CN113388525A - Application of monascus in treatment of ultrahigh-concentration white spirit wastewater - Google Patents
Application of monascus in treatment of ultrahigh-concentration white spirit wastewater Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/347—Use of yeasts or fungi
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/32—Nature of the water, waste water, sewage or sludge to be treated from the food or foodstuff industry, e.g. brewery waste waters
- C02F2103/325—Nature of the water, waste water, sewage or sludge to be treated from the food or foodstuff industry, e.g. brewery waste waters from processes relating to the production of wine products
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention discloses monascus mauritianus and application thereof, and particularly relates to application of monascus mauritianus in treatment of ultra-high concentration white spirit wastewater UHS-LW. The invention also provides monascus mauritianus for treating ultra-high concentration white spirit wastewater UHS-LW, which is obtained from a Maotai-flavor white spirit brewing environment, is numbered YX-1125, is an extreme filamentous fungus, and has the preservation number of CGMCC 21938. According to the invention, through three-dimensional optimization of pretreatment, process and metabolism, the optimal conditions for producing short-chain fatty acid by directly fermenting UHS-LW without saccharification are obtained, and theoretical basis and technical support are provided for resource utilization of high-concentration brewing wastewater.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to application of microorganisms in UHS-LW treatment of ultra-high concentration white spirit wastewater.
Technical Field
Maotai-flavor liquor is also called world's three famous wines together with Scotland whisky and French cognac brandy. The production of the distilled liquor is from selected local organic crops, and is a precise combination of traditional brewing process which is inherited for hundreds of years and local unique microbial flora. However, this unique brewing process and unique microbial flora also results in unique metabolic byproduct buildup. A highly viscous black-brown waste liquid which emits a pungent odor is one of the main by-products in the process of brewing of liquor with sauce flavor. This waste water, also known as Maotai-flavor yellow water, pit bottom water or percolation waste water, is currently not formally referred to uniformly.
Through earlier researches, the invention analyzes the main composition and characteristics of the waste water, and proves that the waste water mainly contains a large amount of soluble organic matters, such as polysaccharides (more than 60g/L, mainly starch and mannan), organic acids (more than 80g/L, mainly lactic acid and acetic acid), ethanol (more than 30g/L), glycerol (more than 50g/L) and the like, and the High-concentration soluble organic matters cause the chemical oxygen demand to be usually more than 350000mg/L, so the waste water is named as Ultra-High-Strength Liquor Waterstewater (UHS-LW). Improper treatment of UHS-LW can lead to serious environmental problems such as groundwater and drinking water contamination, ground vegetation damage, etc. More seriously, environmental changes may lead to irreversible changes in the composition of the unique microbial community on which the brewing industry lives, which in turn leads to deterioration in liquor quality and ultimately to the entire industry.
However, the current treatment of such wastewater is extremely inefficient and costly. According to the standard of liquor sewage treatment charging in Huai city of the Minn Huai province of Guizhou in 2020, UHS-LW belongs to 4-grade wastewater concentration gradient, and the treatment cost is as high as 600 yuan/ton, which is more than 300 times of the treatment cost of common urban sewage. It was found through preliminary studies that the reason for this is that UHS-LW has multiple extreme environmental characteristics of high alcohol degree (> 3%), high acidity (pH < 3.5), and high salinity (greater than 3%) in addition to extremely high COD value, and anaerobic/aerobic microorganisms in conventional sewage treatment are difficult to survive in such extreme environments, resulting in high cost and low treatment efficiency.
The optimal treatment is to convert the large amount of bioenergy and organic matter contained in UHS-LW into high value products. By the method, the environmental pollution is reduced, the waste water treatment cost is reduced, and the precious biological resources can be recycled at the same time. In recent years, the production of Short-Chain fatty acids (SCFAs) from various organic waste water or industrial and agricultural wastes has become a research focus at home and abroad. The short-chain fatty acid is an organic acid with 2-6 carbon atoms, is widely applied to the fields of food, chemical industry, feed and medicine, and is also a precursor for synthesizing various chemical products. In particular, in recent years, studies on the short-chain fatty acids and human health have been actively conducted, and the short-chain fatty acids produced by microbial fermentation are considered as prebiotics capable of treating various diseases, and are considered to have extremely high economic and health values. However, very few microorganisms survive UHS-LW due to the presence of high concentrations of inorganic salts, organic acids and fermentation inhibitors such as ethanol. If a microorganism can be screened out, the microorganism can tolerate multiple environmental pressures in UHS-LW and convert high-concentration organic matters in the microorganism into short-chain fatty acids, which has great significance for resource utilization of the UHS-LW.
Microorganisms capable of growing in extreme environments are collectively referred to as extreme microorganisms. Due to the unique gene type, the special physiological mechanism and the special metabolite, the extreme microorganisms have huge application potential in the fields of industry, agriculture, environmental protection and the like. However, at present, related reports at home and abroad are mostly limited to research on extreme microorganisms (such as thermophilic bacteria, acidophilic bacteria, alkalophilic bacteria, halophilic bacteria and the like) under single-factor environmental stress, and the reports on the extreme microorganisms under multi-factor environmental stress are rare. The unique brewing environments of sauce-flavor liquor, such as extreme high-temperature starter propagation, multiple rounds of stacking fermentation, high-temperature anaerobic fermentation and the like, domesticate brewing microorganisms for a long time, and the enrichment of abundant extreme microorganisms such as salinity resistance, high acidity resistance, high alcohol resistance and the like in the brewing microbial ecological environment is facilitated through succession of microbial communities such as heredity, variation, growth elimination, derivatization and the like of various microorganisms.
Disclosure of Invention
The invention aims to provide application of monascus purpureus in treatment of ultra-high concentration white spirit wastewater and a monascus mauritianus YX-1125 strain for realizing the application, so as to overcome the defects of the prior art.
The invention is realized by the following steps:
the invention provides application of monascus in treatment of ultrahigh-concentration white spirit wastewater. In particular to application of monascus mauritianus in treating ultra-high concentration white spirit wastewater UHS-LW. Wherein the Monascus fungus includes Monascus ruber ATCC 16246(AY498574), Monascus aurantiacas CICC 5014(AY629435), Monascus spurpureus ATCC 16379(AY498573), Monascus spurlosus ATCC 16363T(AY498581)、 Monascus sanguineus ATCC 200613T(AY498586)、Monascus lunisporas ATCC 204397 (AY498583)、Monascus eremophilus ATCC 62925T(AY498584)、Monascus pallens ATCC 200612T(AY498585), Monascus floridanus IMI 282587(AY 629418).
The invention provides a monascus mauritianus for treating ultra-high concentration white spirit wastewater UHS-LW, which is obtained from a maotai-flavor white spirit brewing environment, is numbered YX-1125, is an extreme filamentous fungus, has the preservation number of CGMCC 21938, and has the preservation unit name of: china general microbiological culture Collection center; the address of the preservation unit is as follows: western road No. 1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 03 month 29 in 2021.
In early work, the extreme filamentous fungus with the number of YX-1125 which can grow well in UHS-LW is obtained from a Maotai-flavor liquor brewing environment through screening and purification. The strain is determined to be Monascus pilosus (Monascus pilosus) by fungus morphological identification and ITS and BenA sequence analysis of China center for industrial microorganism preservation. The metabolite composition was analyzed by GC-MS and found to be predominantly short chain fatty acids (80% or more), with by-product alcohols (butanol, propanol and ethanol).
The preliminary research finds that compared with other documents reporting that short-chain fatty acid producing strains or floras are produced by using wastewater, the monascus mauritianus YX-1125 has three main advantages: multiple extreme environmental tolerance: can tolerate multiple extreme environments caused by environmental stress factors such as high salt, high acid and high alcohol; broad spectrum carbon source utilization: can directly utilize various carbohydrates including starch, glucose, mannan, glycerol, lactic acid and the like for fermentation; ③ the high butyric acid ratio: the short-chain fatty acid mainly comprises butyric acid and propionic acid, wherein the content of butyric acid is more than 85%. The result shows that by using the strain YX-1125, only 10g/L of urea is needed to be added, various carbohydrates in UHS-LW can be directly fermented into short-chain fatty acid under the condition of no pretreatment and no exogenous addition, the final concentration of the short-chain fatty acid can reach 19.8g/L, and the method is the highest value for producing the short-chain fatty acid by using wastewater reported in the prior literature. And the butyric acid with the highest added value in the short-chain fatty acid accounts for 85.3 percent, and the COD in the UHS-LW is reduced by 85.1 percent, thereby showing good application potential.
However, the final short chain fatty acid concentration at present is still a certain distance away from the requirements for commercial organic acid production. As the conversion rate of the short-chain fatty acid is only about 7 percent in the current result, the efficiency of producing the short-chain fatty acid by fermenting UHS-LW by using the strain YX-1125 still has great promotion space. In earlier work, applicants found that YX-1125 is more or less tolerant to fermentation environmental stress factor pairs such as salinity, acidity and alcoholicity, and that environmental stresses of varying intensity can affect the final short chain fatty acid make-up. In addition, the invention discovers that the final fermentation result can be greatly influenced by changing partial fermentation conditions. For example, by reducing the dissolved oxygen degree in the fermentation process by half, the yield of the byproduct ethanol is improved by 14 times, namely from 0.77g/L to 10.82g/L, and the yield of the short-chain fatty acid is reduced by more than 50%. The applicants also found that YX-1125, although resistant to the extreme environment of UHS-LW, also caused a large accumulation of bacterial cells (50g/L or more), indicating that the cells grew compensatory to cope with the extreme environment. The compensatory growth will cause more metabolic flows to enter thallus to construct related metabolism such as protein anabolism and polysaccharide anabolism, and less metabolic flows to enter a Reversed Beta-Oxidation (r-BOX) cycle, which is the main synthetic pathway for biologically producing short-chain fatty acids, and finally, the yield of the short-chain fatty acids is lower. And by adding exogenous vitamin C in a preliminary experiment, the compensatory growth of YX-1125 can be effectively relieved, and the conversion rate of short-chain fatty acid is improved, which seems to suggest that the redox imbalance is one of the inherent reasons of compensatory growth of thalli and lower yield of the short-chain fatty acid. The above results indicate that the yield and conversion rate of short-chain fatty acids in YX-1125 fermented UHS-LW can be increased by changing the intensity of environmental stress factors, optimizing fermentation process conditions, and improving redox imbalance in short-chain fatty acid metabolism.
The strain can tolerate multiple extreme environments in UHS-LW and convert complex carbohydrates in wastewater into short-chain fatty acids with high butyric acid ratio. However, the fermentation mechanism of the strain is yet to be further clarified, and the fermentation process needs to be further optimized. Obtaining a pretreatment strategy most beneficial to the generation of the short-chain fatty acid by researching the relationship between the removal and weakening of different environmental stresses and the generation amount of the short-chain fatty acid; from the perspective of fermentation technology, the mechanism of influencing YX-1125 to ferment UHS-LW by oxygen supply, thallus morphology and fermentation mode is respectively researched; from the perspectives of transcriptomics, metabonomics and intracellular redox balance, the influence of multiple extreme environments on the main synthetic pathway r-BOX of the short-chain fatty acid is researched, on the basis, a rational exogenous addition strategy capable of reducing metabolic burden is provided, and the conversion rate of the short-chain fatty acid is improved. Finally, through three-dimensional optimization of pretreatment, process and metabolism, the optimal condition for producing the short-chain fatty acid by directly fermenting UHS-LW without saccharification is obtained, and theoretical basis and technical support are provided for resource utilization of the high-concentration brewing wastewater.
Drawings
FIG. 1 is a macro-morphogram of YX-1125;
FIG. 2 is a microscopic morphology of YX-1125;
FIG. 3 is a graph showing the utilization of the major carbohydrates in UHS-LW by strain YX-1125. A. Lactic acid, glycerol and starch are used as unique carbon sources for independent culture; B. mixed culture of lactic acid, glycerol and starch;
FIG. 4 is a graph showing the tolerance of strain YX-1125 to major environmental stress factors in UHS-LW. A. Ethanol; b, organic acid; C. an inorganic salt; D. fermenting under multiple environmental stresses;
FIG. 5 shows the optimization of fermentation conditions of strain YX-1125. A.b. graph of the effect of temperature on short chain fatty acid yield and COD removal; c.d. influence of urea addition on short chain fatty acid yield and COD removal;
FIG. 6 is a schematic representation of YX-1125 in a repeated batch fermentation mode.
Detailed Description
The present invention is further illustrated by the following examples, which should not be construed as limiting the invention in any way.
Example 1:
(1) screening and identification of extreme microorganisms
A short-chain fatty acid production strain which can grow in UHS-LW and can tolerate multiple extreme environments is screened from a sauce-flavor white spirit brewing production line similar to the UHS-LW environment. Through fungus heterogeneous morphology identification (shown in figures 1 and 2) and filamentous fungus BenA gene detection in China industrial microorganism preservation, filamentous fungus ITS rDNA is detected, and finally, the filamentous fungus ITS rDNA is identified as Monascus pilosus (Monascus pilosus) through phylogenetic analysis.
2.1 the macroscopic morphology of YX-1125 is shown in FIG. 1. Culturing on MEA culture medium at 25 deg.C for 7 days to obtain gray white colony with diameter of 49-53 mm; the surface is flat; velvet-shaped texture; light yellow around the back, middle gray; no exudate and no soluble pigment are produced.
2.2 the microscopic morphology of YX-1125 is shown in FIG. 2. Hypha is irregularly branched, transparent and colorless, has smooth wall and transverse septum, and has a width of 2.5-5.0 μm; conidium is planted on small peduncle or hypha top, and is single-grown or chain-grown, colorless, smooth in wall, inverted pear-shaped or nearly spherical, and the diameter of long axis is 4.5-10 μm; the capsule shell is approximately spherical and has the diameter of 26-30 mu m.
2.3 Strain "YX-1125" phylogenetically develops trees with the ITS rDNA sequences of related species. Adopting MEGA5.0 software, adopting an adjacent connection method to display the strain YX-001 and an ITS rDNA sequence phylogenetic tree of related species, carrying out similarity repeated calculation for 1000 times, wherein the nodes of the phylogenetic tree only display Bootstrap values larger than 70% and the superscript T represents a model strain.
2.4 Strain "YX-1125" phylogenetically tree with the BenA gene sequence of the related species. MeGA5.0 software and an adjacent position connection method are adopted to display a phylogenetic tree of the strain YX-001 and a BenA gene sequence of a related species, 1000 times of similarity repeated calculation and inspection are carried out, nodes of the phylogenetic tree in the graph only display Bootstrap values larger than 70% of numerical values, and the superscript T represents a model strain.
(2) Pre-experiment: strain YX-1125 uses the major carbohydrates in UHS-LW
The main carbon compounds present in UHS-LWW were detected as lactic acid, glycerol and starch. As shown in part A of FIG. 3, YX-1125 can produce short chain fatty acids using the major carbohydrates in UHS-LW. Wherein, the concentration of short-chain fatty acid produced by using lactic acid is highest, the starch absorption is fastest, but the yield of short-chain fatty acid is lowest, and the glycerol is between the two. Notably, when these three carbohydrates were mixed and cultured (part B in FIG. 3), a glucose metabolism repression effect was found, demonstrating that YX-1125 preferentially hydrolyzes starch to glucose in the presence of multiple carbohydrates simultaneously and then serves as a preferential carbon source.
(3) Pre-experiment: tolerance of strain YX-1125 to major environmental stress factors in UHS-LW
The major environmental stress factors in UHS-LW were detected to be ethanol, organic acids (lactic acid + acetic acid) and inorganic salts (sodium chloride + potassium phosphate). Among them, ethanol is considered to be a microbial inhibitor that causes irreversible damage by cell membranes, mitochondria, key metabolic enzymes; organic acids, particularly acetic acid, are believed to directly inhibit microbial growth by disrupting the permeability of the microbial cell membrane; high concentrations of inorganic salts can inactivate microorganisms by allowing the microorganisms to lose water through high osmotic pressure. The tolerance of strain YX-1125 to these environmental stress factors was evaluated by adding ethanol, organic acids and inorganic salts to the simulated wastewater. As a result, YX-1125 was able to tolerate ethanol, organic acids and inorganic salts at high concentrations, respectively, and showed good tolerance to multiple extreme environments, with the dry weight of both short-chain fatty acids and bacterial cells rising in the presence of multiple environmental stress factors, as shown in FIG. 4.
(4) Pre-experiment: fermentation condition optimization
The fermentation temperature and urea addition are regulated and controlled through a pre-experiment, the influence of the urea addition on the removal of short-chain fatty acid and COD in the production of the real UHS-LW wastewater by YX-1125 fermentation is inspected, and the optimized result is used in the invention. As shown in FIG. 5, the strain YX-1125 is selected from the self-high-temperature stacking fermentation in the production of Maotai-flavor liquor, so that the strain YX-1125 can adapt to a higher fermentation temperature, but the short-chain fatty acid yield is not remarkably increased when the temperature is continuously increased, and the COD removal rate is also reduced to a certain extent, so that the optimum temperature is determined to be 35 ℃. The nitrogen source addition has very obvious influence on both the yield of the YX-1125 short-chain fatty acid and the removal of COD, the final nitrogen source addition amount is 6.0g/L, and the removal rate of the COD can reach 80.5 percent.
(5) Optimization pre-experiment of fermentation process: repeated batch fermentation
The project applicant first tested a repeated batch fermentation mode to see if fermentation process control would have an impact on short chain fatty acid production. As shown in FIG. 6, when the repeat batch reached the 5 th time, the short chain fatty acid production reached a maximum of 19.8g/L, corresponding to a 4.95g/L/d yield with butyric acid at a percentage of up to 89.5%. The yield and the productivity of the method reach the highest level of the wastewater produced short-chain fatty acid reported in the literature at present, and the method shows great optimization potential.
Three reasons are preliminarily presumed to lead to the production of high concentrations of short chain fatty acids, namely, high concentrations of fermentation substrate, compared with the wastewater used for producing short chain fatty acids by using wastewater reported in other documents, the carbohydrate content of the wastewater is generally below 30g/L, and the carbohydrate concentration of our substrate reaches above 260g/L, so that YX-1125 can still obtain higher final concentrations of short chain fatty acids even under the condition of lower conversion rate (about 7%). The second reason may be that a single strain fermentation strategy is adopted, and most of the experiments reported in the literature adopt mixed strains collected from a sludge pond or the field for fermentation, wherein the wild mixed strains not only contain short-chain fatty acid production strains, but also contain short-chain fatty acid consumption strains (such as methane bacteria), so that the concentration of the short-chain fatty acid in the fermentation is in a dynamic change. The single strain fermentation strategy adopted by the invention avoids the existence of short-chain fatty acid consumption strains, so that the short-chain fatty acid can be accumulated to a higher concentration, and the final concentration of the short-chain fatty acid with higher concentration reported in the literature at present adopts a single strain fermentation mode. The third reason is the aerobic fermentation strategy. While most of the literature uses anaerobic fermentation mode. It is known that aerobic microorganisms have a much higher metabolic strength than anaerobic microorganisms due to the presence of the tricarboxylic acid cycle. In contrast, when we reduced the oxygen supply during fermentation, the by-product ethanol increased 14-fold and the production of short chain fatty acids also decreased significantly, as shown in part B of figure 6. This result suggests that YX-1125 has a higher dependence of short-chain fatty acid synthesis on aerobic respiration, and that the production of short-chain fatty acids can be further improved by optimizing the fermentation process control.
The above are only specific application examples of the present invention, and other embodiments of the present invention are within the scope of the present invention as claimed by using equivalent alternatives or equivalent variations.
Claims (4)
1. Application of Monascus purpureus in treating waste water of ultra-high concentration white spirit.
2. Application of Monascus pilosus in treating UHS-LW (ultra high concentration white spirit) wastewater.
3. The use of monascus purpureus of claim 1 in the treatment of ultra-high concentration white spirit wastewater, wherein: said Monascus fungus includes Monascus ruber ATCC 16246(AY498574), Monascus aurantiacas CICC 5014(AY629435), Monascus purpureus ATCC 16379(AY498573), Monascus pilosus ATCC 16363T(AY498581)、Monascus sanguineus ATCC 200613T(AY498586)、Monascus lunisporas ATCC 204397(AY498583)、Monascus eremophilus ATCC 62925T(AY498584)、Monascus pallens ATCC 200612T(AY498585), Monascus floridanus IMI 282587(AY 629418).
4. A monascus mauritianus for treating ultra-high concentration white spirit wastewater is characterized in that: the Monascus pilosus is obtained from brewing environment of Maotai-flavor Chinese liquor, is YX-1125, is an extreme filamentous fungus, and has a preservation number of CGMCC 21938.
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| CN116162552B (en) * | 2023-01-10 | 2023-10-20 | 成都宝鹿生物科技有限公司 | Preparation method and application of extremely red aspergillus sorghum fermentation product |
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| CN113388525B (en) | 2023-07-18 |
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