RU2209250C2 - Method for microbiological processing waste in fractionating alcohol purification to yield protein biomass - Google Patents

Abstract

FIELD: microbiological industry. SUBSTANCE: method involves aerobic culturing microbiological strains in nutrient medium containing up to 0.8-1.5% of a mixture of ethyl alcohol and fusel oil head fraction as carbon-containing raw taken in the amount from 1:0.5 to 0.5:1, respectively. Invention proposes to feed carbon- containing raw in cyclic regime in the process of biomass culturing. Time of cycle is 0.5-1.5 h in amplitude of cyclic feeding from 0.3% to 1% of flow and time of maximal feeding is 0.2-1 h. Invention uses yeast Candida lipolytica VKPM Y-2206, Candida utilis VSB-651 and bacterium Rhodococcus erythropolis VKPM S-1379. Aerobic culturing is carried out in continuous regime at the rate of nutrient medium flow D = 0.1-0.3 h^-1, at temperature 28-33 C, pH = 4.5-5.0 and aeration level 0.2-1.0 rev/vol/min. Method provides to obtain practically complete utilization of esters and aldehydes (up to 95%). EFFECT: improved processing method. 3 cl, 1 dwg, 4 ex

Description

 The invention relates to the microbiological industry, and in particular to methods for producing biomass from carbon-containing raw materials, and can be used to obtain a protein feed product from waste from the alcohol industry - the head fraction of ethyl alcohol and fusel oil.

 Alcohol distillation refining waste, the head fraction and fusel oil in their composition contain up to 75% ethyl alcohol. The amount of waste is approximately 1.5% of the total ethanol produced by the microbiological method. Currently, the volume of ethyl alcohol produced in Russia is growing, and consequently, the amount of waste that needs to be processed efficiently is increasing.

There are a large number of patents relating to the synthesis of biomass of yeast or bacteria on ethanol. It is known to grow the yeast Hansenula anomola at a temperature of T = 35-38 ^{° C.} in ethanol (Information Chimie Helppdo, 1976, 423, p. 12). The biomass yield from the substrate is 75%, the crude protein content in the biomass is 60-66%. The best known method for producing unicellular protein in ethanol, in which C. utilis yeast is recommended, is used as a producer. The average biomass yield from the substrate is 70-75% (British Patent 1375189, 1974).

 A known method of producing citric acid on the head fraction of ethyl alcohol and fusel oil (N. A. Filochenov, T. E. Arzumanov, V. A. Samoilenko, T. V. Finogenova. "Synthesis of citric acid by Yaro vialipolytice yeast using various types of ethanol-containing raw materials. "Biotechnology, 1998, 1, pp. 57-61). It was shown that yeast consumes a substrate and accumulate protein on all media containing ethanol, including the ether-aldehyde fraction (a mixture of the head fraction of ethyl alcohol and fusel oil). However, the yield from the substrate was low, 50%. The protein content in the product was 45%. The degree of utilization is 89-90%.

 Thus, at present, there is no effective way to recycle alcohol production waste: the head fraction of ethyl alcohol and fusel oil, the protein yield is low, 50%.

 The problem solved by the use of the present invention is to develop an industrial method for microbiological processing of waste from alcohol production - the head fraction of ethyl alcohol and fusel oil.

 The technical result obtained by the implementation of the invention is to obtain a protein feed product from waste from alcohol production - the head fraction of ethyl alcohol and fusel oil - with a high yield from the substrate and a high degree of waste disposal.

 To achieve the specified technical result, it is proposed to cultivate microorganisms continuously on a rectification waste alcohol in a nutrient medium containing up to 0.8-1.5% carbon-containing raw materials using as a raw material a mixture of the ethyl alcohol and fusel oil head fraction taken in a ratio of 1 : 0.5 to 0.5: 1, respectively, and the substrate is fed cyclically during the growing process, and the cycle time is 0.5-1.5 hours with a cyclic feed amplitude of 0.3 to 1% of the duct, while the feed time maximum the total amount is 0.5-1 hours.

 As microbiological cultures, yeast Candida utilis BSC-651, Candida lipolityca VKPM-Y-2206, and bacteria Rhodococus erythropolis VKPM-S-1379 are used.

Aerobic growth is carried out in a continuous process with a flow rate of D = 0.1-0.34 h ^-1 , T = 28-33 ^o C, pH 4.5-5.0, aeration level of 0.5-1 rev / rev • min

 It is possible that the ratio of the head fraction and fusel oil fluctuates in the range from 1: 0.5 to 1: 1, respectively, which allows maintaining the concentration of aldehydes, esters, and methanol below the critical level on fermentation. The substrate is proposed to be fed cyclically, the cycle is 1 hour, the time of positive amplitude is from 0.5 hours to 1 hour, the amplitude of the oscillations is maintained from 0.3 to 1% of the duct. The selected cyclic feed parameters allow for more complete utilization of the head fraction and fusel oil, causing activation of enzyme systems in the glyoxylate shunt cell. An increase in the concentration of acetyl-CoA in the cell at the stage of maximum substrate supply leads to an acceleration of constructive metabolism in the cell, respectively, to an increase in the content of proteins, vitamins in the cell and an increase in the growth rate of the culture, and to an increase in yield.

 The above strains of yeast and bacteria: Candida lipolytica VKPM-Y-2206, Candida utilis BCB-651, Rhodococcus erythropolis VKPM-S-1379 are known strains deposited in the collections of GOSNIISintezbelok and in the All-Russian collection of industrial microorganisms.

 Yeast Candida lipolytica VKPM-Y-2206 - aerobes, on a solid nutrient medium (wort agar) 1-2 days after the start of cultivation form a creamy-colored colony with smooth edges. They utilize glucose, ethyl alcohol, hydrocarbons, sucrose, and require the presence of biotin sources in the environment for their growth. Able to synthesize citric acid.

Yeast Candida utilis VSB-651 on solid nutrient medium, 2 days after the start of growth, form colonies with a diameter of 3 mm in white. Utilize hydrocarbons, a wide range of sugars. Characterized by a high specific growth rate of 0.26 h ^-1 .

 Bacteria Rhodococcus erythropolis VKPM-S-1379 on a solid nutrient medium (meat-peptone agar) form 2 days after the start of colony growth with uneven yellowish edges. When growing on a liquid nutrient medium, they form chains of cocci, and single cells are also found. They utilize hydrocarbons, sugars, do not have amylolytic activity.

Information confirming the possibility of the invention
Example 1
The strain of yeast Candida utilis VSB-651 was grown in the apparatus: apparatus volume Vp = 20 l, temperature Tsreda = 30-320 ^o C, pHenvironment = 4.5-5.0, air flow 1000-1100 l / h, stirrer speed 1000 r / min Cultivation is carried out on a nutrient medium containing ammonium sulfate 6 g / l, monosubstituted potassium phosphate 2 g / l, magnesium sulfate 0.5 g / l, ferrous sulfate, aqueous 0.002 g / l, manganese sulfate 0.005 g / l, zinc sulfate 0.005 g / l

 A diagram of the cyclic process is shown in the drawing.

 The amplitude of the cyclic feed (SA +, SA-) is 0.5% of the duct. The cycle time (TC) is 1 hour, the feed time of TA + and TA- is 0.5 hours, the pH is maintained by titration with 6% ammonia water, the concentration of the carbon source is 1.5%, the ratio of the head fraction and fusel oil is 0.5: 1.

The process was carried out continuously, the samples determined the concentration of biomass, the concentration of crude protein, (% C) P, the concentration of residual ethanol (%), the concentration of residual esters, (%), the concentration of residual aldehydes. As a result of the cultivation, the yield from the substrate was 71.2%, the average specific growth rate of the culture was 0.25 h ^-1 , the residual ethanol content was 0.01. Concentration of aldehydes, esters - traces. The concentration of crude protein was 54.1%. The utilization rate is 92%.

Example 2
The growing process was carried out in the same way as in example 1, the value of the positive amplitude (SA ⁺ ) was 0.8%, the value of the negative amplitude (SA-) was 0.3%. The cycle time is 0.5 hours. As a result of cultivation, the yield from the substrate was 73%, D = 0.25l h ^-1 , the residual ethanol content was 0.01%, esters, aldehydes - traces. The protein content of 53.9%. The ratio of the head fraction and fusel oil is 1: 1, the degree of utilization is 95%.

 Example 3

 The bacterial strain Rhodococcus erythropolis VKPM-S-1379 was grown in the apparatus in the same way as in example 1, while the concentration of crude protein was 58.3%, and the degree of utilization of rectified waste was 94%.

 Example 4

The strain of yeast Candida lipolytica VKPM-Y-2206 was grown in the same way as in example 1, while the concentration of crude protein was 54.2%, the degree of utilization of rectification purification waste was 92%
The control
In the control experiment, cultivation was carried out continuously without cyclic supply of the substrate. The specific growth rate of 0.24 h ^-1 , the carbon source was 1.5%. The yield was 57%, the crude protein content was 53.2%, the residual ethanol content was 0.17%, the esters were 0.095%, and the traces of aldehydes. The utilization rate is 80%. Thus, the use of a cyclic feed of the etheroaldehyde fraction allows one to increase the biomass yield from the carbon substrate to 73%.

 So, as a result of the implementation of the proposed method of microbiological processing of alcohol production waste - the head fraction of ethyl alcohol and fusel oil - it is possible to achieve almost complete utilization of esters, aldehydes. The degree of disposal is 95%. The protein content in biomass is 54%. The yield of biomass from the carbon-containing substrate is 73%.

Claims (3)

 1. The method of microbiological processing of waste rectification purification of alcohol, providing for aerobic growth of microbiological cultures in a nutrient medium containing sources of N, P, Mg, Zn, Fe, K, up to 0.8-1.5% of carbon-containing raw materials, which use a mixture the head fraction of ethyl alcohol and fusel oil, taken in a ratio of 1: 0.5 to 0.5: 1, respectively, moreover, the carbon-containing raw material is fed cyclically during the growing process, and the cycle time is 0.5-1.5 hours, with an amplitude of cyclic feed from maximum t he 0.3 to 1% of the duct, wherein the feeding time is 0.5-1 hours.  2. The method according to claim 1 is characterized in that the strains of yeast Candida lipolytica VKPM-Y-2206, Candida utilis VSB-651 and bacteria Rhodococcus erythropolis VKPM-S-1379 are used as microbiological cultures. 3. The method according to PP.1 and 2, characterized in that aerobic growth is carried out in a continuous process with a flow rate of the nutrient medium D = 0.1-0.3 h ^-1 at a temperature of T = 28-33 ^o C, pH 4.5 -5.0 and aeration level of 0.2-1.0 rpm (rpm).