptor group transferase.

Hydrolases catalyze the hydrolytic cleavage of C-O, C-N, C-C, and some other bonds. The recommended name often consists simply of the substrate name with the suffix -ase. The systematic name always includes hydrolase.

4. Lyases

Lyases catalyze the cleavage of C-C, C-O, C-N, and other bonds by elimination. The recommended name is, for example, decarboxylase, aldolase, dehydratase (elimination of CO₂, aldehyde, and water, respectively). The systematic name is formed according to substrate group-lyase.

5. Isomerases

Isomerases catalyze geometric or structural rearrangements within a molecule. The different types of isomerism lead to the names racemase, epimerase, isomerase, tautomerase, mutase, or cycloisomerase.

6. Ligases

Ligases catalyze the joining of two molecules, coupled with the hydrolysis of a pyrophosphate bond in ATP or another nucleoside triphosphate.

Until 1983, the recommended name often included synthetase, but the current recommendation is that names of the type X-Y ligase be used instead, to avoid confusion with the name synthase (which is not confined to enzymes of class 6). The systematic name is formed according to X: Y ligase (ADP-forming). [4]

Table 2. Classification of enzymes

	Group	Reaction catalyzed	Examples
11	Oxidoreductases	To catalyze oxidation/reduction reactions; transfer of H and O atoms or electrons from one substance to another	Dehydrogenases Oxidases
22	Transferases	Transfer of a functional group from one substance to another. The group may be methyl-, acyl-, amino- or phosphate group	Transaminase, Kinases
33	Hydrolases	Formation of two products from a substrate by hydrolysis	Lipase, amylase, peptidase
44	Lyases	Non-hydrolytic addition or removal of groups from substrates. C-C, C-N, C-O or C-S bonds may be cleaved	Decarboxylase
55	Isomerases	Intramolecule rearrangement, i.e. isomerization changes within a single molecule	Iomerase, mutase
66	Ligases (synthesases)	Join together two molecules by synthesis of new C-O, C-S, C-N or C-C bonds with simultaneous breakdown of ATP	Synthetase

Hydrolases

1.1.3 Characteristics of glucoamylase

Glucoamylase (б-1,4-glucane-glucanehydrolase) is a glucose forming amylase, exoenzyme that attacks starch from the nonreducing end of polysacharide chain, consistently detaching glucose residues and fully converting starch into glucose. Some general properties at majority of glucoamylases of microbal origin are distinguished. As a rule, it is acid stable enzymes with the optimum of рН 4.5-5.0 and the optimum of temperature 55-60^oС.

Glucoamylase is widely widespread in the nature. It is synthesized by many microorganisms [Appendix B1] and forms in animal tissues, especially in a liver, kidney, placenta of bowels etc. An enzyme in literature is known under the different names: amyloglucosidase, б-amylase, lysosomal б-glucosidase, sour glucase, matulase and and exo-1, 4 -б-glucosidase. The distinctive feature of glucoamylase is ability in ten of one times quicker to hydrolyze high-polymerised subtrate, than olygo- and disaccharides. [5]

Besides specific б-1,4- glucane bonds glucoamylases hydrolyse б-1,6 bonds both in polysaccharides and low molecular olygosaccharides. Under their action starch is fully hydrolyze to glucose.

Ability of glucoamylase to hydrolyze both б- 1,4 and б- 1,6 bond connections with splitting off of glucose ( it takes place only in that case, when б -1,4-linkage follows б -1,6-linkage, therefore dextrane by them is not hydrolyze) put by this enzyme into first place due to efficiency of hydrolysis of starch with the purpose of further fermentation of appearing sugar. The deep hydrolysis of starch is needed in a spirit, starch and treacly, bakery industry.

Usually a hydrolysis is carried out in two stages: on the first stage conduct treatment with б -amylase (stage of dilution of starch), after this gelatinized and diluted suspension at a temperature not higher 60^oС and рН 5.0-5.5 process with glucoamylase (stage of saccharification of starch). As a result of the united action of enzymes the degree of hydrolysis of starch reaches 98-99%. [6]

A mechanism of attack of substrate with glucoamylase can be of two types: either one-chained or multiple attack, and active center has ubcentered structure. Almost all glucoamylases are glycoproteins, containing from 5 to 35% carbohydrates which consist of olygo-, di- and monosaccharides. A carbohydrate component can be an integral fragment or broken on individual compounds which attach to the protein through a threonine and serine. [7]

As a rule, natural microorganisms form the complex of amylolytic enzymes, able to hydrolyze plant substrates on the basis of starch carbohydrates. This complex includes б-amylases, and glucoamylases hydrolyzing molecules of starch to glucose and dextrins of different molecular mass; proteases, destroying the proteins of raw material to amino acid, being a valuable nitrous feed for yeasts; glucanases, which intensify the process of fermentative treatment of raw material due to the hydrolysis of unstarch polysaccharidess; pectinases, destroying a pectin, other enzymes of lytic action.

Received nowadays polyenzymatic preparations have different enzymatic composition and differentiate on the level of activity of separate enzymes. On the whole the known polyenzyme preparations with amylolytic activity need improvement of their functional descriptions, that will promote efficiency of their industrial application. Particular interest is presented by enzymatic preparations with the overactivity of glucoamylase. [8]

1.2 Characteristics of microorganisms producers of glucoamylase. Aspergillus awamori

As producers of amylolytic enzymes most often use the molds of genera Aspergillus species oryzae, usamii, awamori, batatae; of genera Rhizopus species delemar, fonkinsis, neveus, tonnensis, japonicum, topnineusis, and also separate representatives of Neurospora crassa and Mucor. Yeasts and yeast-like microorganisms of genera Candida, Saccharomyces, Endomycopsis and Endomyces also able to synthesize the enzymes of amylolytic action. Presently at the industrial receipt of foods of hydrolysis of starch - decstrose, glucose and fructose syrups, on the stage of saccharification mainly use glucoamylases of producers, related to the genus Aspergilllus: Asp. niger. Asp. awamori, Asp. oryzae, optimal conditions of action of which рН 5.0 and temperature 55^oС.

Aspergillus are typical obligate aerobs, therefore they can develop only on the surface of solid or liquid medium or in a liquid, aerated enough medium. Optimal temperature for majority of Aspergillus 25-30 °С, for some it is to 35 °С. The majority of molds at surface cultivation can undergo short-term increase of temperatures to 40 °C and even 45 °C without the noticeable loss of enzymes activity. Optimal humidity of medium for them is about 65 %.

The recombinant and mutant strains of glucoamylase producers' molds Aspergillus niger are known. Such strains are described: Asp. niger, synthesizing 150 unit/ml of glucoamylase; Asp. niger N 402, got on the basis of natural strain, contains 20 copies of gene of glucoamylase; Asp. niger B0-1. Asp. oryzae is a mutant that synthesizes both glucoamylase and amylase. The use of recombinant strains is related to the necessity of realization of permanent researches on maintenance of strains in the stable and active state, by creation of the special conditions of cultivations which not always are accessible at the industrial conduct of process.

Molds of species Asp.awamori for the saccharification of starch-containing raw material at the industrial receipt of dextroses, glucose and fructose syrups, ethanol are known and widely used.

Active enough in regard to the synthesis of glucoamylase from the known mold Asp. awamori is Asp. awamori 466, synthesizing 183 units/ml of enzyme at growth on medium with a corn-flour at the use of saccharification by malt milk and malt mash with diammonium phosphate during184 h of growth. Mycelium is strongly branched, with swelling, septate; a diameter of hyphae is 10-12 мm, the form of conidium is rounded cylindrical or irregular; diameter of conidium 4.4-6.4 мm, color - from olive-yellow to darkly-olive.[Appendix. A1]

Macroscopic descriptions: colonies on the Dox's agar of with a yeast extract, at 25^oС, have a diameter of a 70-71 mm/7days, radially grooved, surface velvety, edge thin, a conidial area is an umber; an exudate absent, back is dim-yellow.

Microscopic descriptions: conidial heads are spherical, disintegrating on separate columns, conidiophores weakly tinctured in terminal part, apical expansions are spherical 20-45 мm in a diameter, sterigmas are covered on all surface. Sterigmas are mainly double-level, metulas 6-16 х 3,5 -7 мm . Conidium is spherical, 3,5-6 мm.

The culture of strain assimilates glucose, saccharose, arabinose well, and poorly - maltose, lactose, lactoglucose and ramnose. Starch hydrolyze to glucose. Well assimilates ammoniacal salts of inorganic acids. It consumes a peptone, casein, amino acid, peptonizes milk.

The disadvantages of the described strain is a necessity for the receipt of high enough activity of cultural liquid, use of multiphase preparation of inoculum and enriched cultural medium for the basic fermentation process.[9]

The enzyme glucoamylase is commercially valuable biological product that is widely used in food and agricultural industry, that is for beverages and feed additives production. The most feasible and efficient method of this enzyme production is microbial synthesis. According to reviewed literature the best microorganism for glucoamylase production is mold Asp. awamori because of its high activity for biosynthesis.

general notion microorganism glucoamylase

2. Technological process

2.1 Grounds for choosing technological scheme

The biotechnological production of enzymes is realized by two methods - surface and submerged. The first method, applied for cultivation of molds is characterized by development of mycelium on a surface of solid or liquid substrate. The film of mycelium, producing not only amylolytic enzymes but also organic acids, inactivating them appears on liquid substrate, therefore solid substrates with the developed surface - wheat bran, pellet of grains, potato fiber and others are used. Maximal activity of enzymes is reached at cultivation of molds on wheat bran. The pellet of grains is poor in nutrient substances, and activity of enzymes in the cultures of molds, grown on it in 4-5 times lower, than on bran. The mature culture of molds in result of bran particles covering with mycelium looks like dense felt-like mass.[10]

Solid surface fermentation consists in growth of producer on the surface of thin layer of solid loose medium. Submerged fermentation in a liquid medium can be realized both in the conditions of batch process and with the use of the flowing systems.

During surface fermentation for the receipt of inoculum spore material is propageted by a superficial method or museum culture is grow in the conditions of submerged liquid culture. Further inoculum is sent to the stage of fermentation, which comes true on the surface of loose medium in metallic trays or vertical perforated cuvettes. A culture develops on the surface of solid loose medium, the basis of which is wheat bran, grain-growing husk, being the source of growth substances. For loosening of medium the arboreal sawdusts (5-10 %), and oat husk is added in brans. Mixture before autoclaving is moistened to 20-40 % humidity and is acidified for the improvement of sterilization conditions.

In medium a sterile termolabile components, inoculum (0.02-0.1 % from mass of medium) are added, quickly mix manually and lay out in trays with thickness 2-3 cm, which set in the impermeable aerated chambers, preliminary sterilized. Initial humidity of medium is 58-60 %, temperature of cultivation 28- 32°, duration of fermentation about 36-48 hours. [7]

Mycelium obduce and firmly bind solid particles of medium, therefore for a normal transport and oxidation of substances the medium must be loose enough and moist. The effective transport of oxygen from a gas phase and dissolution in a medium takes place on condition of good aeration of thin layer of solid loose medium. It results contribution large volumes of floorspaces. A surface method of fermentation is an extensive method with large volumes of manual work.

Surface fermentation with the use instead of trays of cuvettes is more perfect. A construction provides more effective aeration and allows partially mechanize a process. The column vehicles with volume aeration applied in industry yet more improve the process of solid state fermentation. Such apparatus is divided on a section by the perforated plates, fixed on turning axes. A medium during fermentation is loosened by means of the revolving agitating devices. It allows to increase the height of layer to 30 cm. Mode of overload of medium on plates is set automatically. The productivity of apparatus reaches at a 1 ton of culture per day.

In workshops on the production of surface culture in order to avoid an infection by extraneous microorganisms is maintained the special cleanness. The prepared culture always contains the small amount of spores because of the unsimultaneous maturation in separate areas, that's why workers, directly contiguous with it, must put on gauze bandages, and on hands - rubber gloves. Unloading of cuvettes and crushing of molds culture must be conducted in bottletight chambers, provided with aspiration devices.

The surface method of molds growth has a number of advantages. Because during the growth of molds bran are not mixed, extraneous microorganisms do not spread on all their mass and cause the only insignificant local infecting, which, as a rule, does not influence on activity of enzymes. It, however, does not eliminate the necessity of careful sterilization of medium and equipment. A culture on bran is dried out to content of moistures 10-11%. In such state it can be kept long time without a considerable loss of activity.[11]

It allows organize the centralized providing of biotechnological plants with the dry culture of molds that is one of advantages of surface method of fermentation. A disadvantage of surface method of cultivation is a necessity of setting of great number of cuvettes, work with which it is difficult to mechanize. The prime price of culture of mold-producer is high, thus mainly from the expense of plenty of hand labour. Mechanization of process of cultivation is possible by creation of continuous-action apparatus or cuvettes free vehicles with the vertical thick layer of nutrient medium and intensive blowing of air through this layer.

The submerged culture of microorganisms grow on a liquid nutrient medium at the vigorous aeration in bottletight apparatus and in sterile conditions. A process is fully mechanized. Sterility of submerged culture of microorganism-producer of enzymes positively affects results. The next methods of submerged cultivation are known: periodic, continuously-cyclic and continuously-flowing.

A periodic method is characterized by irremovability of nutrient medium in a fermenter, composition of which in the process of development changes gradually. At continuously-cyclic method microorganisms, located on immobile attachment in a fermenter, are washed by medium, flowing in the reserved contour, to the complete consumption by them nutritives. Enriched with nutritives medium during such cyclic fermentation is gradually exhausted; at times medium stays in the area of reaction this process is more long, than periodic.

The continuously-flowing method of cultivation of microorganisms is more perfect. Essence of it consists in that microbial population develops in a flowing nutrient medium. A method has two varieties: homogeneously-continuous and gradient-continuous. In first case growth conduct in one fermenter; at careful interfusion and aeration of medium the identical state of culture is provided in all volume of liquid. In a fermenter continuously fresh medium is supplied and from it continuously flows out an excess of cultural liquid.

Gradient-continuous cultivation is carried out in the battery of fermenters, connected by downpipes. The inoculated medium with large content of carbohydrates and other components continuously flows from one fermenter in other and also continuously flows out as the finished culture.

By continuous cultivation in flowing mediums it is possible to grow microorganisms in conditions optimal for their stages of development. Thus such important factors, as concentration of nutritives, amount of products of exchange, рН, content of dissolved oxygen, sharply changing at a periodic method of cultivations, are maintained permanent on set level or change by worked out program. [12]

A nutrient medium for fermentation is prepared based on physiological necessities of the used microbial culture, and also from the type of aimed enzyme.

The synthesis of enzyme in a submerged culture flows during a 3-4 days at the continuous supply of sterile air, stabilizing of рН and temperatures of medium on strictly certain levels. The insignificant changes of values of these parameters can cause the frequent decline of fermentation activity. After completion of fermentation for prevention of inactivation of enzymes cultural liquid is cooled and is directed to down stream.

Basic difficulty in realization of continuous cultivation is a large danger of infecting, and necessity of frequent shutoff for realization prophylactic sterilization.

Table 3. Comparison of surface and submerged cultivations

Surface	Submerged
Requires much space for trays Requires much hand labor Uses lower pressure air blower Little power requirement Minimum control necessary Little contamination problem Recovery involves extraction with aqueous solution, filtration or centrifugation, and perhaps evaporation and/or precipitation	Uses compact fermenters Requires minimum of labor Requires high pressure air Needs considerable power for air compressors and agitators Requires careful control Contamination frequently a serious problem Recovery involves filtration or centrifugation, and perhaps evaporation and/or precipitation

At submerged cultivation microorganisms develop in all volume of liquid nutrient medium. Because majority of producers of enzymes is obligate aerobs, a medium is intensively aerated. In microorganisms occures two indissolubly constrained processes that is a synthesis of biomass and synthesis of enzymes. [1]

For the maximal accumulation of enzymes certain composition of nutrient medium, providing of air with Oxygen, timely taking off of metabolites and physiological heat, optimal values of рН and temperatures is needed. A major condition is also sterility of nutrient medium, supplied air, fermenters, pipelines and fittings.

So, the best method for the production of glucoamylase by cultivation of Asp.awamori is submerged fermentation, because of easier controlling of parameters, minimal requirements of hand labor, low cost of raw material and possibility of sufficient providing medium with Oxygen due to requirements of aerobic culture.

2.2 Description of the technological scheme

Technological process of glucoamylase production in department of biosynthesis include the next main stages:

· Additional works

· Preparation of inoculums

· Fermentation [ Apendix C]

2.2.1 Additional works

Preparation of equipment

At surface cultivation it is necessary sterilize an apparatus for preparation of inoculum (capacities for inoculation, cuvettes, capacity for water, for preparation of inoculum suspension, inoculums communications). Sterilization of cuvettes and glassware in an inoculation department is conducted by dry steam at a temperature 160 °C no less than 60 min Apparatus and communications are sterilized by sharp steam at a temperature 105-120 °C and excess pressure 0,05-0,1 МPа.

Apartments, especially inoculation boxing, is sterilized by irradiation by means of the special bactericidal lamps. Sterilization of apparatus and communications has significant mean at the submerged method of cultivation. The most careful sterilization can not give an effect, if impermeability of equipment is broken. [12]

All valves before setting check up by hydraulic compression at pressure 0,3 МPа. Impermeability of connections is checked up at excess pressure of steam 0,15- 0,2 МPа. The special attention is made to sterilization of apparatus and communication for the serve of the defoamer. Sterilization of these knots is conducted at 125-135°C during 1,5-2 h. On the stage of sterilization permanent microbiological control of sterility of nutrient medium, air supplied to fermenter, defoamer etc is conducted.

In process of fermentation for defoaming in apparatus liquid defoamer is supplied. For receiving 0,05% emulsion of defoamer, in a capacity bring in its concentrate, then dilute it to necessary concentration. Emulsion of defoamer is sterilised in the special vehicle of batch-type at temperature 123±2°C during 30 min in order to avoid bringing with it infections to medium. After sterilization defoamer is cooled in the same apparatus to temperature 30-32°C, then supply through a metering device in a fermenter and inoculator.[7]

On a microbiological sterility check up the department of sterilization, its walls and floor, apparatus, communications, and also hands of workers.

Preparation of air

The producer of glucoamylase enzyme Asp.awamori is an aerobe, and for its normal development in the process of cultivation it is necessary to give sterile air in a sufficient amount. Especially high demands to sterility is required at preparation of air for aeration of submerged culture.

There are a few methods of cleaning and sterilization of air, based on two principles: killing of microorganisms and their mechanical separation. Preparation of air for aeration is conducted as follows:

-cleaning air from rough mechanical suspended particles (viscin filters)

-preliminary conditioning to the necessary temperature

- air supply in a compressor

-thin cleaning of air from microorganisms (head filter)

-final cleaning in individual filter.

On the stage of pre-cleaning of air the bulk of large dust particles with the diameter 5-10 мm is removed. As filters of pre-cleaning use oily filters. For a compression and injection of air use turbo-compressors in which the compression of air takes place under the action of centrifugal force. The compression of air is accompanied by its heating to 220^oС. Therefore after compressors air enters refrigerator. To delete excess moisture from air, it must be cooled.

Further air enters head filter of КБ ВНИИФСа, that is a steel cylinder with a spherical bottom and sectional lid. Inside it the nets between which fiberglass filter material is fixed are located. A filter is sterilized by steam with pressure 0,2 МPа at 133°C during 3 hours. Reupholstering of head filter is made once per 2-3 months. [13]

Then cleared air enters individual filters for the thin cleaning and given for aeration of growing culture in an inoculator and fermenter. For a fermenter the filter of ЛАИК СП6/ 15 is used, for inoculums - filter ФТО - 60. Filter material, used for the filters of the thin cleaning, has a coefficient of skip 1?10⁹ %, that provides the required sterilization of air, necessary for development of microorganisms. Filters are sterilized with steam.[7]

Preparation of water

For preparation of medium water is taken from water supply system, artesian mining holes or open reservoirs after corresponding treatment. It must be biologically clean (ГОСТ 2874-82), colourless, without taste and smell, must not give precipitate. The dry residue of water must not exceed 1000 mg-eq/l, general hardness must not be more than 7 mg-eq/l. Too hard water slows the growth of microorganisms, because dissolved in it substances are not taken into account in composition of nutrient medium for a certain culture. [13]

It is necessary to check up chemical composition of water. Maintenance of gypsum is undesirable if there are more than 0,5 g/l and presence of salts of ammonium, because they testify that decaying processes take place.

General quantity of microorganisms in 1 ml of water must not be more than 100. In microbiological industry water is used not only for preparation of mediums but also for washing of apparatus, systems of cooling, etc. [14]

Values must not exceed next concentrations:

Table 4. Contents of harmful substances in water

Substance	Content, g/l
Lead	0.1
Arsenic	0.05
Fluorine	1.5
Zink	5.0
Copper	3.0

In the real time by a basic normative document, qualificatory quality of drinking-water, there is ГОСТ 2874-82 "Water drinkable. Hygienic requirements and control after quality". From 01.01.2000 in Ukraine a new normative document is put in an operation the State sanitary rules and norms (ДСанПіН) №383 (186/1940) "Water drinkable. Hygienic requirements to water quality of centralized household water use". In a microbiological production presence of clean water in large quantity has a great value.[15]

Preparation of disinfectants

The basic requirement to the desinfectants is the effective removing of all kinds of dirtying and contamination; therefore they must possess moistening, emulsifying and dispersive ability. In addition, they must be easily washed off after cleaning of surface, not corrode equipment and be harmless for operating personnel.

In the production of enzyme preparations for washing of equipment surfactants are used, mainly. From alkaline cleansers most widely used: caustic soda, calcinated soda, tripotassium sulphate.

Caustic soda (ГОСТ 2263-59) is used for washing of apparatus and alkalizing of medium. Solid caustic soda must contain no less than 92-96% of caustic soda, liquid - no less than 42-50%.'

Caustic soda is an effective mean for removing of organic admixtures. It is a colourless crystalline substance which dissolves in water good, forming solutions with high pH. Hot 2 - 3-% solutions of caustic soda hydrolyze proteins well, slit carbohydrates, possess a bactericidal action. For washing of equipment it is possible to use 1 - 2% solutions. The lack of caustic soda is its strong corrodible action. For preparation of 1 - 2% solution of caustic soda 1 - 2 kg of it dissolve in a hot water and bring to a volume to 100 l.

Soda calcinated (sodium carbonate). Used for washing of apparatus and alkalizing of environment. A synthetic soda (ГОСТ 5100-64) is used in a microbiological production, containing 96,8% chemically clean substances.

The calcinated soda is weaker alkaline mean in comparison with a caustic soda. It is white fine-crystalline powder, good water-soluble. Hot solutions of the calcinated soda saponify fats and hydrolyze proteins well. For washing of equipment apply 1 - 2% solutions with a temperature 70 - 80 °C (1 - 2 kg of soda dissolve in hot water and bring to a volume 100 l).

As desinfectant large distribution in food industry have the next groups of chemicals: phenols, chlorine containing preparations, formaldehyde and quaternary ammonium compounds (QAC).[16]

Formalin is 35 - 40% aquatic solution of gas of formaldehyde. Possesses bactericidal, sporicidal and fungicide action. In 5-% solution of formalin spores perish through 30 min, in a 2-% - through 60 min, in a 1-% - through 2 h.

Antiformin is the combined disinfectant, contains in 1 m³ of 100 kg solution of chloric lime, 75 kg of the calcinated soda and 10 kg of caustic soda. Solution of chloric lime (100 kg per 400 l of water) at 60°C pour in solution of the calcinated soda (75 kg per 500 l of water) and to this mixture add solution of caustic soda (10 kg per 75 l of water). Mixture is settled by 12-24 hours and before the use dissolve with water in ratio 1: 30.

Limewater is applied for disinfection of walls of shopfloors and storages. Sometimes 0,5 - 2% solution of chloric lime or 3-%t solution of formalin is added to it. For the receipt of limewater one part of quicklime is dissolved in nine parts of water. Ammonia or ammoniac water is used as a source of nitrogen and regulator of medium рH. The ammonia of I brand contains no less than 25%, and an ammonia of II brand is no less than 20% nitrogen. [17]

Preparation of nutrient medium

Nutrient medium is prepared, using corn mash with the concentration of dry substances 18-20%, which is received due to the next scheme: corn-flour through a portion automatic scales are loaded in a mixer, in which simultaneously and during permanent work of mixer water with a temperature no more than 45° С is supplied. Ratio of flour and water 1: (2,5-3,0).



Received mass is pumped with a pump in a boiling apparatus, working under the pressure, mass in a vehicle is heated by sharp steam, which is entered through the bottom. Boiling soft is produced at temperature148-154°C and pressure 0,36-0,44 МPа 15-20 min.[12]

To boil soft a flour is possible both in the vehicles of batch-type and by continuous method in the boiling apparatus of tube-type, to which steam is supplied by pressure no less than 0,5 МPа.

The boiled soft mass enters saccharifier, equipped with a worm-pipe for cooling. Before blowing of mass in a saccharifier water in an amount 5% to the volume of mass is added.

The boiled soft mass is cooled to 63°C, then saccharify with malt milk, which is received by mixing in the malt vats of the malt ground up on a maltcrusher and water. Correlation of malt and water 1: (6-8). Duration of saccharification at a temperature 58-60^oC during 30 min.

Got wort with the concentration of dry substances 18-20% and рН 5,3-5,6 by a pump is feeded on a contact head, pumped through it, warmed up by sharp steam to 85°C and given in a sterilizer 15.



Sterilization of nutrient medium is carried out on setting, including a contact head, tubular holder and heat-exchanger.Before sterilization of medium the system is checked for impermeability by steam under the pressure 0,20- 0,25 МPа. At finding out leaking of steam pressure is reduced to zero, defects are removed and the system is again checked up on hermeticity. During the complete pressurizing the system is sterilized by sharp steam during 30-40 min at pressure 0,2-0,25 МPа. After termination of sterilization of the systems proceed the sterilization of medium. Nutrient medium with temperature 75-80°C by a piston pump given through a contact head, where heated to the temperature 120-125°C, in a tubular holder-sterilizer, where 30-40 minutes is maintained, then cools down in heat-exchanger to 35°C and enters the fermenter.

During work of heat-exchanger the by-pass line, valves of water and medium supply must be under steam defence. In absence of heat-exchanger a nutrient medium directly from holder enters fermenter. In the process of its filling pressure 0,10-0,12 МPа is supported in a vehicle. Sharp steam at this time is given

only through the system of aeration. Medium cools down directly in fermenter. [18]

After filling of fermenter all system is released from medium, pump with a water for removing of suspended particles of nutrient medium and sterilize with sharp steam during 30-40 min at pressure 0,20-0,25 МPа. Liberation and puming of the system is carried out in an agitation tank. Water discharge must make 2-3 volumes of the system.

2.2.2 Technological processes

Preparation of inoculum

For inoculation of production nutrient medium at submerged cultivation inoculum is also prepared by a submerged method.

The receipt of inoculum is carried out by the phasic increase of mass culture of producer. At small productivity of workshops it is taken to one or two operations, and for the plants of the large productivity there is a sequential process.

For Аsp. awamori there are such stages of inoculums preparation:

1. Test tube with an initial culture on agar nutrient medium.

2. Subculturing of water suspension of culture in retorts with a liquid nutrient medium containing 5% of corn-flour and 0,5% of the autolyzed yeast, cultivation on shake-flask propagator during 48 h.

3. Subculturing of culture in vessels with the capacity 6 l (quantity of inoculum 10-12% to the volume of nutrient medium), cultivation during 48 h.

4. Subculturing of culture in an inoculator on corn wort with concentration 6%, cultivation during 48 h. at a temperature 27°C, interfusion with mixer with frequency of rotation 950 rotations/minute and with the air supply 16 m³/(m³/h)

5. Subculturing of culture into productive fermenter (3% of inoculums to the volume of nutrient medium)

The inoculums material of Asp. awamori mold is prepared in laboratory in test tubes on agar nutrient medium with the next content (in %):

Glucose	2.0
Sodium nitrate	0.91
Potassium chloride	0,05
Magnesium sulphate	0.05
Dihydroorthophosphate	0.10
Iron sulphate	0.001
Distilled water	96.84

Medium is sterilized during 40 minutes at pressure 0,1 МPа.

The culture of mold is inoculated on slant medium in test tubes and grow during 12 days in a thermostat at 25° С. The prepared culture must have a characteristic for species color and folded surface. It is used for preparation of liquid inoculating material.[19]

Preparation of liquid inoculume.

A liquid inoculum prepares in a few stages. By a three-phasic scheme on the first stage prepare the nutrient medium of the next content (in %) :

Corn flour	5.0
Yeast autolysate	0.5
Water	94.5

With the dilute solution of sulphuric acid рН of medium is reached to 4,8. The prepared medium is poured out in shake-flask propagators on 500 ml per 300 ml in each and sterilize at a temperature 121-125°C during 40-60 min. After sterilization medium is cooled to 26°C and is inoculated with the suspension of conidia received at growing of culture on the slant agar medium in tests tubes. Flasks stand on shake-and-flask propagator. Growing is conducted at a temperature 24-26°C during36-48 h at frequency of vibrations of shake and-flask propagator 200-220 min^{- 1}.

After 36-48 h (due to the conclusion of microbiologist) a liquid culture is passed on the second stage. Incubated liquid inoculum of the second stage is the inoculums for incubation of sowing culture. The volume of inoculums should be 0.5 - 1.0 % to volume of nutrient medium in inoculator.

Inoculation of medium in fermenter

Before sowing from fermenter take the tests of medium through the sampler for the microbiological inoculation and biochemical analysis.

For sowing close a valve on an output air-track at a sowing vehicle and lift pressure to 0,06-0,08 МPа, and in fermenter remains pressure 0,02-0,03 МPа. After this open a valve on the line of pressing at a sowing vehicle and fermenter and due to the difference of pressure press inoculum into a fermenter.

Close a valve on the line of pressing, turn on a mixer and begin the process of culture growing in fermenter. After termination of cultivation the line of pressing is steamed thoroughly. The amount of inoculum is 3% to the volume of nutrient medium in fermenter. [1]

Receive of Asp. awamori culture

Process of growing of submerged culture of Asp.awamori in a production is carried out in fermenters from stainless steel in sterile conditions at continuous agitation and aeration of medium. Fermenter in which grows the submerged culture is provided with a shirt for heating and cooling, with an aerating device, jets for steam supply, inoculating and drain lines, bushing for a manometer and thermometer, sampling knot, anti-foaming tank and individual air filter.[20]

For microbiological and biochemical control of development of culture with the observance of all terms of sterility tests are taken from fermenter after 72 h after inoculation, and then every twenty-four hours of growth. In tests glucoamylase activity, рН, concentration of dry substances, state of culture and absence of extraneous microflora at microscopy is determined. [21]