Project of glucoamylase production by submerged cultivation of Aspergillus awamori
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
Тема курсової роботи:
Проект виробництва глюкоамілази шляхом глибинного культивування Aspergillus
awamori. Відділення біосинтезу
. Термін виконання
роботи: з 7.10.2011р. до 16.12.2011р.
. 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
. 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.)
. 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
Ecological Safetyof
Biotechnologythe execution of yearly projectSuslova Violettatheme of course
work: Project of glucoamylase production by submerged cultivation of
Aspergillus awamori. Department of biosynthesis.
. The term of work
execution: from 7.10.2011- 16.12.2011
. 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_____.yearly project is defended with a mark__.of commission: Doctor
Garkava K.G. 6.12.2011.of commission: associate professor, Karpenko V.I..
ABSTRACT
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.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.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
. LITERATURE REVIEW
.1 Characteristics of final product
.1.1 General notion about enzymes
.1.2 Classification of enzymes
.1.3 Characteristics of glucoamylase
`1.2 Characteristics of microorganisms
producers of glucoamylase. Aspergillus awamori
. TECHNOLOGICAL PROCESS
.1 Grounds of choosing technological
scheme
.2 Description of technological scheme
.2.1 Additional works
.2.2 Technological processes
2.2.4 Processing of waste water and air
2.3 Description of equipment scheme.
Specification of equipment
. DESCRIPTION AND CALCULATION OF HEATER
FOR NUTRIENT MEDIUM
.1 Description of heater
.2 Calculation of heater
INTRODUCTION
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.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.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.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.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.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.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 55oС. 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.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 Characteristics
of final product
.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.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.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]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”.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
|
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.of their specificity, enzymes can differentiate
between chemicals with closely related structures and can catalyse reactions
over a wide range of temperatures (0-110oC) 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.technology embraces production, isolation,
purification and use in soluble or immobilised form. [2]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.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:
. the number first
indicates to which of the six classes the enzyme belongs,
. the second indicates
the subclass,
. the third number
indicates the sub-subclass, and
. the fourth is the
serial number of the enzyme in its sub-subclass.six classes are distinguished
in the following manner:
. Oxidoreductasesclass
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.
. Transferasescatalyze
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.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.
. Lyasescatalyze 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.
. Isomerasescatalyze
geometric or structural rearrangements within a molecule. The different types
of isomerism lead to the names racemase, epimerase, isomerase, tautomerase,
mutase, or cycloisomerase.
. Ligasescatalyze the
joining of two molecules, coupled with the hydrolysis of a pyrophosphate bond
in ATP or another nucleoside triphosphate.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
<#"555834.files/image001.jpg">
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]a rule, natural microorganisms form the complex of
amylolytic enzymes, able to hydrolyze plant substrates on the basis of starch
carbohydrates. This complex includes α-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.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]
.2 Characteristics of microorganisms producers of
glucoamylase. Aspergillus awamori
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 55oС.
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.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.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 μm, the form of
conidium is rounded cylindrical or irregular; diameter of conidium 4.4-6.4 μm, color - from
olive-yellow to darkly-olive.[Appendix. A1]descriptions: colonies on the Dox’s
agar of with a yeast extract, at 25oС, 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.descriptions: conidial heads are spherical, disintegrating on
separate columns, conidiophores weakly tinctured in terminal part, apical
expansions are spherical 20-45 μm in a diameter, sterigmas are covered on all surface. Sterigmas
are mainly double-level, metulas 6-16 х 3,5 -7 μm . Conidium is spherical, 3,5-6 μm.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.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]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
.1
Grounds for choosing technological scheme
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]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.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.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]
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.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.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.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.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.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.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
|
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., 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 Description of the technological scheme
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
equipmentsurface 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., 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]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.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]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.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.the stage of pre-cleaning of air the bulk of large dust
particles with the diameter 5-10 μm 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 220oС. Therefore after
compressors air enters refrigerator. To delete excess moisture from air, it
must be cooled.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]
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]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.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]must
not exceed next concentrations:
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.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.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%.'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.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.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).desinfectant large distribution in food industry have the next
groups of chemicals: phenols, chlorine containing preparations, formaldehyde
and quaternary ammonium compounds (QAC).[16]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.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.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).
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]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.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 58-60oC 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.
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.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
giventhrough 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 Technological processes
Preparation of inoculum
For inoculation of
production nutrient medium at submerged cultivation inoculum is also prepared
by a submerged method.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.Аsp. awamori there are such stages of inoculums preparation:
. Test tube with
an initial culture on agar nutrient medium.
. 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.
. 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.
. 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)
. 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
|
0.10
|
Iron sulphate
|
0.001
|
Distilled water
|
96.84
|
is sterilized during 40
minutes at pressure 0,1 МPа.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.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
|
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- 1.36-48 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.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.
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]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]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]
process
of fermentation proceed at next conditions:
Temperature of medium
in fermenter, oC
|
35
|
Method of agitation
|
double-level turbine
stirrer
|
Frequency of mixer
rotations, min-1
|
150-170
|
Quantity of air
supplied, m3 /m3 of medium/hour
|
no less than 30-60
|
Pressure under the lid
of fermenter during the growth, MPa
|
0.02-0.03
|
Temperature of air at
inlet to fermenter , oC
|
35-40
|
prepared
culture must satisfy the next requirements:
Glucoamylase activity
units/ml
|
no less than 200
|
Concentration of dry
substances in the filtrate of culture, %
|
8-10
|
pH
|
3.0-3.5
|
presence of extraneous
microflora
|
absent
|
.2.3 Micribiological and chemical control
Regardless of method of
cultivation from the moment of inoculation with producer a sterile nutrient
medium the control for growth of culture and production of enzymes is
conducted. For every species of producer and method of cultivation, the
periodicity of selection of middle tests of growing culture is set. The
selected tests are exposed to microscopy and visual examination. With the
purpose of exposure of possible infections the periodic inoculation of tests on
agar media with introduction of factors, repressing the growth of producer is
made. Determination of accumulation in the culture of fermentative activity is
constantly conducted. At submerged cultivation control for the consumption of
basic limiting substrates of medium (carbohydrates, N, Р) is conducted, рН of
culture is measured.indexes of growth of culture, the changes of composition of
medium and accumulation of enzymes, etc. are added to the laboratory
magazine.all stages of selection of enzymes conduct the analyses of activity,
the sizes of losses and output of commodity product is determined. The prepared
preparations of enzymes expose to especially careful research, especially those
which are used in medicine and in foods. Spores or cells of producer must be
absent in the prepared product, and a maximum norm of semination by microflora
is determined in every case.liquid and dry enzymatic preparations are analysed
on activity of enzymes, contents of dry substances, presence of microbial
contamination. At control of the high-cleared preparations besides
determination of contamination with microbes and activity of enzymes analyses
on content of protein, ash elements, carbohydrates and other specific
properties of enzymes is conducted. [1]
2.2.4
Processing of waste water and air
At submerged cultivation
of glucoamylse producer Asp. awamori the waste products are air and water,
which contain spores of the cultivated microorganism. At submerged cultivation
of microorganisms in fermenters amount of air, outgoing for fermenter per 1
hour hesitates from 20 to 60 m3 per 1 m3 of medium.air
with relative humidity 90-95% and temperature 35-36°C it is necessary to render
harmless in scrubbers with an antiseptic. The variant of rendering of air
harmless is possible by the use of it in heating of steam-boiler.of major
measures, reducing emission of microorganisms in an environment is pressurizing
of fermenters, flotators and equipment of separation knot. On the row of
enterprises of fermentation type the high-efficiency cleaning of exhaust air
from fermenters, flotators, knots, drying settings and packing department is
carried out by means of Venturi’s scrubbers. The final separation of liquid
from gas is carried out in centrifugal scrubber. The cleared gas is thrown out
in an atmosphere, and water with solid particles is taken out from an inertia
vehicle and scrubber in collector. Cleaning of air to the clean or sterile
state is possible to carry out by means of filters of the rough and thin
cleaning or by incineration. In a number of cases decrease of harmful
discharges in an atmosphere is possible to attain by perfection of technology.
[1]at the production of submerged culture are divided by two categories:
working water from heat-exchangers, from the shirts of manifolds of fermenters
etc. and productively polluted waters.from washing of technological equipment
(of inoculating apparatus, fermenters and product communications) gathers in
collector and before supply on a biological purification is exposed to
sterilization. Water is given by a centrifugal pump on a contact head, where
heated to 126-130°C, and maintained during 1 h in a pipe holder, after cools
down to 30°C in heat-exchanger and is directed for a bioscrubbing.from washing
of boiling apparatus, discharge vats, saccharifier, agitation tank sterilizer,
heat-exchanger, washings of floors are directed on a bioscrubbing. Flows from
water closets and shower-bath are sent in a sewerage network.infected culture
of mold, the amount of which must not exceed 5% from the general amount of
fermentation or 0,05 m3 per 1 m3 of culture, is exposed
to sterilization at pressure 0,18- 0,20 МPа during 2 h directly in fermenter,
whereupon through balancing tank is given on a bioscrubbing. The amount of
polluted water is about 22-35 m3 per 1 m3 of
culture.decrease of contaminations amount can be attained at introduction of
new technological methods and processes, for example at introduction of cycles
for the secondary use of sewages, in particular use of exhaust cultural liquid
on preparation of solutions of nutrient salts and limewater. As a result the
amount of exhaust cultural liquid decrease twice. [22]
.3 Description of equipment scheme. Specification
of equipment
equipment
scheme of the production of glucoamylase by submerged cultivation of Asp.
awamori is shown on figure 6.saccharified corn mash enters collecting tank 1
from where by a centrifugal pump 2 it is pumped to the agitation tank 8 for
preparation of nutrient medium. Other components for medium such as water and
salts such as potassium phosphate and ammonium sulphate is supplied from
collecting tank 3 are added in agitation tank 8.agitation tank components of
nutrient medium is carefully mixed and рН of solution bring to 4.8 with sodium hydroxide. A medium is
sterilized then, for what by a pump 9 it is given in a contact head 10, heat
from 75-80 °C to 125°C, maintain in a pipe-type holder 11 during 30-40 mines
and cool to 30-32 °C in surface heat-exchanger 12.sterilized and cooled medium
enters fermenter 13 that is a vertical cylindrical vessel with radial aerators
or with two-level turbine stirrer and bubbler for air supply.the process of
filling of fermenter an excess pressure 0,25 MPа is supported in it by a steam, supplied through the air duct
through an aerating device. Fillfactor of fermenter is 0,75-0,85. At its less
value a volume is taken to the norm by the supply of medium from agitation tank
8 through the system of sterilization. After filling of fermenter all system is
released from a medium, water is pumped and sterilize with sharp steam. A
nutrient medium in fermenter is cooled to 33-35°С.fermenter medium is inoculated by the culture of molds from
manifold 14. Before inoculation from fermenter take samples through the sampler
for microbiological control and biochemical analyses. Inoculation is carried
out through a pressing line preliminary sterilized from manifold to fermenter
by sharp steam during 1 hour. For this purpose valve on an output airline of
manifold close and lift in it pressure to 0,06-0,08 MPa, leaving in fermenterе pressure 0,02-0,03 MPa,
whereupon open a valve on the pressing line in manifold and fermenter and in
result of pressure differences inoculum from manifold is pressed into a
fermenter. After this close valves on the pressing line, in fermenter drive to
the rotation a stirrer and begin the process of growing of culture.pressing of
all inoculum culture from manifold let the air out, open a lid and carefully
wash internal surface. Then manifold is sterilized and fill with a nutrient
medium for the next cycle of preparation of inoculum.medium for manifold is
prepared in an agitation tank 5, equipped with a stirrer. In the beginning in
agitation tank add water, then turn stirrer and gradually supply salts and corn
wort and vegetable oil if necessary. Stirring of medium is made not only by a
stirrer, but also as a result of its circulation by pump 6.the same pump medium
is given through a contact head 7 into manifold, where it is maintained during
1,5-2 h at 125оС, cool to 33-35°C and
inoculate with Asp. awamori spores, through sowing actuator acces with the
maintenance of sterility and at minimum motion of air in a workshop. After
inoculation open valves for inlet and outlet of air. Expense of air 30-60 m3/(m3-h),
its temperature 35-40°С. Duration of cultivation 36 h.of air, supplied in manifold and
fermenters, conduct as follows. Before pumping to the rotary liquid-packed ring
compressor 17 air is purified from mechanical admixtures on a viscin filter 16,
and after a compressor is released from moisture consequently in dehydrator 18
and moisture separator 19. The compressed and dried air is heated in
heat-exchanger 20 to the temperature 60-80°C and then purify from a microflora
on a general head filter 22, filled with a basaltic fibre. After a head filter
air is additionally purified on individual filters 22 at manifold and 23 at
fermenter, which are also filled with a basaltic fibre.filters sterilize
simultaneously with manifolds and fermenters by sharp steam during 2 h at excess
pressure about 0.2 - 0.3 MPa. Moisture is removed from filters by blowing air
through them.cultivation of molds in fermenter temperature of nutrient medium
35 °C is supported by automatic control of water supply in the shirt of
apparatus. Aeration and agitation with stirrer (frequency of rotation about 150
- 170 rotations/minute) is conducted continuously from the moment of
inoculation completion and to the end of fermentation. Amount of the air
supplied is 30-60 m3/(m3-h). Sampler and lower draining
communication are under steam defence. Duration of the fermentation is 120-160
h.
air
from fermenter and manifold through the nozzle in a lid is thrown out through
scrubber 24 in an atmosphere. In scrubber air is purified from spores and other
suspended particles.liquid by a pump 15 is pumped for further processing and
obtaining of enzyme preparation. Empty fermenter is washed and sterilized at
120°C during 2 hours.the process of fermentation for microbiological and biochemical
control of development of culture take samples (with the maintenance of
sterility) in 24 hours after inoculation and then every 12 hours of growth. The
prepared culture must have activity of glucoamylase no less than 200 units/ml.
[20]of the equipment is given in the table 5.
Table 5.
Specification of equipment of technological scheme for glucoamylase production
Format
|
Zone
|
Position
|
Notation
|
Names
|
Quantity
|
Note
|
|
|
|
|
Documentation
|
|
|
|
|
|
AD 04.000
|
Assembly drawing
|
|
|
|
|
|
|
|
|
|
|
|
1 3
|
04.001
|
Collecting tank
|
2
|
|
|
|
2 4 15
|
04.002
|
Centrifugal pump
|
3
|
|
|
|
6 9
|
04.006
|
2
|
|
|
|
5 8
|
04.005
|
Agitation tank
|
2
|
|
|
|
7 10
|
04.007
|
Contact head
|
2
|
|
|
|
11
|
04.011
|
Pipe-type holder
|
1
|
|
|
|
12
|
04.012
|
Surface heat-exchanger
|
1
|
|
|
|
13
|
04.013
|
Fermenter
|
1
|
|
|
|
14
|
04.014
|
Manifold
|
1
|
|
|
|
16
|
04.016
|
Viscin filter
|
1
|
|
|
|
17
|
04.017
|
Rotary liquid-packed
ring compressor
|
1
|
|
|
|
18
|
04.018
|
Dehydrator
|
1
|
|
|
|
19
|
04.019
|
Moisture separator
|
1
|
|
|
|
20
|
04.020
|
Heat-exchanger
|
1
|
|
|
|
21
|
04.021
|
Head filter
|
1
|
|
|
|
22
|
04.022
|
Individual filter at
manifold
|
1
|
|
|
|
23
|
04.023
|
Individual filter at
manifold
|
1
|
|
|
|
24
|
04.024
|
Scrubber
|
1
|
|
3. DESCRIPTION AND
CALCULATION OF HEATER
.1 Description of heater for nutrient medium
supplied to fermenter
of the conditions of
influence on biologically active substances biosynthesis is ensuring of
production sterility and as well as nutrient medium components sterility. At
biologically active substances production different multicomponent nutrient
mediums in which foreign microorganisms may be present are used. These
microorganisms should be fully disrupted or killed. Sterilization is the
process of complete disruption or elimination of microorganisms.of
sterilization is destruction of all microflora in the nutrient medium,
different liquid admixtures. Necessity of sterilization is caused because
cultures those are producers of enzymes or other biologically active substances
very sensitive to other organisms’ presence.of molds for production of enzymes
are cultivated on sterile nutrient mediums.process can be divided into three
stages: medium or apparatus heating to the temperature of sterilization,
holding this temperature during some time providing death of all microorganisms
and the last - cooling of sterilized object to the temperatures available for
inoculation of medium by pure culture of producent. Here is described apparatus
for heating of medium to necessary temperature of sterilization.[18]the
sterilization of medium it is heated to 130°С and is held at this temperature for 15 minutes. Heating proceeds
in contact head that is built in the pipeline through which from agitation tank
medium enters fermenter or manifold.cylindrical body of apparatus (diameter
159/149 mm) the pipe is built in (diameter 112 mm) with apertures with diameter
2mm,in screw facet, turn across external surface of glass with height 290 mm.
In circular space between body and inner pipe through the connecting branch
(diameter 57/70 mm) inject steam (P = 4kg/cm2), which is after
passing through the aperture penetrates product heating it to temperature of
sterilization.sterilization of nutrient medium supplied to manifold sterilizers
of similar construction is applied. [23]
.2. Calculation of
heater of medium supplied to fermenteris heated in the column of continuous
action by sharp steam at p=4 kg/cm2 from 30 to 130 oC.
Steam is injected to mass through the orifices. Volume of nutrient medium for
filling of fermenter - 18m3. Duration of sterilization processes,
holding and cooling of medium - 2 hours.
Initial data
|
sharp steam pressure
|
4 kg/cm2
|
temperature initial
|
30 оС
|
130 оС
|
quantity of nutrient
medium
|
27 m3
|
duration of
sterilization process
|
3 h
|
of sterilizing column is
the volume of heated mass
s.c. = 27/3 = 9 m3/h
of heat necessary for
heating of mass
h = Vs.cρc (t2 - t1) = 9⋅1065⋅1 (130-30) = 959500
kcal/h
h = ((9⋅1065)/3600)⋅4186(130-30) = 1115⋅103 W
ρ - density of medium (volumetric mass);
ρ = 1065 kg/m3;
с - heat capacity of medium;
с=1 kcal/(kg⋅grad) [4186 J/(kg⋅grad)];
1 and t2 - initial and final temperature of medium;1 = 30 and t2 =
= 130° С.of heat on
sterilization subject to losses of heat into environment in quantity 2%ster=1,02Qh
= 1,02.959 500 = 978 690 kcal/hourster= 1,02⋅115⋅103 =
1137⋅103 W.of steam on sterilization
где Ist - enthalpy of heating steam;st = 657,3
kcal/kg (2758 KJ/kg);c - enthalpy of condensate at 130°С; at 130° Сc= 130,5 kcal/kg (547,2 KJ/kg).discharge of steam on sterilization
v-specific volume of
saturated water steam at
р = 4 kg/cm2
υ = 0,3818 m3/kg.of steam outflow from orifices in
heating column
φ - velocity coefficient, φ = 0.9;' - enthalpy of steam at inlet to orifice;' = 657,3 kcal/kg
(2755 KJ/kg);"- enthalpy of steam at outlet from orifice and mixing with
heated mediumр= 1,755 kg/cm2 (tsat = 130°С),"=650,6 kcal/kg
(2720 KJ/kg).square of orifices in medium heater necessary for inlet of heating
steam
of orifices with
diameter 2 mm for inlet of steam into medium
of one orifice with
diameter 2 mm
the inner cylinder of heater with diameter 12 mm consider six rows of orifices - 294/6 = 49 orifices per row.of medium flow in heater
section for passage of
medium through heater
din - inner
diameter of sleeve for passage of mass;in = 104 mm.of medium stay in
heating head
where l - length of
heating cylinder= 0.4 m.
Results of calculations
of heater with given parameters:of heat necessary
for heating of mass 959500 kcal/hdischarge of steam on sterilization 710 m3/hof
steam outflow from orifices in heating column 213.5 m/secof orifices with
diameter 2 mm for inlet of steam into medium 294of medium stay in heating head
1.36 sec.
CONCLUSIONS
. 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.
. The best method
for the production of glucoamylase by cultivation of Asp.awamori is submerged
fermentation on the corn mash substrate, 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.
. Technological
process of glucoamylase production in department of biosynthesis includes the
several stages, such as additional works (preparation of equipment, water, air
sterilization, disinfectants and nutrient medium preparation), preparation of
inoculums and actually fermentation. And according to these stages
technological and equipment schemes of glucoamylase production by submerged
cultivation were developed.
. Time of medium
holding in heating head for sterilization 27 m3 of nutrient medium
from 30 to 130oC with volumetric discharge of steam 710 m3/h
is 1.36 sec and quantity of orifices with diameter 2 mm for inlet of steam into
medium is 294.
REFERENCES
1. (Microbiological Process Report Production of Microbial
Enzymes and Their Applications,L A. UNDERKOFLER, R.R. BARTON, AND S.S.
RENNERT.Takamine Laboratory, Division of Miles Laboratories, Inc., Clifton, New
Jersey, October 1, 1957)
2. Biotechnology,Fourth Edition. John E. Smith Emeritus
Professor of Applied Microbiology, University of Strathclyde, Glasgow and Chief
Scientific Adviser to MycoBiotech Ltd, Singapore, Cambridge University Press
2004
3. Industrial Enzymes. Structure, Function and Applications
Edited by Julio Polaina and Andrew P. MacCabe, 2007 Springer)
4. Enzymes in Industry.Production and Applications,Edited by
Wolfgang Aehle, Third, Completely Revised Edition, 2007)
5. ://www.nezachetovnet.ru/free/organicheskaya_himiya/?id=f16052
6. http://chemanalytica.com/book/novyy_spravochnik_khimika_i_tekhnologa/06_syre_i_produkty_promyshlennosti_organicheskikh_i_neorganicheskikh_veshchestv_chast_II/5426
7. Грачева И.М. Технология ферментных препаратов. - Москва:
Агропромиздат, 1987. - 335с.
8. Wongwicham A et. al.: Biotechnol. Bioeng, Nov 20; 65(4),
1999.
9. <http://ru-patent.info/21/95-99/2196821.html>
10. Биотехнология. Т.Г. Волова. - Новосибирск: Изд-во
Сибирского отделения Российской Академии наук, 1999
11. Гореликова Г.А. Основы современной пищевой биотехнологии:
Учебное пособие. - Кемеровский технологический институт пищевой промышленности.
- Кемерово, 2004. - 100 с.
12. http://www.sergeyosetrov.narod.ru/Projects/Enzym/Production_microbial_ferments.htm
13. Сидоров Ю.І., Влязло Р.Й., Новіков В.П. Процеси і апарати
мікробіологічної та фармацевтичної промисловості. Технологічні розрахунки.
Приклади і задачі. Основи проектування: Навчальний посібник. - Львів:
«Інтелект-Захід», 2008
14. ГОСТ 2874-82 «Вода питьевая. Гигиенические требования и
контроль за качеством».
15. Беккер М.Е. Введение в биотехнологию. - перевод на русский
язик, изд-во Пищевая промышленность, 1978.
16. ДСанПіН №383 Про затвердження Державних санітарних правил і
норм “Вода питна. Гігієнічні
вимоги до якості води централізованого господарсько-питного водопостачання»
17. http://www.pravoteka.ru/pst/88/43830.html
18. Калунянц К.А., Голгер Л.И., Балашов В.Е. Оборудование
микробиологических производств. - М.: Агропромиздат, 1987. - 304 с.
19. http://www.sergeyosetrov.narod.ru/Projects/Enzym/Grow_cultures_microorganism.htm
20. Яровенко В.Л, Устинников Б.А., Богданов Ю.П., Громов С.И.
Справочник по производству спирта. Сырье, технология и технохимконтроль. - М.:
Легкая и пищевая промышленность, 1981
21. Biotechnology,Fourth Edition. John E. Smith Emeritus
Professor of Applied Microbiology, University of Strathclyde, Glasgow and Chief
Scientific Adviser to MycoBiotech Ltd, Singapore, Cambridge University Press
2004
22. http://www.eurolab.ua/encyclopedia/3863/34236/
23. Колосков С.П. Оборудование предприятий ферментной
промышленности. - М.: Пищевая промышленность, 1969. - 383 с.
Appendix.
Characteristics of different producers of glucoamylase
B1 Characteristics of
different producers of glucoamylase [5]
Sources of
glucoamylase Optimal conditions Molecular weight,
kDa Isoelectric
point (рI) Content of
carbohydrates, % Degree of soluble starch
hydrolysis, % рН temperature,
С Endomycopsis sp. 20-9 5,7-5,9 50 53,0 3,8-3,82 8,5 98-99
Endomyces JF 00111 4,8-5,0 - 55,0 4,8-5,5 present - Rhizopus javanicus
5,0-5,2 - 48,0 7,5-8,0 10,5 - Aspergillus awamori 4,5 60 83,7-88,0 3,7 - 90
Aspergillus niger: I 4,5-5,0 - 99,0 3,4 - 95 II 4,5-5,0 - 112,0
4,0 - - Mucor rouxianus I 4,6 55 59,0 8,4 present 100 Rhizopus delemar 4,5
40 100,0 - - 95 Penicillium oxalicum 5,0 55-60 84,0 7,0 - 88 Aspergillus
phoenicis 4,5 60 69,0 - 17,0 - Aspergillus awamori X-100 4,7-5,0 60-62 62,0
4,4 presen -
|