CN109762767B - Sewage composite microbial treatment agent and preparation method and application thereof - Google Patents
Sewage composite microbial treatment agent and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a sewage composite microorganism treating agent and a preparation method and application thereof. The sewage composite microorganism treating agent can obviously reduce the viscosity of high-concentration aquaculture sewage after being used. After the sewage is treated by the treating agent for a period of time, the viscosity is reduced by over 60-95%; the reduction of the sewage viscosity obviously reduces the risks of pipeline blockage and unsmooth flow in the subsequent treatment steps, and improves the sewage treatment efficiency; and the competitive inhibition effect among the four bacteria is not obvious, and the extracellular enzymes generated by the four bacteria respectively have the overlapping and synergistic effect on reducing the viscosity.
Description
Technical Field
The invention relates to a sewage composite microorganism treating agent, a preparation method and application thereof, belonging to the technical field of livestock breeding sewage treatment.
Background
In recent years, with the rapid development of the economy of China and the increasing improvement of the living standard of people, the market demands for meat, eggs, milk and other livestock and poultry products are more and more large, and the vigorous market demands and the innovation of breeding varieties and breeding technologies drive the vigorous development of the livestock and poultry breeding industry of China.
In the world, livestock developed countries represented by Australia, New Zealand, Canada and the like have abundant grassland and natural resources due to the fact that vast numbers of people are rare, the livestock breeding industry mainly adopts large-scale grazing management and is assisted by modern product deep processing, the livestock carrying capacity of unit land is low, and the environmental pressure is low; however, the population of China is large, natural resources such as land, water and the like are relatively short, the livestock breeding industry is mainly developed in a large-scale and intensive manner at the present stage, and although the unit production efficiency is relatively high, the problems of huge total amount, high concentration, difficulty in treatment and the like of livestock breeding waste are caused, and the environmental pressure is prominent.
The large-scale breeding of live pigs, cows, beef cattle and the like consumes huge water, and besides the water consumption for animal drinking, a large amount of water resources are consumed for flushing excrement, cleaning a milking parlor, keeping the environment of a breeding area sanitary and the like. Correspondingly, the sewage generated by the large-scale farm comprises animal urine, excrement mixture, field flushing water and other sources, the components are complex, the concentration is higher, the COD can reach 5000-. At present, the harmless treatment of high-concentration culture sewage is still difficult, and the cost is huge.
Because the breeding sewage is often mixed with a large amount of animal wastes, feed, body hair and other substances, the solid content is high, the viscosity is high, and when the breeding sewage is treated by adopting conventional sewage purification modes such as an oxidation pond, a biological wetland and the like, pipelines are easily blocked, and the breeding sewage is attached and retained in treatment equipment, so that the treatment efficiency is seriously influenced, and even the purification equipment is failed. Although solid matters can be removed primarily by increasing and improving a primary sedimentation tank and a secondary sedimentation tank in the production process, the viscosity of the sewage is difficult to reduce, and the treatment of the high-viscosity breeding sewage is one of the problems troubling the development of the industry.
The aquaculture sewage is rich in components such as polysaccharide, protein, filamentous fibers and the like, and the components are crosslinked with each other, so that the viscosity of the sewage is greatly increased. Some kinds of lactic acid bacteria, bacillus and the like have high protease, polysaccharase and cellulase activity, can efficiently decompose protein, polysaccharide, filamentous fibers and the like in the sewage, and may help to reduce the viscosity of the sewage to some extent.
Chinese patent document CN109010883A (application No. 201810616325.0) discloses an environment-friendly microbial deodorant, wherein active ingredients dispersed in a culture medium include lactobacillus plantarum, bacillus subtilis, enterococcus faecalis, and yeast, and the ratio of effective viable count of the lactobacillus plantarum, the bacillus subtilis, the enterococcus faecalis, and the yeast is 1:1:1: 1. the invention selects and matches active bacteria, the active components contain four bacterial combinations which have similar contents and are mutually adaptive, and the invention is suitable for the complex and changeable deodorization requirements of urban and rural domestic garbage and domestic wastewater.
The microorganisms in the deodorant can be deodorized by N, S and other substances in the wastewater, but the problem of high viscosity in the culture wastewater cannot be solved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a sewage composite microorganism treating agent and a preparation method and application thereof.
Technical term description of the present invention:
a microbial treatment agent: the live bacteria preparation is prepared with one or several kinds of microbial strains and other supplementary material and through certain process, and may be used widely in food production, feed processing, sewage treatment and other fields.
Breeding sewage: the sewage generated and discharged by large-scale livestock and poultry breeding generally comprises animal urine, excrement, flushing water of a farm and other different sources, and has high concentration, high viscosity and a large amount of organic pollutants. The harmless treatment of the breeding sewage is one of the keys for realizing the green sustainable development of the large-scale livestock and poultry breeding industry.
Viscosity: also known as viscosity, is the resistance that a fluid exhibits to flow. When fluid flows, one part of the fluid is subjected to resistance on the other part of the surface, namely the internal friction of the fluid.
Bacillus: generally, it refers to a microorganism of the genus Bacillus, gram-positive, capable of producing spores with particular resistance to adverse conditions. Widely distributed in soil, plant surface, air, animal intestinal tract and other environment, has strong resistance to external bad stimulation, is facultative anaerobe, and can survive well in both anaerobic and aerobic environment.
Lactic acid bacteria: lactic acid bacteria are not taxonomic units and generally refer to a class of gram-positive bacteria capable of fermenting sugars to produce lactic acid. The lactobacillus is widely distributed in nature and has good application prospect in different fields of medicine, industry, agriculture and the like.
Colony Forming Units (CFU, Colony-Forming Units): the total number of colonies in a unit area counted by a plate counting method is used for representing the total number of viable bacteria.
The technical scheme of the invention is as follows:
a sewage composite microorganism treating agent comprises a microorganism thallus and an adsorption stabilizer, wherein the microorganism thallus at least contains bacillus megaterium, bacillus subtilis, lactobacillus plantarum and lactobacillus brevis, wherein:
bacillus megaterium with viable count not less than 1.0 × 1010CFU/g；
Bacillus subtilis with viable count not less than 1.0 × 1010CFU/g；
Lactobacillus plantarum with viable count not less than 7.5 × 109CFU/g；
Lactobacillus brevis with viable count not less than 2.5 × 109CFU/g。
According to the invention, the components of the adsorption stabilizer are as follows, and the components are all in parts by weight:
1-2 parts of trehalose, 3-6 parts of agar powder and 4-8 parts of skim milk powder.
According to a further preferred embodiment of the present invention, the adsorption stabilizer comprises the following components in parts by weight:
1 part of trehalose, 4 parts of agar powder and 5 parts of skim milk powder.
According to the invention, the adsorption stabilizer accounts for 40-60% of the total mass of the treating agent.
Preferably, according to the invention, the Bacillus megaterium is from the American type culture Collection, with the strain number ATCC 14945 or ATCC 11562.
Preferably, the bacillus subtilis is from American type culture collection with the strain number of ATCC 15134, or from China general microbiological culture collection center with the strain number of CGMCC 1.821.
Preferably, the lactobacillus plantarum is derived from American type culture Collection (ATCC 8014) or from China general microbiological culture Collection center (CGMCC) 1.568.
Preferably, according to the invention, the Lactobacillus brevis is derived from the American type culture Collection, and the strain number is ATCC367 or ATCC 13648.
According to the invention, the ratio of viable count of the bacillus megaterium, the bacillus subtilis, the lactobacillus plantarum and the lactobacillus brevis is (2-4): 1-2.
More preferably, the ratio of the viable count of the bacillus megaterium, the bacillus subtilis, the lactobacillus plantarum and the lactobacillus brevis is 4:4:3: 1.
According to the invention, the microbial thallus is preferably a microbial thallus acclimated by culture sewage.
The preparation method of the sewage composite microorganism treating agent comprises the following steps:
(1) respectively carrying out activation culture, shaking shake culture, seed culture and fermentation culture on bacillus megatherium, bacillus subtilis, lactobacillus plantarum and lactobacillus brevis to prepare each viable bacteria fermentation liquor;
the viable bacteria concentration of the bacillus megaterium fermentation liquor and the bacillus subtilis fermentation liquor is 2.0-3.0 multiplied by 109CFU/mL；
The viable bacteria concentration of the lactobacillus plantarum fermentation liquor and the lactobacillus brevis fermentation liquor is 1.0-2.0 multiplied by 109CFU/mL；
(2) Mixing bacillus megaterium fermentation liquor, bacillus subtilis fermentation liquor, lactobacillus plantarum fermentation liquor and lactobacillus brevis fermentation liquor to prepare mixed bacterial liquor; adding an adsorption stabilizer into the mixed bacterial liquid, and carrying out solid-liquid separation and drying to obtain the composite microbial treatment agent.
Preferably, the culture conditions of Bacillus megaterium and Bacillus subtilis in step (1) are as follows:
the activation culture conditions are as follows: static culture is carried out for 24 hours at 37 ℃, and a culture medium for activation culture is an LB solid culture medium;
shake flask shake culture conditions were all: culturing for 24 hours at 37 ℃ and the rotating speed of 180r/min, wherein the culture medium for shake flask culture is LB liquid culture medium;
the seed culture conditions are as follows: culturing for 24 hours at 37 ℃ and the rotating speed of 180r/min, wherein the culture medium for seed culture is LB liquid culture medium;
the fermentation culture conditions are as follows: culturing for 24 hours at 37 ℃ and 180r/min of rotation speed, wherein the culture medium for fermentation culture is LB liquid culture medium added with 2 percent of glucose by mass.
Preferably, the culture conditions of lactobacillus plantarum and lactobacillus brevis in the step (1) are as follows:
the activation culture conditions are as follows: static culture is carried out for 24 hours at the temperature of 30 ℃, and a culture medium for activation culture is an MRS solid culture medium;
the triangular flask static culture conditions are as follows: static culture is carried out for 24 hours at 30 ℃, and a culture medium for static culture in a triangular flask is an MRS liquid culture medium;
the seed culture conditions are as follows: static culturing at 30 deg.c for 24 hr in MRS liquid culture medium;
the fermentation culture conditions are as follows: and (3) performing static culture at 30 ℃ for 24 hours, wherein the culture medium for fermentation culture is an MRS liquid culture medium added with 1% glucose by mass.
According to the present invention, before the preparation of the mixed bacterial liquid, the method further comprises an acclimatization step:
(i) taking culture sewage, carrying out solid-liquid separation, and taking liquid; sterilizing, and carrying out solid-liquid separation to obtain sterilized sewage;
(ii) respectively mixing the bacillus megaterium fermentation liquor and the bacillus subtilis fermentation liquor with the sterilized sewage in the step (i) according to the volume ratio of 1 (2-6), and carrying out aerobic culture at 37 ℃ for 24 hours; solid-liquid separation, namely respectively collecting bacillus megatherium and bacillus subtilis thalli;
mixing lactobacillus plantarum and lactobacillus brevis fermentation liquor with the sterilized sewage in the step (i) according to the volume ratio of 1 (2-6), and carrying out anaerobic culture at 37 ℃ for 24 hours; performing solid-liquid separation, and collecting the lactobacillus plantarum and lactobacillus brevis thalli;
(iii) mixing the wet thalli and distilled water according to the mass-volume ratio of 1 (20-25), and shaking for resuspension in unit g/ml.
According to the invention, the solid-liquid separation is further preferably carried out for 15-20 min under the condition of 5000-7000 g/min.
According to the invention, the adsorption stabilizer in the step (2) is preferably prepared by mixing trehalose, agar powder and skim milk powder in proportion and then sterilizing.
According to the invention, the solid-liquid separation in the step (2) is preferably low-temperature solid-liquid separation; the drying is low-temperature drying.
The application of the sewage composite microorganism treating agent in reducing the viscosity of high-concentration aquaculture sewage.
According to the invention, the high-concentration breeding sewage is preferably dairy farm breeding sewage, pig farm sewage and/or beef farm sewage.
The invention has the following function principle:
the breeding sewage has complex components, and animal excrement, feed, animal hair and other substances are mixed in the breeding sewage, so that the problems of high solid content and high viscosity are caused. Although the addition of a settling step can reduce a portion of the solids in the wastewater, it is difficult to effectively reduce the viscosity of the wastewater. The breeding sewage is rich in components such as polysaccharide, protein, filamentous fibers and the like, and the components are mixed together to form cross-linked colloid, so that the viscosity of the sewage is increased, and the difficulty is increased for the subsequent treatment steps.
The bacillus megaterium, the bacillus subtilis, the lactobacillus plantarum and the lactobacillus brevis have strong enzymatic activities of extracellular and intracellular proteases, beta-glucosidase and the like, and can quickly degrade components such as polysaccharide, protein, filamentous fibers and the like in sewage, thereby breaking a colloid structure and effectively reducing the viscosity of the aquaculture sewage; after the bacillus megaterium, the bacillus subtilis, the lactobacillus plantarum and the lactobacillus brevis are subjected to early culture and effective domestication of sterilized culture sewage, the bacillus megaterium, the bacillus subtilis, the lactobacillus plantarum and the lactobacillus brevis can well survive and grow in the culture sewage with high salinity, high organic components and high ammonia nitrogen content, the high flora concentration is maintained, and the effect of reducing the sewage viscosity can be effectively exerted.
Compared with the prior art, the invention has the following beneficial effects:
1. the use effect is good. The sewage composite microorganism treating agent can obviously reduce the viscosity of high-concentration aquaculture sewage after being used. After the sewage is treated by the treating agent for a period of time, the viscosity is reduced by over 60-95%; the reduction of the sewage viscosity obviously reduces the risks of pipeline blockage and unsmooth flow in the subsequent treatment steps, and improves the sewage treatment efficiency; the competitive inhibition effect among the four bacteria is not obvious, and the ectoenzymes generated by the four bacteria respectively have the overlapping and synergistic effects on reducing the viscosity;
2. the application range is wide. The sewage composite microorganism treating agent is suitable for various large-scale farms with different breeding types, such as large-scale dairy cow farms, large-scale beef cattle farms, large-scale pig farms and the like, and can also be matched with various currently used breeding sewage treatment processes, such as different sewage treatment processes of an ecological wetland method, an aerobic aeration method, an anaerobic biogas method and the like, so that the sewage treatment efficiency is improved, and the cost is reduced;
3. the product has long effective period. The compound sewage microbial treatment agent prepared by the invention is added with trehalose, agar powder and skim milk powder, is easy to store and transport, has long effective viable count retention time, and has initial viable count as high as 2.5-3.5 multiplied by 1010CFU/g, after 24 months of normal temperature storage, the viable count is still more than 1.5 multiplied by 1010CFU/g, and the ratio of active bacillus/active lactobacillus can maintain the initial ratio and can still be normally used; the product can be stored and transported at normal temperatureThe transmission has no special low-temperature requirement;
4. the preparation process is simple. The preparation method of the sewage composite microorganism treating agent is simple, the adopted technologies are common technologies of microbiology and environmental engineering, and the used equipment is common microorganism fermentation equipment, so that the sewage composite microorganism treating agent is easy to popularize;
5. meets the requirements of green environmental protection. The sewage composite microorganism treating agent prepared by the invention is an active microorganism preparation, the source microorganism is safe and nontoxic, and the product does not contain toxic and harmful chemical substances and cannot cause secondary pollution. The using amount of the product is small, and a good treatment effect can be achieved only by adding 80-160 g of microbial inoculum into each cubic meter of culture sewage. The microbial inoculum is added once without repeated addition, and the method is easy to master; compared with methods such as a chemical flocculation method and the like, the method has the advantages of reducing the cost and meeting the requirements of environmental protection.
Detailed Description
The technical solution of the present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto.
Source of biological material
Bacillus megaterium (Bacillus megaterium) is from the American type culture Collection, and the strain number: ATCC 14945; or Bacillus megaterium (Bacillus subtilis) is from American type culture Collection, and the strain number is as follows: ATCC 11562.
The Bacillus subtilis (Bacillus subtilis) is from American type culture Collection, and the strain number is as follows: ATCC 15134; or Bacillus subtilis (Bacillus subtilis) is from China general microbiological culture collection center, and the strain number is as follows: CGMCC 1.821.
Lactobacillus plantarum (Lactobacillus plantarum) originated from the American type culture Collection, strain number: ATCC 8014; or the Lactobacillus plantarum (Lactobacillus plantarum) is derived from China general microbiological culture collection center, and the strain number is as follows: CGMCC 1.568.
Lactobacillus brevis (Lactobacillus brevis) is from the American type culture Collection, and the strain number: ATCC 367; or Lactobacillus brevis (Lactobacillus brevis) is from American type culture Collection, and the strain number is as follows: ATCC 13648.
Culture medium
LB solid culture medium, per liter composition as follows:
10g of peptone, 5g of yeast extract, 10g of NaCl and 20g of agar, wherein the pH is natural, and the volume of water is up to 1L.
LB liquid culture medium, per liter composition as follows:
10g of peptone, 5g of yeast extract and 10g of NaCl, wherein the pH is natural, and the water volume is up to 1L.
MRS solid culture medium, per liter of components as follows:
20g of glucose, 10g of tryptone, 5g of yeast powder, 10g of beef extract, 2g of diammonium citrate, 3.12g of sodium acetate, 1.63g of dipotassium hydrogen phosphate, 2.25g of potassium acetate, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate, Tween-801 mL, 20g of agar, and sterilizing at 115 ℃ for 30min, wherein the volume of distilled water is 1000 mL;
MRS liquid culture medium, per liter component as follows:
20g of glucose, 10g of tryptone, 5g of yeast powder, 10g of beef extract, 2g of diammonium citrate, 3.12g of sodium acetate, 1.63g of dipotassium phosphate, 2.25g of potassium acetate, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate, Tween-801 mL, distilled water with the constant volume of 1000mL, and sterilization at 115 ℃ for 30 min;
example 1
A composite microbial sewage treating agent comprises:
bacillus megaterium (Bacillus megaterium) ATCC 14945, viable count not less than 1.0 × 1010CFU/g；
Bacillus subtilis ATCC 15134 with viable count not less than 1.0 × 1010CFU/g；
Lactobacillus plantarum ATCC 8014 with viable count not less than 7.5 × 109CFU/g；
Lactobacillus brevis (ATCC 367), the viable count of which is not less than 2.5 × 109CFU/g；
And the ratio of the viable count of the bacillus megaterium, the bacillus subtilis, the lactobacillus plantarum and the lactobacillus brevis is 4:4:3: 1.
The microbial treatment agent also comprises an adsorption stabilizer, wherein the mass percentage of the adsorption stabilizer is 50%, and the adsorption stabilizer comprises the following components in parts by weight:
1 part of trehalose, 4 parts of agar powder and 5 parts of skim milk powder.
The preparation method of the composite microorganism treating agent capable of reducing the viscosity of the high-concentration culture sewage comprises the following steps:
(1) inoculating Bacillus megaterium (Bacillus megaterium) to LB solid culture medium, standing at 37 deg.C for activation culture for 24 hr, transferring to LB liquid culture medium, shaking at 37 deg.C and 180r/min for shake culture for 24 hr, transferring to LB liquid culture medium at 5 vol%, seed culturing at 37 deg.C and 180r/min for 24 hr, transferring to fermentation culture medium (LB liquid culture medium containing 2 wt% glucose), fermenting at 37 deg.C and 180r/min for 24 hr to obtain viable bacteria with concentration of 2.5 × 109CFU/ml of Bacillus megaterium fermentation broth;
inoculating Bacillus subtilis to LB solid culture medium, standing at 37 deg.C, activating, culturing for 24 hr, transferring to LB liquid culture medium, shaking at 37 deg.C and 180r/min for shake culture for 24 hr, transferring to LB liquid culture medium at 5 vol%, culturing for 24 hr, transferring to fermentation culture medium (LB liquid culture medium containing 2 wt% glucose) at 5 vol%, fermenting at 37 deg.C and 180r/min for 24 hr to obtain active bacteria with concentration of 2.5 × 10%9CFU/ml of Bacillus subtilis fermentation liquor;
(2) inoculating Lactobacillus plantarum (Lactobacillus plantarum) to MRS solid culture medium, performing static activation culture at 30 deg.C for 24 hr, transferring to MRS liquid culture medium, performing static culture at 30 deg.C for 24 hr in triangular flask, transferring to MRS liquid culture medium at 5 vol%, performing static culture at 30 deg.C for 24 hr, transferring to fermentation culture medium at 5 vol% (adding 1 wt%)MRS liquid culture medium of glucose), and static fermentation culture is carried out for 24 hours at 30 ℃, and the concentration of the prepared viable bacteria is 2.5 multiplied by 109CFU/ml lactobacillus plantarum fermentation broth;
inoculating Lactobacillus brevis (Lactobacillus brevis) to MRS solid culture medium, static activating at 30 deg.C for 24 hr, then transferring to MRS liquid culture medium, static culturing at 30 deg.C for 24 hr in triangular flask, then transferring to MRS liquid culture medium at 5 vol%, static culturing at 30 deg.C for 24 hr, then transferring to fermentation culture medium (MRS liquid culture medium containing 1 wt% glucose) at 5 vol%, static fermenting at 30 deg.C for 24 hr to obtain viable bacteria concentration of 2.5 × 109CFU/ml Lactobacillus brevis fermentation liquor;
(3) centrifuging high-concentration breeding sewage of a dairy farm for 15min under the condition of 7000g/min, and removing most solid matters; sterilizing at 115 deg.C under high temperature and pressure for 30min, centrifuging again at 5000g/min for 20min, removing precipitated solid, and cooling to obtain sterilized sewage;
(4) respectively mixing the bacteria-containing fermentation liquor in the steps (1) and (2) and the sterilized sewage in the step (3) according to the proportion of 1 (2-6), and culturing for 24 hours at 37 ℃; centrifuging for 20min at the speed of 5000g/min, and respectively collecting thalli of four kinds of bacteria;
(5) mixing the thalli obtained in the step (4) with distilled water according to the mass-to-volume ratio of 1 (20-25), shaking and resuspending the thalli in unit g/ml to obtain a heavy suspension of four kinds of bacteria;
(6) and (3) mixing the bacillus megaterium heavy suspension, the bacillus subtilis heavy suspension, the lactobacillus plantarum heavy suspension and the lactobacillus brevis heavy suspension prepared in the step (5) according to a volume ratio of 4:4: 2:1 to obtain mixed bacteria liquid;
(7) and (4) adding an adsorption stabilizer into the mixed bacterial liquid obtained in the step (6), and performing low-temperature solid-liquid separation and low-temperature drying to obtain the composite microbial treatment agent.
The adsorption stabilizer is prepared by mixing trehalose, agar powder and skim milk powder in proportion and sterilizing. After the adsorption stabilizer is added, the viable bacteria yield can reach 97%.
Example 2
The sewage composite microorganism treating agent as described in example 1, which is different in that microorganism thalli are selected:
bacillus megaterium ATCC 11562, viable count not less than 1.0 × 1010CFU/g；
Bacillus subtilis CGMCC 1.821, and viable count not less than 1.0 × 1010CFU/g；
Lactobacillus plantarum CGMCC 1.568, viable count not less than 7.5 × 109CFU/g；
Lactobacillus brevis ATCC 13648 with viable count not less than 2.5 × 109CFU/g。
The procedure for preparing the adsorption stabilizer was the same as in example 1.
Example 3
The sewage composite microorganism treating agent of the embodiment 1 is different from the following components in that:
the ratio of the viable count of the bacillus megaterium, the bacillus subtilis, the lactobacillus plantarum and the lactobacillus brevis is 1:1:1: 1.
Example 4
The sewage composite microorganism treating agent of the embodiment 1 is different from the following components in that:
the ratio of the viable count of the bacillus megaterium, the bacillus subtilis, the lactobacillus plantarum and the lactobacillus brevis is 4:4:1: 1.
Example 5
The sewage composite microorganism treating agent of the embodiment 1 is different in that the adsorption stabilizer comprises the following components in parts by weight:
2 parts of trehalose, 3 parts of agar powder and 8 parts of skim milk powder.
Example 6
The sewage composite microorganism treating agent of the embodiment 1 is different in that the adsorption stabilizer comprises the following components in parts by weight:
1 part of trehalose, 6 parts of agar powder and 4 parts of skim milk powder.
Comparative example 1
The composite microbial treatment agent as described in example 1, except that Bacillus megaterium was replaced with Bacillus licheniformis (Bacillus licheniformis), and the treatment agent specifically contained the following strains:
bacillus licheniformis ATCC 9945 with viable count not less than 1.0 × 1010CFU/g；
Bacillus subtilis ATCC 15134 with viable count not less than 1.0 × 1010CFU/g；
Lactobacillus plantarum ATCC 8014 with viable count not less than 7.5 × 109CFU/g；
Lactobacillus brevis ATCC367 with viable count not less than 2.5 × 109CFU/g。
The procedure for preparing the adsorption stabilizer was the same as in example 1.
Comparative example 2
The composite microbial treatment agent of example 1, except that Lactobacillus plantarum was replaced with Lactobacillus acidophilus (Lactobacillus acidophilus), the treatment agent contained the following strains:
bacillus megaterium ATCC 14945 with viable count not less than 1.0 × 1010CFU/g；
Bacillus subtilis ATCC 15134 with viable count not less than 1.0 × 1010CFU/g；
Lactobacillus acidophilus ATCC 4356, the viable count is not less than 7.5 × 109CFU/g；
Lactobacillus brevis ATCC367 with viable count not less than 2.5 × 109CFU/g。
The procedure for preparing the adsorption stabilizer was the same as in example 1.
Comparative example 3
The composite microbial treatment agent of example 1, except that trehalose was replaced with sucrose.
The strain composition, preparation procedure and the like were the same as in example 1.
Comparative example 4
The composite microbial treatment agent of example 1, except that the agar powder was replaced with diatomaceous earth.
The strain composition, preparation procedure and the like were the same as in example 1.
Experimental example 1
The beneficial effects of the compound microorganism sewage treatment agent of the present invention are further described in detail in the following experimental examples, which are experiments on the effect of sewage treatment.
Taking the sewage composite microorganism treating agents obtained in example 1, example 2, example 3, example 4 and example 5, example 6 and comparative example 1, comparative example 2, comparative example 3 and comparative example 4 respectively, the effect of reducing the viscosity of the sewage was tested by taking the high-concentration breeding sewage of the dairy farm, the high-concentration breeding sewage of the pig farm and the high-concentration breeding sewage of the beef farm as experimental subjects.
The basic physicochemical properties of the high-concentration breeding sewage in the dairy farm, the high-concentration breeding sewage in the pig farm and the high-concentration breeding sewage in the beef farm are shown in table 1. Wherein, the COD (chemical oxygen demand) measuring method of the sewage is according to the industry standard of the bichromate method for measuring the chemical oxygen demand of water quality (HJ 828-2017); the ammonia nitrogen determination method is according to the industry standard of determination of ammonia nitrogen in water quality continuous flow-salicylic acid spectrophotometry (HJ 665-2013); the total nitrogen determination method is according to the industry standard of determination of total nitrogen of water quality continuous flow-naphthyl ethylenediamine hydrochloride spectrophotometry (HJ 667-2013); viscosity determination methods were in accordance with the national Standard "methods of viscosity measurement" (GBT 10247-2008).
All data are the average of three determinations.
TABLE 1 basic physicochemical Properties of Experimental culture Sewage
The experimental process is as follows:
(1) viscosity reduction treatment experiment of high-concentration breeding sewage in dairy farm:
the experiments were divided into 11 groups, each set of 3 replicates. Taking high-concentration breeding sewage of 16.5m from a sewage primary sedimentation tank of a dairy farm3And the average part is 33 parts (0.5 m each part)3) Push-buttonAccording to 100g/m3(mass to volume ratio) the sewage composite microbial treatment agents described in example 1, example 2, example 3, example 4 and example 5, example 6 and comparative example 1, comparative example 2, comparative example 3 and comparative example 4 were added, respectively, and, at the same time, comparative example 5 was set to add 100ml of distilled water to sewage instead of the microbial treatment agent. After uniformly mixing, the treatment temperature is 25-40 ℃, and the treatment time is 24 hours; at the beginning and end of the experiment, the viscosity values of the wastewater were measured, and the results are shown in Table 2.
(2) The experiment of viscosity reduction treatment of high-concentration breeding sewage in a pig farm:
the experiments were divided into 11 groups, each set of 3 replicates. Taking high-concentration breeding sewage of 16.5m from a sewage primary sedimentation tank of a pig farm3And the average part is 33 parts (0.5 m each part)3) According to 100g/m3(mass to volume ratio) the sewage composite microbial treatment agents described in example 1, example 2, example 3, example 4 and example 5, example 6 and comparative example 1, comparative example 2, comparative example 3 and comparative example 4 were added, respectively, and, at the same time, comparative example 5 was set to add 100ml of distilled water to sewage instead of the microbial treatment agent. After uniformly mixing, the treatment temperature is 25-40 ℃, and the treatment time is 24 hours; at the beginning and end of the experiment, the viscosity values of the wastewater were measured, and the results are shown in Table 3.
(3) The beef cattle farm high-concentration breeding sewage viscosity reduction treatment experiment comprises the following steps:
the experiments were divided into 11 groups, each set of 3 replicates. Taking high-concentration breeding sewage of 16.5m from a sewage primary sedimentation tank of a beef cattle farm3And the average part is 33 parts (0.5 m each part)3) According to 100g/m3(mass to volume ratio) the sewage composite microbial treatment agents described in example 1, example 2, example 3, example 4 and example 5, example 6 and comparative example 1, comparative example 2, comparative example 3 and comparative example 4 were added, respectively, and, at the same time, comparative example 5 was set to add 100ml of distilled water to sewage instead of the microbial treatment agent. After uniformly mixing, the treatment temperature is 25-40 ℃, and the treatment time is 24 hours; at the beginning and end of the experiment, the viscosity values of the wastewater were measured, and the results are shown in Table 4.
TABLE 2 viscosity reduction treatment experiment for high-concentration breeding sewage of dairy farm
Note: viscosity reduction degree (%) - (viscosity after treatment-viscosity before treatment)/viscosity before treatment × 100%
TABLE 3 viscosity reduction treatment experiment for high concentration breeding sewage in pig farm
Note: viscosity reduction degree (%) - (viscosity after treatment-viscosity before treatment)/viscosity before treatment × 100%
TABLE 4 viscosity reduction treatment experiment for high-concentration breeding sewage of beef cattle farm
Note: viscosity reduction degree (%) - (viscosity after treatment-viscosity before treatment)/viscosity before treatment × 100%
The experiments show that the sewage composite microorganism treating agent has good viscosity reducing treatment effects on high-concentration breeding sewage of a dairy farm, high-concentration breeding sewage of a pig farm and high-concentration breeding sewage of a beef farm:
under experimental conditions, the sewage composite microorganism treating agent of the examples 1 to 6 is used for treating high-concentration breeding sewage of a dairy farm, the viscosity of the sewage can be respectively reduced by 90.0%, 74.5%, 83.5%, 83.3% and 82.5%, and the treatment effect is remarkably better than that of comparative example 1, comparative example 2 and comparative example 5, namely 14.5%, 9.6% and 1.1%; particularly, the composite sewage microorganism treating agent comprises bacillus megaterium, bacillus subtilis, lactobacillus plantarum and lactobacillus brevis which are mixed according to a ratio of 4:4:3:1, and the adsorption stabilizer comprises trehalose, agar powder and skim milk powder according to a ratio of 1:4:5, and the obtained treating effect is optimal in the sewage composite microorganism treating agent of the embodiment 1 and the embodiment 2; similar experimental results are obtained by taking high-concentration breeding sewage of a live pig farm and high-concentration breeding sewage of a beef cattle farm as experimental objects.
The sewage treatment effect experiments of comparative examples 1-2 show that the sewage treatment effect of the treatment agent is greatly reduced after replacing bacillus megaterium with bacillus licheniformis (comparative example 1) or replacing lactobacillus plantarum with lactobacillus acidophilus (comparative example 2). The compound microorganism treating agent consisting of bacillus megatherium, bacillus subtilis, lactobacillus plantarum and lactobacillus brevis can effectively reduce the viscosity of the aquaculture sewage.
In contrast, in comparative examples 3 and 4, when trehalose is replaced by sucrose in the adsorption stabilizer (comparative example 3) or agar powder is replaced by diatomite (comparative example 4), the treatment effect is slightly worse than that of examples 1 and 2, and further, the selection of the adsorption stabilizer also has a certain influence on the viscosity reduction effect.
The experiments show that the bacillus megaterium, the bacillus subtilis, the lactobacillus plantarum and the lactobacillus brevis contained in the composite sewage treatment agent can efficiently decompose proteins, polysaccharides, filamentous fibers and the like in sewage by secreting protease, polysaccharidase, cellulase and other activities under the conditions of specific flora proportion and adsorption stabilizer, and the strains have weak competitive inhibition effect and can act synergistically, so that the effect of well reducing the viscosity of high-concentration aquaculture sewage is exerted; on the other hand, when the composition is changed in the types of active microorganisms contained in the treatment agent and the type of the adsorption stabilizer, the treatment effect is relatively poor due to a strong competitive inhibition effect between strains and the like.
Experimental example 2
This section tests the beneficial effect of the adsorption stabilizer of the present invention on maintaining microbial activity of the microbial agents at a specific ratio.
For the sewage composite microorganism treating agents obtained in example 1, example 2, example 3, example 4 and example 5, example 6 and comparative example 1, comparative example 2, comparative example 3 and comparative example 4, respectively, the total viable bacteria density, the initial viable bacteria density and the initial viable bacteria density were measured immediately after the completion of the preparation; each microbial inoculum was stored in the laboratory at ambient temperature for 24 months. Then respectively measuring the density of total live bacteria, the density of initial active bacillus and the density of initial active lactic acid bacteria; meanwhile, according to the method described in the example, the mixed dry powder of bacillus megatherium, bacillus subtilis, lactobacillus plantarum and lactobacillus brevis was prepared without adding any adsorption protective agent, and the viable bacteria data just after the preparation and 24 months of storage were measured as comparative example 6.
The method for determining the active strain is as follows:
(1) active bacillus density determination:
respectively taking the sewage composite microorganism treating agent under aseptic conditions, carrying out gradient dilution by using sterile distilled water, coating an LB solid flat plate with proper gradient diluent, putting the flat plate into a constant-temperature incubator, culturing at the culture temperature of 37 ℃ for 24 hours, and counting the number of bacterial colonies to obtain the number of viable bacillus.
(2) Active lactobacillus density determination:
respectively taking the sewage composite microorganism treating agent under the aseptic condition, carrying out gradient dilution by using sterile distilled water, coating an MRS solid culture medium (additionally adding 0.5% calcium carbonate) flat plate with a proper gradient diluent, putting the flat plate into an anaerobic glove box, culturing for 24 hours at the culture temperature of 37 ℃ under the anaerobic condition, and counting colonies forming transparent rings to obtain the viable count of the lactic acid bacteria.
(3) And (3) measuring the total viable bacteria density:
adding the viable count of the bacillus and the viable count of the lactobacillus to obtain the total viable count of the sewage composite microorganism treating agent.
All data are the average of three determinations and the results are shown in table 5.
TABLE 5
Through long-term storage experiments of the sewage composite microorganism treating agent, the initial total viable count of the sewage composite microorganism treating agents prepared in the examples 1, 2, 3, 4 and 5, 6 and the comparative examples 1, 2, 3, 4 and 6 can reach 3.0 multiplied by 1010CFU/g, but after 24 months of normal temperature storage, the total viable count and the ratio of active bacillus/active lactic acid bacteria of each experimental group may change greatly.
When the adsorption stabilizer consisting of trehalose, agar powder and skim milk powder in a ratio of 1:4:5 is used, the total viable count of the sewage composite microorganism treating agent in the embodiments 1, 2, 3 and 4 can still reach 1.5 multiplied by 10 after the sewage composite microorganism treating agent is stored for 24 months10CFU/g, and the ratio of active bacillus/active lactobacillus can maintain the initial ratio of the microbial inoculum. The sufficient total viable count and the proper ratio of active bacillus/active lactic acid bacteria can ensure that the active bacillus/active lactic acid bacteria can play an active role in reducing the viscosity of sewage; when the ratio of trehalose, agar powder and skim milk powder in the adsorption protective agent is 2:3:8 (example 5) or 1:6:4 (example 6), the total viable count is 1.3 × 10 after 24 months of storage10CFU/g and 1.2X 1010On the left and right, the ratio of the active bacillus/the active lactic acid bacteria is changed from 2:1 to 4:1 and 10:3, the total viable count is reduced compared with the examples 1 to 4, and the ratio of the active bacillus/the active lactic acid bacteria is greatly changed relative to the initial state, which shows that the ratio of trehalose, agar powder and skim milk powder is more suitable for the adsorption stabilizer when being mixed according to the ratio of 1:4: 5.
In comparative examples 1 and 2, the total viable count can still reach 1.5 × 10 after 24 months of storage10CFU/g, and ratio of Bacillus sexually active lactobacillus to Lactobacillus can be maintainedThe initial ratio of the microbial inoculum indicates that the adsorption stabilizer consisting of trehalose, agar powder and skim milk powder in a ratio of 1:4:5 has an outstanding activity protection effect on other types of bacillus and lactic acid bacteria.
In contrast, in comparative examples 3, 4 and 6, when trehalose was replaced with sucrose (comparative example 3), or agar powder was replaced with diatomaceous earth (comparative example 4) in the adsorption stabilizer, or no adsorption stabilizer was used (comparative example 6), the total viable cell count decreased to 5.0X 10 after 24 months storage, respectively8CFU/g、5.0×108CFU/g and 2.0X 108CFU/g, corresponding to only 1.67%, 1.67% and 0.67% of the initial chamber, respectively, and the lactic acid bacteria had all died, and had lost their value of use completely.
The experiment shows that the adsorption stabilizer consisting of trehalose, agar powder and skim milk powder (1:4:5) can effectively protect the survival of microbial agents and has corresponding technical effects in a specific proportion, and if the adsorption stabilizer is replaced by other similar components, the activity of the microbes and the effective viable bacteria proportion are seriously influenced.
Claims (13)
1. The sewage composite microorganism treating agent is characterized by comprising a microbial thallus and an adsorption stabilizer, wherein the adsorption stabilizer accounts for 40-60% of the total mass of the treating agent; the microbial thallus consists of bacillus megaterium, bacillus subtilis, lactobacillus plantarum and lactobacillus brevis, wherein:
bacillus megaterium with viable count not less than 1.0 × 1010CFU/g; the bacillus megaterium is from American type culture Collection, and the strain number is ATCC 14945 or ATCC 11562;
bacillus subtilis with viable count not less than 1.0 × 1010CFU/g; the bacillus subtilis is derived from American type culture collection center, and the strain number is ATCC 15134, or derived from China general microbiological culture collection center, and the strain number is CGMCC 1.821;
lactobacillus plantarum with viable count not less than 7.5 × 109CFU/g; the lactobacillus plantarum is derived from the American type culture Collection,the strain number is ATCC 8014, or the strain number is CGMCC 1.568 from China general microbiological culture Collection center;
lactobacillus brevis with viable count not less than 2.5 × 109CFU/g; the Lactobacillus brevis is from American type culture Collection, and the strain number is ATCC367 or ATCC 13648;
the ratio of the viable count of the bacillus megatherium, the bacillus subtilis, the lactobacillus plantarum and the lactobacillus brevis is (2-4): 1-2;
the adsorption stabilizer comprises the following components in parts by weight:
1-2 parts of trehalose, 3-6 parts of agar powder and 4-8 parts of skim milk powder.
2. The composite microbial sewage treatment agent of claim 1, wherein the adsorption stabilizer comprises the following components in parts by weight:
1 part of trehalose, 4 parts of agar powder and 5 parts of skim milk powder.
3. The composite microbial wastewater treatment agent according to claim 1, wherein the ratio of viable count of bacillus megaterium, bacillus subtilis, lactobacillus plantarum and lactobacillus brevis is 4:4:3: 1.
4. The composite microbial wastewater treatment agent according to claim 1, wherein the microbial cells are domesticated microbial cells from aquaculture wastewater.
5. The preparation method of the sewage composite microorganism treating agent of claim 1, which is characterized by comprising the following steps:
(1) respectively carrying out activation culture, shaking shake culture, seed culture and fermentation culture on bacillus megatherium, bacillus subtilis, lactobacillus plantarum and lactobacillus brevis to prepare each viable bacteria fermentation liquor;
the viable bacteria concentration of the bacillus megaterium fermentation liquor and the bacillus subtilis fermentation liquor is 2.0-3.0109 CFU/mL；
The viable bacteria concentration of the lactobacillus plantarum fermentation liquor and the lactobacillus brevis fermentation liquor is 1.0-2.0 multiplied by 109 CFU/mL；
(2) Mixing bacillus megaterium fermentation liquor, bacillus subtilis fermentation liquor, lactobacillus plantarum fermentation liquor and lactobacillus brevis fermentation liquor to prepare mixed bacterial liquor; adding an adsorption stabilizer into the mixed bacterial liquid, and carrying out solid-liquid separation and drying to obtain the composite microbial treatment agent.
6. The method according to claim 5, wherein the culture conditions of Bacillus megaterium and Bacillus subtilis in step (1) are as follows:
the activation culture conditions are as follows: static culture is carried out for 24 hours at 37 ℃, and a culture medium for activation culture is an LB solid culture medium;
shake flask shake culture conditions were all: culturing for 24 hours at 37 ℃ and the rotating speed of 180r/min, wherein the culture medium for shake flask culture is LB liquid culture medium;
the seed culture conditions are as follows: culturing for 24 hours at 37 ℃ and the rotating speed of 180r/min, wherein the culture medium for seed culture is LB liquid culture medium;
the fermentation culture conditions are as follows: culturing for 24 hours at 37 ℃ and 180r/min of rotation speed, wherein the culture medium for fermentation culture is LB liquid culture medium added with 2 percent of glucose by mass.
7. The method according to claim 5, wherein the culture conditions of Lactobacillus plantarum and Lactobacillus brevis in step (1) are as follows:
the activation culture conditions are as follows: static culture is carried out for 24 hours at the temperature of 30 ℃, and a culture medium for activation culture is an MRS solid culture medium;
the triangular flask static culture conditions are as follows: static culture is carried out for 24 hours at 30 ℃, and a culture medium for static culture in a triangular flask is an MRS liquid culture medium;
the seed culture conditions are as follows: static culturing at 30 deg.c for 24 hr in MRS liquid culture medium;
the fermentation culture conditions are as follows: and (3) performing static culture at 30 ℃ for 24 hours, wherein the culture medium for fermentation culture is an MRS liquid culture medium added with 1% glucose by mass.
8. The method according to claim 5, wherein the step (2) further comprises an acclimatization step of, before the preparation of the mixed bacterial liquid:
(i) taking culture sewage, carrying out solid-liquid separation, and taking liquid; sterilizing, and carrying out solid-liquid separation to obtain sterilized sewage;
(ii) respectively mixing the bacillus megaterium fermentation liquor and the bacillus subtilis fermentation liquor with the sterilized sewage in the step (i) according to the volume ratio of 1 (2-6), and carrying out aerobic culture at 37 ℃ for 24 hours; solid-liquid separation, namely respectively collecting bacillus megatherium and bacillus subtilis thalli;
mixing lactobacillus plantarum and lactobacillus brevis fermentation liquor with the sterilized sewage in the step (i) according to the volume ratio of 1 (2-6), and carrying out anaerobic culture at 37 ℃ for 24 hours; performing solid-liquid separation, and collecting the lactobacillus plantarum and lactobacillus brevis thalli;
(iii) mixing the wet thalli and distilled water according to the mass-volume ratio of 1 (20-25), and shaking for resuspension in unit g/ml.
9. The preparation method according to claim 8, wherein in the step (i), the solid-liquid separation is performed by centrifugation for 15-20 min at 5000-7000 g/min.
10. The method according to claim 5, wherein the adsorption stabilizer in step (2) is prepared by mixing trehalose, agar powder and skim milk powder in proportion and sterilizing.
11. The production method according to claim 5, wherein the solid-liquid separation in the step (2) is a low-temperature solid-liquid separation; the drying is low-temperature drying.
12. The use of the composite sewage treating agent of claim 1 for reducing the viscosity of aquaculture sewage.
13. The use according to claim 12, wherein the aquaculture wastewater is dairy farm aquaculture wastewater, pig farm wastewater and/or beef farm wastewater.
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