SU786917A3 - Method of microorganisms biomass production - Google Patents

Description

The invention relates to microbiological activity and includes a method for producing biomass by culturing microorganisms on starch-containing raw materials previously liquefied and hydrolyzed with amylolytic enzymes. There is a method of liquefying starch-containing raw materials using dextrinogenic amylolytic enzymes l. The resulting liquefied feedstock after hydrolysis can be used to cultivate microorganisms. The closest technical solution to the invention is a method of obtaining biomass of microorganisms, involving the enzymatic hydrolysis of starch-containing raw materials, the cultivation of yeast and the separation of biomass 2. The disadvantage of this method is the low biomass yield in the process of growing microorganisms. The purpose of the invention is to increase the biomass yield. The goal is achieved by the fact that, prior to enzymatic hydrolysis, starch-containing raw materials are liquefied by adding dextrinogenic enzyme -1-4 glucan-4-glucan hydrolase obtained from Bacillus subtil is var Blotecus, and the yeast growth is carried out directly during the enzymatic hydrolysis of the raw material, moreover Yeast species of Candida utilis or Rhodotorala grac-1 II s are cultivated. The method is carried out as follows. Microorganisms utilize mono oligosaccharides. High-molecular-weight carbohydrates, which include starch, are hydrolyzed with the aid of amylolytic saccharifying enzymes before growing microorganisms, and a mixture of sugars is obtained. This mixture is used as a carbon source. The intensity of starch hydrolysis increases if the starch-containing raw materials are subjected to liquefaction by amylolitic dextrinogenic Lermenta before hydrolysis. Various plant sources of starch are used as raw materials, preferably potato starch or waste liquid from starch production plants. Raw materials containing starch, cassava or other potato varieties are ground, sieved and mixed with water to a certain concentration of starch in the mixture. When used as a raw material for waste liquid from production facilities, the collapse of the raw material pre-extracts the protein by known methods; The pH of the starch suspension is adjusted to a certain value, and the dextrinogenic enzyme O6-1-4-glucan-4-glucan hydrolase is added to it. The enzyme is fed continuously to the two-stage liquefaction apparatus. The mixture is heated to a temperature of 85-8. The duration of the liquefaction process is from 60 to 90 minutes. The liquified mass is diluted with water to a sugar content of 1-8%, preferably 4%. Then, in addition, the amount of inorganic nutrient salts containing nitrogen, phosphorus, magnesium, and potassium is added to the mixture. The pH of the resulting nutrient medium is adjusted to 4, 6-6. One or several inorganic compounds are used as a nitrogen source: ammonium sulfate, ammonium phosphate, and ammonium nitrate. In some cases, organic nitrogen is used in the form of urea. Potassium dihydrogen phosphate, sodium hydrogen phosphate, magnesium sulfate, and ferrous sulfate are used as other sources of microorganisms. The obtained nutrient medium is sterilized at a temperature of 120-135 ° under pressure for 1060 minutes. The sterile nutrient medium enters the fermenters and is inoculated with microorganisms: yeast, bacteria or other microorganisms, preferably yeasts of the genus Candida, synthesizing a protein, or yeast of the genus Rhodotorula, preferably synthesizing lipids. At the same time as the inoculant, the required amount of sugar into the fermenter is added. preferably gluco amylase. The enzyme is added under sterile conditions. In the fermenter, saccharification of liquefied starch and microbial growth take place simultaneously. Microorganisms using the resulting glucose and ma for growth. Toza. The solution of the sucrogenic amylase Ylucuzyme N (from the company Amane Seyku, Japan) is sterilized by passing through a microfilter q with a pore size of about 0.2 microns, for example, an EX filter from Millipor Corp., USA. Then amylase is served on Wednesday. Microbial cultivation of the wire under aerobic conditions with mixing of the SRI of the medium of 4.0-7.0, temperature 30-45 .. The cultivation is carried out in a batch and continuous manner in several stages on an installation consisting of several Fermenters connected in series. The time of growing the yeast in a nutrient medium containing liquefied starch, which is continuously saccharified by saccharogenic amylase, is 14-21 hours. The biomass is separated from the medium by decanting or centrifuging. Then the biomass is dried. The biomass yield is 45-52% of the injected sugar. The amount of unused sugar is 0.10, 2%. Figure 1 shows the flow chart of the proposed method; Figures 2 and 3 show examples of the implementation of the enzymatic saccharification stage and the cultivation of crops. The method includes the following process steps: preliminary preparation of the raw material 1 / enzymatic dilution of starch 2, obtaining a nutrient medium for growing culture 3, sterilizing the nutrient medium 4, Enzymatic saccharification and growing culture 5, separating microbial cells 6, drying 7 and obtaining the finished product 8. . The fermentation plant (see FIG. 2) contains three fermenters 9-11 arranged in series. The volume of the third fermenter is twice the volume of the first or second fermenters. The culture medium enters through conduit 12, and the succinic amylase through conduit 13. Three fermenters are sequentially connected via conduits 14 and 15. A portion of the culture medium is returned from the second fermenter to the first via pipe 16. The amount of medium returned is determined by the growth rate of the microorganisms used, the effective capacity of the fermenters, the concentration of microorganisms, and the concentration and amount of medium for growing into the fermenters. for growing the culture from the second fermenter, returns to the first in order to plant in the first fermenter a culture of microorganisms with a high growth rate. This ensures the stabilization of the concentration of microorganisms in the first fermenter. At the same time, the phenomenon of the so-called cell leaching is prevented if a larger amount of culture medium is supplied to the first fermenter. In a continuous multistep growing process, the medium is returned from the last apparatus. one. . In a continuous three-stage growth system, in the first fermenter, an increase in the activity of microorganisms and an acceleration of the saccharification process is observed. The second fermenter contains microorganisms in the logarithmic growth phase. In the third apparatus, the microorganisms are in the stationary growth phase. Due to the assimilation of sugar in the latter fermenter, the biological oxygen demand of the spent aquatic environment is significantly reduced. The plant for fermentation (see Fig. 3) also consists of three Fermenters 17-19, arranged in series, as in the plant shown in Figure 2, but with significantly improved indicators of the fermentation process. The process of growing aerobic microorganisms requires aeration of the environment. This leads to the formation of abundant foam. In addition, molasses, spent starch solution, or waste effluents from the food industry contain foaming agents that cause abundant foam formation. The fluid flow from the first fermenter to the second or the fluid flow from the second fermenter to the third contains foam and air bubbles, which makes this stream unstable. Variations in flow rate and dilution rate in known continuous flow systems or recycled systems lead to disruption of microbial growth. and undesirable decrease in product yield. The fermentation plant shown in FIG. 3 is intended to overcome foaming. It is characterized in that over-pressure is maintained between the first, second and third fermenters. The process of continuous growth of aerobic microorganisms proceeds calmly. The liquid level in the fermenters is easily balanced by controlling the pressure drop P, and P in enzymes ax 17 and 18, and P and P in fermenters 18 and 19. If the liquid level in the first apparatus starts to rise due to the strong formation of foam, it can be reduced by using a small increase in pressure level P. At the same time, at the same time the pressure P and P, slightly increase. This is achieved by a stable flow of continuous culture. The fermenters are interconnected by pipelines 20 and 21. The starting material is fed through inlet 22 into the first fermenter. Air is fed through pipe 23 into each apparatus and exits through opening 24. In the first fermenter, a pressure of 1000–2500 mm of water is maintained, in the second, 600–1800 mm of water and in the third 300–1500 mm of water. If necessary, the pressure in the first fermenter can be raised above 2500 mm water column. Sky; 1 increase in pressure in the apparatus increases the rate of oxygen transfer, which leads to an increase in the growth rate of microorganisms and an increase in the productivity of the process. With continuous cultivation of Candida's yeast in a medium containing inorganic nutrient salts and a carbon source prepared from agricultural products in the fermentation plant shown in Fig. 3, the biomass yield is 50% or more in terms of total raw material costs. The biomass is separated from the spent medium by decanting, then concentrated on a centrifugal separator with a jet or on other separators. The biomass is then dried in a spray dryer, drum dryer, or flash apparatus. In some cases, a special type of dryer can be used to obtain a product in the form of granules or tablets. The method allows processing of large quantities of the raw material for a short period of time. The process of growing microorganisms proceeds calmly. The control over the process is carried out automatically. You can recycle starting material of high concentration. Since the enzymatic saccharification and the cultivation of microorganisms are carried out simultaneously, there is no need for additional saccharification. Pre-dilution helps to increase the rate of hydrolysis. The method is illustrated by the following examples. . Example 1. Cassava roots (tapioca) were crushed and diluted with water to form a slurry with a starch content of about 16%, the slurry pH was adjusted to 6.0-6.2, a dextrinogenic enzyme was added (Spigaz K, Nagase Industrial Corporation, Japan), 2% in terms of starch content. The slurry was continuously diluted at a temperature of 85-88 ° C. After removing the impurities, the liquefied solution was diluted with water to a sugar concentration of 1.5-6%. The required amount of inorganic nutrient salts was added to the solution, the pH was adjusted to 4.4-5. The resulting culture medium was continuously sterilized

by heating in a sterilizer, and then fed to the first fermenter. The medium was inoculated with Candida utilis. At the same time and continuously, sucrogenic amylase was added under sterile conditions to the growth medium at a given rate. The total growing time was 4-8 hours.

The biomass was separated using a centrifuge and dried. The yield of dry microbial cells was 45-53% in terms of the starch content in tapioca.

Example 2. Cassava roots (tapioci) were treated and unclenched in the same manner as described in Example 1. The liquefied solution was diluted with water so that the starch content was 4%. The required amount of inorganic salts, such as inorganic compounds of nitrogen, phosphorus, potassium and others, was added to the solution. The solution was sterilized by heating and continuously fed to a fermenter. The medium was inoculated with Saccharomyces cerevisial yeast. At the same time, 0.3-0.5% of the sugar-forming enzyme was added to the sterile environment, calculated on the total sugar content of the medium. The growing time was 6-10 hours.

Yeast cells were separated, concentrated and dried. The yield of yeast cells was 45-50% based on the weight of the starch.

Example 3. Waste effluent from a potato starch production plant (solids content about 8%, content of nitrogen-free soluble substances, 3-4%) was processed to obtain a pH of 6. Then the mass was diluted by adding a predetermined amount of thinning enzyme. Then the amount of inorganic nutrient salts and was added to the solution. a culture medium was obtained. The medium was heat sterilized and continuously fed to the fermenter. Cultivated yeasts of the genus Candida. C-medium, under sterile conditions, was added sucrogenic amylase continuously at a given rate. After cultivation, the yeast cells were separated from the medium, concentrated and dried. About 45% of the soluble nitrogen-free substances were transferred to yeast cells.

Example 4: Raw cassava roots were treated and liquefied by analogy with Example 1. The diluted solution was diluted so that the sugar concentration was 2-5%. The necessary inorganic salts were added to the solution. In the resulting culture medium, the C / D concentration ratio was

The medium was sterilized and fed to the fermenter. A periodic or continuous cultivation of yeast, of the genus Rhodotorula, which produce lipids, has been carried out. At the same time, the sucrogenic amylase was added to the medium at a given rate under sterile conditions. After separation and concentration, the biomass was dried. The yield of dry cells was 40-48% in terms of the starch content in raw cassava. Containing lipids in dry yeast cells was 15-50%.

Lipid had a composition similar to that of vegetable oil. In addition, the residue resulting from lipid extraction was a protein rich mass.

Example 5. Continuous cultivation of microorganisms was carried out on the unit for aerobic fermentation, shown in FIG. The pressure in the first fermenter was 1800 mm water column, in the second 1500 mm water column and in the third 1000 mm water column. The intensity of aeration of the BLA in the first fermenter is 0.5 rev / min, in the second 1, 0 rev / min and 0.5 rev / min) in the third fermenter.

The total volume of the first, second, and third fermenters was 500 L, 500 L, and 1000 L, respectively. The volume of culture medium was 300 liters, 300 liters and 600 liters, respectively. The feed rate of the medium was 250 l / h. Used the Candida genus yeast. The culture medium contained the following inorganic salts: NH NO-j - 6 g 3 g, Na2HPOx 12H2 O, 5 g, MgSO47H2O - 0.5 g, FeSO4 - 0.5 g, tap water - 1000 ml, pH 5.5

The carbon source was a solution obtained by grinding and diluting raw sweet potatoes, which were added to the medium so that the glucose concentration was 4%, the pH of the medium was maintained at a given level using. The continuous growth in three stages was 500 hours. The biomass yield was 50% in terms of carbon source, and the growth rate of the yeast was 0.6 hours,

Example 6. A medium was used for growing the culture, the composition of which is given in Example 5. Molasses with a concentration of 4% was added to the medium as a carbon source.

(w / w) in terms of sugar. Continuously grow the strain of Candida uti) is. In the first, second and third fermenters, a pressure of 2500 mm water column was maintained.

(P), 2000 mm of water column (Р J and 1500 mm of water, column (Р), respectively. At that, the intensity of aeration

were 1, Oh, rev / min, 1.0 rev / min and 0.5 rev / min, min, respectively. The three-way continuous cultivation method was carried out using the fermentation plant shown in Figure 3. The sizes of the fermenters and the amount of medium loaded in each were similar to the sizes and quantities given in example 5. Continuous cultivation was carried out for more than 300 hours. The output of microbial cells was 52% in terms of sugar content.

Example 7. An inorganic medium having a composition similar to that shown in Example 5 was used for growing the culture. A spent solution of potato starch was used as a carbon source. Cultivated culture of Candid utills. The carbon source had a concentration of 4%.

Continuous cultivation proceeded under the same conditions as described in example 6 for 400 hours or more without any difficulties caused by the contamination of the medium by other microorganisms. The output of microbial cells was 48% in terms of sugar concentration. The protein content in microbial cells was 46-52%.

Example 8. Molasses was used as a carbon source. Lipids-producing Rhodotorula gracilis were grown. The carbon source concentration was la-4% (w / w). The inorganic grafting medium contained 75 mg / l of urea and 25 mg / l, the pH of the medium was 5, 0. The intensity of aeration was 5.0 v / v min. Continuous yeast cultivation was carried out at a temperature. The medium was swept over at 200 rpm. The cultivation was carried out on a plant for fermentation J shown in Fig. 3. In the first, second and third fermenters the pressure, mm in a water column, was maintained: 1200, 800 and 500, respectively. As a result of the continuous process of growing for 250 hours, the lipid content reached 39.6% in terms of dry yeast cells.

Claims (2)

1. A method for producing microorganism biomass, which involves fergmentative hydrolysis of a starch-containing raw material, cultivating yeast and individual biomass, characterized in that, in order to increase the biomass yield, prior to enzymatic hydrolysis, the starch-containing raw material is diluted by adding dextrogramean ventricular carcidians. -4-glucan hydrolase obtained 5 from Bacillus subtilis var Biotecus, A leveling of yeast is carried out directly during the enzymatic hydrolysis of the raw material. 2. The method according to claim 1, characterized in that the species of Candida utilis or Rhodotorula gracllic are cultivated. Sources of information received by 9H .HH during examination five 1. US Patent No. 3149049, cl. 195-31, published 1964. 2. The patent of France 2285080, cl. C 12 D 13/06, published 1976 (prototype).