Project of glucoamylase production by submerged cultivation of Aspergillus awamori

The process of fermentation proceed at next conditions:

The prepared culture must satisfy the next requirements:

2.2.3 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.

All indexes of growth of culture, the changes of composition of medium and accumulation of enzymes, etc. are added to the laboratory magazine.

At 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.

Technical 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.

Exhaust 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.

One 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]

Sewages 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.

Water 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.

Sewages 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.

The 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.

The 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]

2.3 Description of equipment scheme. Specification of equipment

The equipment scheme of the production of glucoamylase by submerged cultivation of Asp. awamori is shown on figure 6.

The 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.

In 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.

The 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.

In 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.

In 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.

After 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.

Nutrient 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.

By the same pump medium is given through a contact head 7 into manifold, where it is maintained during 1,5-2 h at 125^оС, 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.

Preparation 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.

Individual 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.

During 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.

Exhaust 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.

Cultural 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.

In 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]

Specification of the equipment is given in the table 5.

Table 5. Specification of equipment of technological scheme for glucoamylase production

3. DESCRIPTION AND CALCULATION OF HEATER

3.1 Description of heater for nutrient medium supplied to fermenter

One 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.

Aim 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.

Cultures of molds for production of enzymes are cultivated on sterile nutrient mediums.

Sterilization 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]

For 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.

In cylindrical body of apparatus (diameter 159/149 mm) the pipe is built in (diameter 112 mm) with apertures with diameter 2mm,

Perforated 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.

For sterilization of nutrient medium supplied to manifold sterilizers of similar construction is applied. [23]

3.2. Calculation of heater of medium supplied to fermenter

Medium is 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.

Productivity of sterilizing column is the volume of heated mass

V_s.c. = 27/3 = 9 m³/h

Quantity of heat necessary for heating of mass

Q_h = V_s.cсc (t₂ - t₁) = 9?1065?1 (130-30) = 959500 kcal/h

or

Q_h = ((9?1065)/3600)?4186(130-30) = 1115?10³ W

where

с -- density of medium (volumetric mass);

с = 1065 kg/m³;

с -- heat capacity of medium;

с=1 kcal/(kg?grad) [4186 J/(kg?grad)];

t₁ and t₂ -- initial and final temperature of medium;

t₁ = 30 and t₂ = = 130° С.

Discharge of heat on sterilization subject to losses of heat into environment in quantity 2%

Q_ster=1,02Q_h = 1,02.959 500 = 978 690 kcal/hour

or

Q_ster= 1,02?115?10³ = 1137?10³ W.

Discharge of steam on sterilization

or

where

где I_st -- enthalpy of heating steam;

I_st = 657,3 kcal/kg (2758 KJ/kg);

I_c -- enthalpy of condensate at 130°С; at 130° С

I_c= 130,5 kcal/kg (547,2 KJ/kg).

Volumetric discharge of steam on sterilization

where v--specific volume of saturated water steam at

р = 4 kg/cm²

х = 0,3818 m³/kg.

Velocity of steam outflow from orifices in heating column

where ц - velocity coefficient, ц = 0.9;

I' - enthalpy of steam at inlet to orifice;

I' = 657,3 kcal/kg (2755 KJ/kg);

I"-- enthalpy of steam at outlet from orifice and mixing with heated medium

at р= 1,755 kg/cm² (t_sat = 130°С),

I"=650,6 kcal/kg (2720 KJ/kg).

Total square of orifices in medium heater necessary for inlet of heating steam

Quantity of orifices with diameter 2 mm for inlet of steam into medium

Square of one orifice with diameter 2 mm

On the inner cylinder of heater with diameter 12 mm consider six rows of orifices - 294/6 = 49 orifices per row.

Velocity of medium flow in heater

Flow section for passage of medium through heater

where d_in -- inner diameter of sleeve for passage of mass;

d_in = 104 mm.

Time of medium stay in heating head

where l - length of heating cylinder

l = 0.4 m.

Results of calculations of heater with given parameters:

Quantity of heat necessary for heating of mass 959500 kcal/h

Volumetric discharge of steam on sterilization 710 m³/h

Velocity of steam outflow from orifices in heating column 213.5 m/sec

Quantity of orifices with diameter 2 mm for inlet of steam into medium 294

Time of medium stay in heating head 1.36 sec.

CONCLUSIONS

1. 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.

2. 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.

3. 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.

4. Time of medium holding in heating head for sterilization 27 m³ of nutrient medium from 30 to 130^oC 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.

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Appendix. Characteristics of different producers of glucoamylase

Table B1 Characteristics of different producers of glucoamylase [5]

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