CN113388525B

CN113388525B - Application of monascus in treatment of ultra-high concentration white spirit wastewater

Info

Publication number: CN113388525B
Application number: CN202110493528.7A
Authority: CN
Inventors: 郑羽西; 宋桐伊; 张锡梅
Original assignee: Moutai University
Current assignee: Moutai University
Priority date: 2021-05-07
Filing date: 2021-05-07
Publication date: 2023-07-18
Anticipated expiration: 2041-05-07
Also published as: CN113388525A; CN116354520A

Abstract

The invention discloses a strain of bacteria Mao Gongqu and application thereof, in particular to application of bacteria Mao Gongqu in treating ultra-high concentration white spirit wastewater UHS-LW. The invention also provides a strain of Mao Gongqu of the strain Mao Gongqu for treating UHS-LW (ultra-high concentration white spirit) wastewater, which is obtained from a Maotai-flavor white spirit brewing environment, and has the number of YX-1125, and is an extreme filamentous fungus with the preservation number of CGMCC 21938. The optimal condition for producing the short-chain fatty acid by directly fermenting UHS-LW without saccharification is obtained through optimization of pretreatment, technology and metabolism in three dimensions, and theoretical basis and technical support are provided for the resource utilization of high-concentration brewing wastewater.

Description

Application of monascus in treatment of ultra-high concentration white spirit wastewater

Technical Field

The invention relates to the technical field of microorganisms, in particular to application of microorganisms in UHS-LW (ultra high concentration white spirit wastewater) treatment.

Technical Field

Maotai-flavor liquor and Scotland whiskey, french family, nike brandy are also called world three-large-name liquor. The distilled liquor is produced by finely selecting local organic crops, and is precisely combined with local unique microorganism flora by a traditional brewing process for hundreds of years. However, this unique brewing process and unique microbial flora also result in a unique accumulation of metabolic byproducts. A dark brown waste liquid which gives off a pungent smell and has a high viscosity is one of the main by-products in the brewing process of Maotai-flavor wine. This waste water is also called soy sauce yellow water, pit water or percolation waste water, and is not formally and uniformly named at present.

Through previous researches, the invention analyzes the main constitution and characteristics of the wastewater, and discovers that the wastewater mainly contains a large amount of soluble organic matters such as polysaccharide (more than 60g/L, mainly starch and mannans), organic acid (more than 80g/L, mainly lactic acid and acetic acid), ethanol (more than 30 g/L) and glycerol (more than 50 g/L), and the like, and the chemical oxygen demand of the soluble organic matters is usually more than 350000mg/L, so the wastewater is named as Ultra-High-Strength Liquor Wastewater, UHS-LW. Improper treatment of UHS-LW can lead to serious environmental problems such as groundwater and potable water pollution, ground vegetation destruction, etc. More seriously, the change of environment may cause irreversible change of unique microbial community constitution which the wine industry depends on, and further cause deterioration of quality of white spirit, ultimately endangering the whole industry.

However, the efficiency of treating such wastewater is currently low and the cost and high. According to the charging standard of white spirit sewage treatment in the Hui city of Guizhou province in 2020, UHS-LW belongs to the grade 4 of the concentration gradient of the wastewater, and the treatment cost is up to 600 yuan/ton and is more than 300 times of that of common urban sewage treatment. Preliminary studies have found that the reason for this is that UHS-LW has multiple extreme environmental characteristics of high alcohol content (> 3%), high acidity (pH < 3.5) and high salinity (more than 3%) in addition to extremely high COD values, 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 organics contained in UHS-LW into high value products. By the method, the environmental pollution is reduced, the wastewater treatment cost is reduced, and the recovery of precious biological resources can be realized simultaneously. In recent years, the production of Short Chain fatty acids (Short-Chain FattyAcids, SCFAs) from various organic waste water or industrial and agricultural wastes has become a research hotspot at home and abroad. Short-chain fatty acid is an organic acid with carbon atoms between 2 and 6, 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, research on short chain fatty acids and human health has been underway, and short chain fatty acids produced by microbial fermentation are considered as a prebiotic capable of treating various diseases and are considered to have extremely high economic and health values. However, due to the simultaneous presence of high concentrations of inorganic salts, organic acids and fermentation inhibitors such as ethanol, few microorganisms are able to survive in UHS-LW. If a microorganism can be screened, the UHS-LW can withstand multiple environmental pressures and convert high-concentration organic matters in the UHS-LW into short-chain fatty acids, which has great significance for the 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, special physiological mechanism and special metabolite, the extreme microorganism has great 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 thermophiles, acidophilic bacteria, alkalophiles, halophiles and the like) which are under single-factor environmental stress, and rare reports of extreme microorganisms which are under multi-factor environmental stress are considered. The unique extremely high temperature starter propagation, multi-round stacking fermentation, high temperature anaerobic fermentation and other brewing environments of the Maotai-flavor white spirit are used for domesticating brewing microorganisms for a long time, and various microorganisms are subjected to succession of microorganism communities such as inheritance, mutation, growth elimination, derivatization and the like, so that enrichment of abundant extremely microorganisms such as salinity resistance, high acidity resistance, high alcohol resistance and the like in the brewing micro-ecological environment is facilitated.

Disclosure of Invention

The invention aims to provide application of monascus in treating ultra-high concentration white spirit wastewater and Mao Gongqu bacteria YX-1125 for realizing the application, so as to overcome the defects of the prior art.

The invention is realized in the following way:

the invention provides application of monascus in treating ultra-high concentration white spirit wastewater. In particular to application of Mao Gongqu bacteria in treating ultra-high concentration white spirit wastewater UHS-LW. Wherein the Monascus include Monascus ruber ATCC 16246 (AY 498574), monascus aurantiacus CICC 5014 (AY 629435), monascuspurpureus ATCC 16379 (AY 498573), monascuspilosus ATCC 16363 ^T (AY498581)、 Monascus sanguineus ATCC 200613 ^T (AY498586)、Monascus lunisporas ATCC 204397 (AY498583)、Monascus eremophilus ATCC 62925 ^T (AY498584)、Monascus pallens ATCC 200612 ^T (AY 498585), monascusfloridanus IMI 282587 (AY 629418).

The invention provides a Mao Gongqu strain for treating ultra-high concentration white spirit wastewater UHS-LW, wherein the Mao Gongqu strain is obtained from a Maotai-flavor white spirit brewing environment, the number is YX-1125, and the strain is an extreme filamentous fungus, the preservation number is CGMCC 21938, and the preservation unit name is: china general microbiological culture Collection center (China Committee for culture Collection); the preservation unit addresses are: the institute of microbiology, national academy of sciences, north chen xi lu 1, 3, the region of the morning sun in beijing; the preservation date is: 2021, 03 and 29.

In the earlier work, the invention obtains a strain of extreme filamentous fungi with the number of YX-1125 from the brewing environment of the Maotai-flavor liquor through screening and purifying, and can grow well in UHS-LW. Fungus morphology identification and ITS and BenA sequence analysis by China center for type industry microbiological collection center confirm that the strain is from Mao Gongqu bacteria (Monascus pilosus). The metabolite composition was analyzed by GC-MS and found to be mainly short chain fatty acids (80% or more), alcohols (butanol, propanol and ethanol) as by-products.

Preliminary researches show that compared with other short-chain fatty acid production strains or flora reported in other documents, the strain or flora produced by utilizing wastewater has three main advantages from Mao Gongqu strain YX-1125: (1) multiple extreme environmental tolerance: can endure multiple extreme environments caused by environmental stress factors such as high salt, high acid, high alcohol and the like; (2) broad spectrum carbon source utilization: can directly utilize various carbohydrates including starch, glucose, mannans, glycerol, lactic acid and the like for fermentation; (3) high butyrate ratio: the short chain fatty acids are mainly composed of butyric acid and propionic acid, wherein the ratio of butyric acid is above 85%. The result shows that by utilizing the strain YX-1125, only 10g/L urea is needed to be added, and various carbohydrates in UHS-LW can be directly fermented into short-chain fatty acids without pretreatment and exogenous addition, and the final concentration of the short-chain fatty acids can reach 19.8g/L, which is the highest value reported in the current literature for producing short-chain fatty acids by utilizing wastewater. And the butyric acid with the highest added value in the short-chain fatty acid accounts for 85.3 percent, and meanwhile, the COD in the UHS-LW is reduced by 85.1 percent, thus showing good application potential.

However, the current final short chain fatty acid concentration is still a distance from the requirements of commercial organic acid production. Because the conversion rate of short chain fatty acid in the current result is only about 7%, the efficiency of fermenting UHS-LW by utilizing the strain YX-1125 still has great improvement. In earlier work, the applicant found that YX-1125 was tolerant to different degrees of fermentation environmental stress factors such as salinity, acidity and alcohol degree, and that environmental stress of different intensities could affect the constitution of the final short chain fatty acid. In addition, the invention finds that changing part of the fermentation conditions can greatly influence the final fermentation result. For example, by reducing the oxygen solubility in the fermentation process by half, the yield of ethanol as a by-product would be increased 14-fold from 0.77g/L to 10.82g/L, while the yield of short chain fatty acids would be reduced by more than 50%. The applicant has also found that although YX-1125 is able to withstand the multiple extreme environments in UHS-LW, it also results in a large accumulation of cells (above 50 g/L), indicating that the cells are compensatory grown in order to cope with the extreme environments. Such compensatory growth will result in more metabolic flux into the bacterial cell to build related metabolism such as protein anabolism and polysaccharide anabolism, and less metabolic flux into the reverse Beta-Oxidation (r-BOX) cycle, which is the primary synthetic pathway for the biological production of short chain fatty acids, ultimately resulting in lower short chain fatty acid production. By adding exogenous vitamin C in the pre-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 redox imbalance is one of the intrinsic reasons of bacterial compensatory growth and lower short-chain fatty acid yield. The above results indicate that the yield and conversion of short chain fatty acids of the YX-1125 fermentation UHS-LW can be increased by varying the intensity of the environmental stress factors, optimizing the fermentation process conditions, and improving the redox imbalance in the short chain fatty acid metabolism.

The strain is able to withstand the multiple extreme environments in UHS-LW and converts complex carbohydrates in wastewater into short chain fatty acids with high butyrate ratios. However, the fermentation mechanism of the strain is still to be further clarified, and the fermentation process is still to be further optimized. The pretreatment strategy which is most favorable for the production of the short-chain fatty acid is obtained by researching the relation between the removal and weakening of different environmental stresses and the production of the short-chain fatty acid; from the perspective of fermentation process, the mechanism of oxygen supply, thallus morphology and fermentation mode affecting the fermentation of the UHS-LW by the YX-1125 is respectively studied; from the aspects of transcriptomics, metabonomics and intracellular redox balance, the influence of multiple extreme environments on the main synthesis pathway r-BOX of short-chain fatty acids is studied, and on the basis, a rational exogenous addition strategy capable of reducing metabolic burden is provided, so that the conversion rate of the short-chain fatty acids is improved. Finally, through optimization of pretreatment, technology and metabolism in three dimensions, 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 recycling of high-concentration brewing wastewater.

Drawings

FIG. 1 is a macroscopic morphology of YX-1125;

FIG. 2 is a microscopic morphology of YX-1125;

FIG. 3 is a graph showing the utilization of primary 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 the major environmental stress factors in UHS-LW. A. Ethanol; b, organic acid; C. an inorganic salt; D. multiple environmental stress fermentation;

FIG. 5 shows the fermentation condition optimization of the strain YX-1125. A.b. graph of the effect of temperature on short chain fatty acid yield and COD removal; c.d. effects 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 invention is further illustrated, but is not intended to be limited, by the following examples.

Example 1:

(1) Screening and identification of extreme microorganisms

From a Maotai-flavor liquor brewing production line similar to the UHS-LW environment, a short-chain fatty acid production strain which can grow in the UHS-LW and can withstand multiple extreme environments is screened. Through the heterogeneous morphological identification of fungi in China industry microbiological culture Collection (see FIGS. 1 and 2) and the detection of the BenA gene of the filamentous fungus, the ITS rDNA detection of the filamentous fungus is finally identified as being from Mao Gong Aspergillus (Monascus pilosus) by phylogenetic analysis.

2.1 macroscopic morphology of YX-1125 is shown in FIG. 1. Culturing on MEA culture medium at 25deg.C for 7 days, wherein colony diameter is 49-53mm, and the colony is off-white; the surface is flat; texture velvet shape; pale yellow surrounding the back face, central grey; no exudates and no soluble pigments are produced.

2.2 the microscopic morphology of YX-1125 is shown in FIG. 2. The mycelium branches irregularly, is transparent and colorless, has smooth wall and transverse partition, and has width of 2.5-5.0 μm; conidium is planted on small stalk or mycelium top, single or chain is planted, colorless, smooth wall, inverted pear shape or nearly spherical, long axis diameter is 4.5-10 μm; the capsule shell is nearly spherical and has a diameter of 26-30 μm.

2.3 Strain "YX-1125" phylogenetic tree with related species of ITS rDNA sequences. Adopting MEGA5.0 software, displaying the phylogenetic tree of the strain YX-001 and the ITS rDNA sequences of related species by adopting an orthotopic connection method, and carrying out similarity repeated calculation for 1000 times, wherein in the figure, the node of the phylogenetic tree only displays the value of Bootstrap which is larger than 70%, and the superscript "T" represents the model strain.

2.4 Strain "YX-1125" phylogenetic tree with the BenA gene sequences of the related species. Adopting MEGA5.0 software, displaying a phylogenetic tree of the gene sequences of the strain YX-001 and the BenA of related species by an orthotopic connection method, performing 1000 times of repeated calculation and detection of similarity, wherein in the figure, a node of the phylogenetic tree only shows a numerical value with a Bootstrap value of more than 70%, and the superscript 'T' represents a model strain.

(2) Pre-experiment: utilization of main carbohydrate in UHS-LW by strain YX-1125

The primary carbon compounds present in UHS-LWW were detected to be lactic acid, glycerol and starch. As shown in FIG. 3, part A, YX-1125 is capable of producing short chain fatty acids using the primary carbohydrate in UHS-LW. Among these, the concentration of short chain fatty acids produced using lactic acid is highest, starch absorption is fastest, but short chain fatty acids yield is lowest, and glycerol is between the two. Notably, upon mixed culture of these three carbohydrates (part B of fig. 3), a glucose catabolism repression effect was found, demonstrating that YX-1125 preferentially hydrolyzes starch to glucose when multiple carbohydrates are present simultaneously as a preferential carbon source.

(3) Pre-experiment: tolerance of strain YX-1125 to major environmental stress factors in UHS-LW

The main environmental stress factors in UHS-LW are ethanol, organic acids (lactic acid+acetic acid) and inorganic salts (sodium chloride+potassium phosphate) as detected. Among them, ethanol is considered as a microbial inhibitor that is irreversibly damaged by cell membranes, mitochondria, key metabolic enzymes; organic acids, particularly acetic acid, are believed to be capable of directly inhibiting microbial growth by disrupting microbial cell membrane permeability; the high concentration of inorganic salts is capable of losing water from the microorganism through high osmotic pressure, thereby losing activity. The tolerance of the strain YX-1125 to these environmental stress factors was evaluated by adding ethanol, organic acid and inorganic salt to the simulated wastewater. As shown in FIG. 4, YX-1125 is capable of tolerating ethanol, organic acid and inorganic salt at high concentrations, respectively, and in the presence of multiple environmental stress factors, both short chain fatty acids and bacterial dry weight are increased, exhibiting good multiple extreme environmental tolerance.

(4) Pre-experiment: fermentation condition optimization

The influence of the fermentation temperature and urea addition on the removal of short-chain fatty acid and COD in the production of the real UHS-LW wastewater by fermenting YX-1125 is examined through pre-experiment regulation and control, and the optimized result is used in the invention. As shown in FIG. 5, the strain YX-1125 was able to adapt to a higher fermentation temperature in the self-high temperature bulk fermentation in the production of Maotai-flavor liquor, but the continuous increase in temperature did not significantly increase the short-chain fatty acid yield, and the COD removal rate was also somewhat decreased, so that the optimum temperature was set at 35 ℃. The addition of the nitrogen source has very obvious influence on the yield of the short chain fatty acid of YX-1125 and the removal of COD, the final addition amount of the nitrogen source is 6.0g/L, and the removal rate of the COD at the moment can reach 80.5 percent.

(5) Fermentation process optimization pre-experiment: repeated batch fermentation

The applicant first tested a repeated batch fermentation mode to investigate whether fermentation process control would have an impact on short chain fatty acid production. As a result, as shown in FIG. 6, when the repeated batch reached the 5 th time, the short chain fatty acid yield reached the highest value of 19.8g/L, corresponding to a yield of 4.95g/L/d, in which the butyric acid content was as high as 89.5%. The yield and the productivity reach the highest level of the short-chain fatty acid produced by the wastewater reported in the current literature, and the method has huge optimizing potential.

There are three primary reasons for the high concentration of short chain fatty acid production, first the high concentration of fermentation substrate, generally below 30g/L of medium carbohydrate content of wastewater used in the production of short chain fatty acids from wastewater reported in other literature, and our substrate carbohydrate concentration above 260g/L, which allows YX-1125 to obtain higher final concentration of short chain fatty acids even at lower conversion (around 7%). The second reason is probably because of the single strain fermentation strategy adopted, and most of the experiments reported in the literature use mixed strains collected from a sludge pond or the field for fermentation, and the wild mixed strains contain not only short-chain fatty acid production strains but also short-chain fatty acid consumption strains (such as methane bacteria), so that the concentration of the short-chain fatty acids 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 short-chain fatty acids can be accumulated to a higher concentration, and the final concentration of the higher short-chain fatty acids reported in the prior literature adopts a single strain fermentation mode. The third reason is the aerobic fermentation strategy. While most literature uses anaerobic fermentation modes. It is well known that aerobic microorganisms have a much stronger metabolic strength than anaerobic microorganisms because of the presence of tricarboxylic acid cycle. In contrast, when we reduced the oxygen supply during fermentation, the ethanol by-product increased by a factor of 14, and the yield of short chain fatty acids was also significantly reduced, as shown in part B of fig. 6. The results suggest that the short chain fatty acid synthesis of YX-1125 is highly dependent on aerobic respiration, and that the short chain fatty acid yield can be further improved by performing fermentation process control optimization.

The above is only a specific application example of the present invention, and other embodiments of the present invention are also possible, and all the technical solutions formed by equivalent substitution or equivalent transformation fall within the scope of protection claimed by the present invention.

Claims

1. From Mao Gong AspergillusMonascus pilosus) The application in treating the ultra-high concentration white spirit wastewater is characterized in that: the aspergillus Mao Gong is obtained from the brewing environment of Maotai-flavor white spirit, and the number is YX-1125, is an extreme filamentous fungus, and has a preservation number of CGMCC 21938.