CN112175866A - Composite biological agent for purifying black and odorous water and preparation method thereof - Google Patents

Abstract

The invention discloses a composite biological agent for purifying black and odorous water and a preparation method thereof, relates to the field of sewage treatment, and aims to solve the problems that the existing chemical agents in the prior art generally cause secondary pollution, the treatment of a river channel is not scientific after long-term consideration, and in actual engineering, the addition amount is large, the cost is high, and the implementation is difficult. The deodorant plant liquid is plant liquid extracted from plant leaf fruits, barks, tree roots, buds and seeds. The releasing degree of the odor is reduced, no secondary pollution is caused, the strength of black odor outbreak is thoroughly controlled, the brown sugar water is not required to be activated, the black odor water can be put into the black odor water body for application only by activating for 3-7 hours at room temperature, and the use is convenient.

Description

Composite biological agent for purifying black and odorous water and preparation method thereof

Technical Field

The invention relates to the technical field of water treatment, in particular to a composite biological agent for purifying black and odorous water and a preparation method thereof.

Background

With the rapid development of economy and the increase of population in China, domestic garbage, domestic wastewater, industrial water and the like are directly discharged into the river channel, so that the pollution concentration of the river channel far exceeds the self-cleaning capacity of the river channel, and in recent years, most of the river channels are in a black and odorous state, and the lives and urban images of residents on the coasts of the river channel are seriously influenced. Black and odorous water is an extreme water pollution state, and the main reasons include exogenous organic load, re-release of endogenous sediments, water thermal pollution, insufficient river hydrodynamic force, metal pollution of iron, manganese and the like. The main mechanism of the black and odorous river channel is that the water body is in an anaerobic state for a long time, so that organic pollutants are decomposed anaerobically to generate odor such as hydrogen sulfide and nonferrous metal sulfides such as iron sulfide.

In the aspect of river black and odor treatment, the current international popular method is to increase the oxygen content in the water body by adding calcium nitrate, calcium peroxide and the like so as to oxidize sulfides and inhibit the release of phosphorus in bottom mud; the method has the advantages that the existing engineering application examples at home and abroad are used for eliminating the black and odorous river channel by adding calcium nitrate, but the method has the defects of long treatment period, easy promotion of excessive release of ammonia nitrogen and nitrate, secondary pollution of nitrogen and the like. The chemical agents generally cause secondary pollution, the treatment of the river is not scientific after long-term consideration, and in actual engineering, the addition amount is large, the cost is high, and the implementation is difficult; therefore, the market urgently needs to develop a compound biological agent for purifying black and odorous water bodies to help people to solve the existing problems.

Disclosure of Invention

The invention aims to provide a composite biological agent for purifying black and odorous water, which aims to solve the problems that the existing chemical agents in the background art generally cause secondary pollution, the treatment of a river is not scientific after long-term consideration, and in actual engineering, the addition amount is large, the cost is high and the implementation is difficult.

In order to achieve the purpose, the invention provides the following technical scheme: a composite biological agent for purifying black and odorous water comprises the following components of 50-60 parts of bacillus subtilis, 30-35 parts of methanogen, 75-100 parts of deodorization plant liquid, 50-75 parts of sulfur bacteria, 50-75 parts of nitrobacteria, 45-50 parts of Pichia pastoris, 100-120 parts of bacillus strains, 1000-2000 parts of culture solution and 500-600 parts of em bacterial liquid.

Preferably, the preparation method of the bacillus subtilis comprises the following steps:

the method comprises the following steps: respectively connecting fresh inclined planes of parent strains T4412 and TT2 to a test tube filled with a liquid complete culture medium, and carrying out shake culture at 36 ℃ for 14 h;

step two: transferring 1ml of bacterial liquid from T4412 and TT2 into a 250ml conical flask filled with 20ml of liquid complete culture medium, and performing shaking culture at 36 ℃ for 3h to enable the cell growth to enter the prophase of logarithm;

step three: adding 25u/ml penicillin into T4412 and TT2 respectively to make the final concentration 0.3u/ml, and continuing shaking culture for 2 h;

step four: taking 10ml of bacterial liquid from T4412 and TT2 respectively, centrifuging for 10min at 4000r/min, discarding supernatant, suspending the thalli in phosphate buffer, centrifuging, repeatedly washing twice, suspending the thalli in 10ml of SMM, wherein each ml of SMM contains 108-109 viable bacteria;

step five: 0.5ml of bacterial liquid is taken from T4412 and TT2 respectively, diluted by normal saline, and 1ml of 10-5, 10-6 and 10-7 respectively is taken, poured into a complete culture medium, cultured for 24 hours at 36 ℃ and counted;

step six: respectively taking 5ml of bacterial suspension of each parent strain, adding 5ml of lysozyme solution, wherein the concentration of lysozyme is 100ug/ml, uniformly mixing, carrying out heat preservation treatment in a water bath at 36 ℃ for 30min, sampling at regular time, observing the formation condition of protoplasts through microscopic examination, centrifuging for 10min at 4000r/min when more than 95% of cells become spherical protoplasts, removing supernatant, washing with hypertonic buffer solution to remove enzyme, and suspending the protoplasts in 5ml of hypertonic buffer solution to obtain bacillus subtilis;

step seven: plates 1/5-1/3 were coated and the coated areas were cross-hatched using an inoculating loop to form single colonies.

Preferably, the methanogen is prepared by a submerged culture method in a liquid with paraffin added on the liquid surface, the methanogen is prepared by an anaerobic glove box, and the methanogen adopts gram-positive bacteria as components.

Preferably, the plant liquid for deodorization is plant liquid extracted from fruits, barks, roots, buds and seeds of plant leaves, and the plant liquid for deodorization comprises terpenes, linear compounds of aldehyde, alcohol and ketone, benzyl acetate, vanillin, cinnamic acid and geranyl formate.

Preferably, the terpenes are pinane and menthane, and the linear compounds of the aldehyde, the alcohol and the ketone are volatile vegetable oil extracted from linalool and lauromacrogol.

Preferably, the sulfur bacteria adopt aerobic energy-conversion nutrient bacteria without producing pigment, the pichia pastoris adopts a milder purification mode to purify target protein, ammonium sulfate is adopted for precipitation, and then the protein is further purified by methods of ion exchange, gel filtration chromatography and hydrophobic chromatography.

Preferably, the bacillus cereus strain is bacillus cereus, and bacillus cereus is separated from various foods at the temperature of 28-37 ℃, and extracted and cultured, wherein the bacillus cereus comprises meat, dairy products, vegetables, fish, potatoes, paste, soy sauce, pudding, fried rice and various desserts.

Preferably, the culture solution is a complete inorganic salt culture solution, and comprises 1g of calcium nitrate, 0.25g of potassium dihydrogen phosphate, 0.25g of magnesium sulfate, 0.25g of ferric chloride, 0.25g of potassium chloride and 1L of distilled water.

Preferably, the em bacteria liquid adopts photosynthetic bacteria, lactic acid bacteria, actinomycetes and yeast.

Preferably, the photosynthetic bacteria are photosynthetic bacteria which contain chlorophyll a, use water as a hydrogen donor and an electron donor, convert light energy into chemical energy through photosynthesis, assimilate CO2 as organic substances, the lactic acid bacteria are plant-derived lactic acid bacteria, the actinomycetes are spore bacteria, and the yeasts are plant-type secretory yeasts.

Compared with the prior art, the invention has the beneficial effects that:

1. according to the invention, through the composition of bacillus subtilis, methanogen, sulfur bacteria and nitrobacteria, all strains are distributed in the water body, various substances such as black odorous sulfide, ammonia nitrogen concentration and organic matters in the water body are degraded, the growth and reproduction of black odorous microorganisms can be inhibited, the odor release degree is reduced, no secondary pollution is caused, the black odorous outbreak strength is thoroughly controlled, the brown sugar water activation is not needed, and the black odorous water can be put into the water body for application only by activating for 3-7 hours at room temperature, so that the use is convenient.

2. The invention arranges the deodorizing plant liquid, the foul gas is often ignored by people, because the foul gas usually does not produce immediate serious harm to human body, but can stimulate the olfactory organ of people to cause discomfort, and the generation of the foul gas is often the mixture of various foul substances, and acts on the olfactory organ of people through air media to become a perception pollution, so that the strategy of deodorization is difficult to determine, so the foul gas is often ignored by people on the premise, but the problems float out of the water again along with the improvement of the living standard of people, the odor in the black foul water can be treated by the deodorizing plant liquid, and the plant liquid is atomized in the space through special deodorizing equipment to form the fog-like particles with small particles, the fog drops have large specific surface area, can absorb the foul molecules in the air, the adsorbed malodorous molecules can react with an acidic buffer solution in the plant liquid to generate tasteless and nontoxic organic salts, for example, hydrogen sulfide reacts under the action of the plant liquid to generate sulfate ions and water, ammonia generates nitrogen and water under the action of the plant liquid, and the adsorbed malodorous molecules can simultaneously act on acidic, alkaline and neutral gases, and can be diluted with water by different times for different odor places and odor substances according to actual conditions for use, so that the environmental effect is improved.

3. Through the arrangement of the pichia pastoris, the pichia pastoris has an alcohol oxidase AOX1 gene promoter which is one of the strongest promoters with the strictest regulation mechanism at present, the pichia pastoris has high expression efficiency, the expressed foreign protein can account for more than 90 percent of the total expressed protein, the separation and purification of target protein are facilitated, the pichia pastoris can realize high-density culture in a simple synthesis culture medium, pichia pastoris expression plasmids can be stably integrated in a single copy or multi-copy mode at a specific site of a genome, and because the pichia pastoris can use methanol as the only carbon source and energy source, most microorganisms can not use methanol as the carbon source, the pollution can be reduced.

4. According to the invention, the em bacterial liquid is arranged to generate a large amount of beneficial substances, such as amino acids, organic acids, polysaccharides, various vitamins, various biochemical enzymes, growth promotion factors, antibiotics, antiviral substances and the like, so that the stress resistance of crops is improved, the dissolved oxygen concentration in water is improved, and the respiration of root systems is facilitated; the soil water retention capacity can be improved, meanwhile, the leaf protection film can be strengthened, the water loss is reduced, the survival and the propagation of harmful microorganisms are inhibited, soil-borne diseases and insect pests and continuous cropping obstacles are reduced and gradually eliminated, the increase of the number of soil micro-animals eating the microorganisms and decomposition products thereof is promoted, the micro-ecosystem composition of the whole plough layer is fundamentally changed, the soil bioactivity and the buffering capacity are improved, the em bacteria liquid can phagocytize, inhibit and kill the harmful bacteria in a bacteria-treating mode, the biological sterilization and disinfection effects are achieved, no medicine harm exists, and the treatment effect of black and odorous water is improved.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.

The embodiment provided by the invention comprises the following steps: a composite biological agent for purifying black and odorous water comprises the following components of 50-60 parts of bacillus subtilis, 30-35 parts of methanogen, 75-100 parts of deodorant plant liquid, 50-75 parts of sulfur bacteria, 50-75 parts of nitrobacteria, 45-50 parts of Pichia pastoris, 100-120 parts of bacillus strains, 1000-2000 parts of culture solution and 500-600 parts of em bacteria liquid, wherein all the strains are distributed in the water, various substances such as black and odorous sulfides, ammonia nitrogen concentration and organic matters in the water are degraded, growth and reproduction of the black and odorous microorganisms can be inhibited, the odor release degree is reduced, secondary pollution is avoided, the black and odorous outbreak strength is thoroughly controlled, and the black and odorous water can be put into the black and odorous water for application and convenient use only by activating for 3-7 hours at room temperature without activation of brown sugar water.

Further, the preparation method of the bacillus subtilis comprises the following steps:

the method comprises the following steps: respectively connecting fresh inclined planes of parent strains T4412 and TT2 to a test tube filled with a liquid complete culture medium, and carrying out shake culture at 36 ℃ for 14 h;

step two: transferring 1ml of bacterial liquid from T4412 and TT2 into a 250ml conical flask filled with 20ml of liquid complete culture medium, and performing shaking culture at 36 ℃ for 3h to enable the cell growth to enter the prophase of logarithm;

step three: adding 25u/ml penicillin into T4412 and TT2 respectively to make the final concentration 0.3u/ml, and continuing shaking culture for 2 h;

step four: taking 10ml of bacterial liquid from T4412 and TT2 respectively, centrifuging for 10min at 4000r/min, discarding supernatant, suspending the thalli in phosphate buffer, centrifuging, repeatedly washing twice, suspending the thalli in 10ml of SMM, wherein each ml of SMM contains 108-109 viable bacteria;

step five: 0.5ml of bacterial liquid is taken from T4412 and TT2 respectively, diluted by normal saline, and 1ml of 10-5, 10-6 and 10-7 respectively is taken, poured into a complete culture medium, cultured for 24 hours at 36 ℃ and counted;

step six: respectively taking 5ml of bacterial suspension of each parent strain, adding 5ml of lysozyme solution, wherein the concentration of lysozyme is 100ug/ml, uniformly mixing, carrying out heat preservation treatment in a water bath at 36 ℃ for 30min, sampling at regular time, observing the formation condition of protoplasts through microscopic examination, centrifuging for 10min at 4000r/min when more than 95% of cells become spherical protoplasts, removing supernatant, washing with hypertonic buffer solution to remove enzyme, and suspending the protoplasts in 5ml of hypertonic buffer solution to obtain bacillus subtilis;

step seven: the bacillus subtilis is coated in a plate 1/5-1/3 area, an inoculating loop is used for cross-scribing and coating the area to form a single colony, active substances such as subtilin, polymyxin, nystatin, gramicidin and the like generated in the growth process of the bacillus subtilis have an obvious inhibiting effect on pathogenic bacteria or conditional pathogenic bacteria infected endogenously, the bacillus subtilis quickly consumes free oxygen in intestinal tracts to cause the intestinal tract hypoxia, promotes the growth of beneficial anaerobic bacteria and indirectly inhibits the growth of other pathogenic bacteria.

Further, methanogenic bacteria are subjected to a submerged culture method in liquid with paraffin added on the liquid surface, the methanogenic bacteria are subjected to a preparation process by using an anaerobic glove box, the methanogenic bacteria adopt gram-positive bacteria as components, the methanogenic bacteria form natural gas and have synergistic effects with hydrolytic bacteria, acid-producing bacteria and the like in the natural world, so that organic matters are methanized, and bioenergy substances with economic value are generated.

Further, the plant liquid extracted from the fruits, barks, roots, buds and seeds of plant leaves is adopted as the deodorizing plant liquid, the deodorizing plant liquid comprises terpenes, linear compounds of aldehyde, alcohol and ketone, benzyl acetate, vanillin, cinnamic acid and geranyl formate, the odor in the black stink water can be treated through the deodorizing plant liquid, the plant liquid is atomized in a space through special deodorizing equipment to form mist particles with small particles, the mist droplets have large specific surface area, can absorb stink molecules in the air, the adsorbed stink molecules can react with an acid buffer solution in the plant liquid, and then odorless and nontoxic organic salt is generated.

Further, the terpenes are pinane and menthane, and the linear compounds of aldehyde, alcohol and ketone are volatile vegetable oils extracted from linalool and lauromacrogol.

Furthermore, sulfur bacteria adopt aerobic energy-transforming nutrient bacteria which do not produce pigments, pichia pastoris adopts a milder purification mode to purify target protein, ammonium sulfate is adopted for precipitation, and then the protein is further purified by ion exchange, gel filtration chromatography and hydrophobic chromatography.

Further, the bacillus cereus strain is bacillus cereus, and is extracted and cultured from various foods at the temperature of 28-37 ℃, wherein the bacillus cereus strain comprises meat, dairy products, vegetables, fish, potatoes, paste, soy sauce, pudding, fried rice and various desserts, the bacillus cereus can secrete various enzymes such as protease with strong activity, can exist in a sporophyte form in the life process, has the characteristics of high temperature resistance, salt and alkali resistance, extrusion resistance, small damage to the bacillus cereus by feed processing and the like, is easy to produce and store, and can be used for beneficially regulating and controlling the water microecology.

Further, the culture solution was a complete inorganic salt culture solution comprising 1g of calcium nitrate, 0.25g of potassium dihydrogen phosphate, 0.25g of magnesium sulfate, 0.25g of ferric chloride, g of potassium chloride, and 1L of distilled water.

Furthermore, the ingredients of the em bacteria liquid adopt photosynthetic bacteria, lactic acid bacteria, actinomycetes and saccharomycetes, and the em bacteria liquid can phagocytose, inhibit and kill harmful bacteria in a bacteria-treating mode, plays a role in biological sterilization and disinfection, has no medicine harm, and improves the treatment effect of black and odorous water.

Further, the photosynthetic bacteria are photosynthetic bacteria containing chlorophyll a, using water as a hydrogen donor and an electron donor, converting light energy into chemical energy by photosynthesis, assimilating CO2 as an organic substance, the lactic acid bacteria are plant-derived lactic acid bacteria, the actinomycetes are spore bacteria, and the yeasts are plant-derived secretory yeasts.

The working principle is as follows: when in use, after 50-60 parts of bacillus subtilis, 30-35 parts of methanogen, 75-100 parts of deodorizing plant liquid, 50-75 parts of sulfur bacteria, 50-75 parts of nitrobacteria, 45-50 parts of pichia pastoris, 100-120 parts of bacillus strains, 1000-2000 parts of culture solution and 500-600 parts of em bacteria liquid are mixed, the black odorous water can be put into the water body with the concentration of 3-10g/M and all strains are distributed in the water body without being activated by brown sugar water, the growth and the propagation of black odorous microorganisms can be inhibited by the activation of the brown sugar water at room temperature for 3-7h, various substances such as black odorous sulfide, the concentration of ammonia nitrogen and organic matters in the water body can be degraded, the odor release degree can be reduced, no secondary pollution exists, the strength of black odorous outbreak can be thoroughly controlled, the odor in the black odorous water can be treated by the deodorizing plant liquid and atomized by special deodorizing equipment, plant liquid is atomized in a space to form mist particles with small particles, fog drops have large specific surface area, odor molecules in air can be absorbed, the absorbed odor molecules can react with acid buffer solution in the plant liquid to generate tasteless and nontoxic organic salt, em bacteria liquid can phagocytize, inhibit and kill harmful bacteria in a bacteria treatment mode, biological sterilization and disinfection effects are achieved, no medicine harm exists, and the treatment effect of black and odorous water is improved.

Example 1

A composite biological agent for purifying black and odorous water comprises, by weight, 50 parts of bacillus subtilis, 35 parts of methanogen, 100 parts of deodorizing plant liquid, 50 parts of sulfur bacteria, 75 parts of nitrobacteria, 45 parts of pichia pastoris, 120 parts of bacillus strains, 1000 parts of culture solution and 600 parts of em bacteria liquid.

The preparation method of the bacillus subtilis comprises the following steps:

the method comprises the following steps: respectively connecting fresh inclined planes of parent strains T4412 and TT2 to a test tube filled with a liquid complete culture medium, and carrying out shake culture at 36 ℃ for 14 h;

step two: transferring 1ml of bacterial liquid from T4412 and TT2 into a 250ml conical flask filled with 20ml of liquid complete culture medium, and performing shaking culture at 36 ℃ for 3h to enable the cell growth to enter the prophase of logarithm;

step three: adding 25u/ml penicillin into T4412 and TT2 respectively to make the final concentration 0.3u/ml, and continuing shaking culture for 2 h;

step four: taking 10ml of bacterial liquid from T4412 and TT2 respectively, centrifuging for 10min at 4000r/min, discarding supernatant, suspending the thalli in phosphate buffer, centrifuging, repeatedly washing twice, suspending the thalli in 10ml of SMM, wherein each ml of SMM contains 108-109 viable bacteria;

step five: 0.5ml of bacterial liquid is taken from T4412 and TT2 respectively, diluted by normal saline, and 1ml of 10-5, 10-6 and 10-7 respectively is taken, poured into a complete culture medium, cultured for 24 hours at 36 ℃ and counted;

step six: respectively taking 5ml of bacterial suspension of each parent strain, adding 5ml of lysozyme solution, wherein the concentration of lysozyme is 100ug/ml, uniformly mixing, carrying out heat preservation treatment in a water bath at 36 ℃ for 30min, sampling at regular time, observing the formation condition of protoplasts through microscopic examination, centrifuging for 10min at 4000r/min when more than 95% of cells become spherical protoplasts, removing supernatant, washing with hypertonic buffer solution to remove enzyme, and suspending the protoplasts in 5ml of hypertonic buffer solution to obtain bacillus subtilis;

step seven: plates 1/5-1/3 were coated and the coated areas were cross-hatched using an inoculating loop to form single colonies.

The methanogen is prepared by a submerged culture method in a liquid with paraffin added on the liquid surface, an anaerobic glove box is adopted for the methanogen, and gram-positive bacteria are adopted as components of the methanogen.

The deodorizing plant liquid is plant liquid extracted from fruits, barks, roots, buds and seeds of plant leaves, and comprises terpenes, linear compounds of aldehyde, alcohol and ketone, benzyl acetate, vanillin, cinnamic acid and geranyl formate.

The terpenes are pinane and menthane, and the linear chain compounds of aldehyde, alcohol and ketone are volatile vegetable oil extracted from linalool and lauromacrogol.

The sulfur bacteria adopts aerobic energy-conversion nutrient bacteria without producing pigment, the pichia pastoris adopts a milder purification mode to purify target protein, ammonium sulfate precipitation is adopted, and then the protein is further purified by methods of ion exchange, gel filtration chromatography and hydrophobic chromatography.

The bacillus strain is Bacillus cereus, and is prepared by separating Bacillus cereus from various foods at 28-37 deg.C, including meat, dairy products, vegetables, fish, potato, paste, soy sauce, pudding, parched rice and various desserts, and extracting and culturing.

The culture solution adopts complete inorganic salt culture solution and comprises 1g of calcium nitrate, 0.25g of monopotassium phosphate, 0.25g of magnesium sulfate, 0.25g of ferric chloride, 0.25g of potassium chloride and 1L of distilled water.

The em bacteria liquid adopts photosynthetic bacteria, lactic acid bacteria, actinomycetes and saccharomycetes as components.

The photosynthetic bacteria comprise chlorophyll a, water serving as a hydrogen donor and an electron donor, can convert light energy into chemical energy through photosynthesis, assimilate CO2 as organic substances, are plant-derived lactic acid bacteria, are spore bacteria, and secrete yeasts by plants.

Example 2

The present embodiment is different from embodiment 1 in that: a composite biological agent for purifying black and odorous water comprises, by weight, 60 parts of bacillus subtilis, 30 parts of methanogen, 75 parts of deodorizing plant liquid, 75 parts of sulfur bacteria, 50 parts of nitrobacteria, 50 parts of pichia pastoris, 100 parts of bacillus strains, 2000 parts of culture solution and 500 parts of em bacteria liquid.

Example 3

The present embodiment is different from embodiment 1 in that: a composite biological agent for purifying black and odorous water comprises, by weight, 55 parts of bacillus subtilis, 33 parts of methanogen, 88 parts of deodorizing plant liquid, 60 parts of sulfur bacteria, 47 parts of nitrobacteria, 47 parts of pichia pastoris, 110 parts of bacillus strains, 1500 parts of culture solution and 550 parts of em bacteria liquid.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (10)

1. The composite biological agent for purifying the black and odorous water comprises the following components in parts by weight: 50-60 parts of bacillus subtilis, 30-35 parts of methanogen, 75-100 parts of deodorization plant liquid, 50-75 parts of sulfur bacteria, 50-75 parts of nitrobacteria, 45-50 parts of pichia pastoris, 100-120 parts of bacillus strain, 1000-2000 parts of culture solution and 500-600 parts of em bacteria liquid.

2. The compound biological agent for purifying black and odorous water according to claim 1, which is characterized in that: the methanogen adopts a submerged culture method in liquid with paraffin added on the liquid surface, the methanogen adopts an anaerobic glove box to carry out a preparation process, and the methanogen adopts gram-positive bacteria as components.

3. The compound biological agent for purifying black and odorous water according to claim 1, which is characterized in that: the deodorizing plant liquid is plant liquid extracted from fruits, barks, roots, buds and seeds of plant leaves, and comprises terpenes, linear compounds of aldehyde, alcohol and ketone, benzyl acetate, vanillin, cinnamic acid and geranyl formate.

4. The compound biological agent for purifying black and odorous water according to claim 3, wherein the compound biological agent comprises: the terpenes are pinane and menthane, and the linear chain compounds of aldehyde, alcohol and ketone are volatile vegetable oil extracted from linalool and lauromacrogol.

5. The compound biological agent for purifying black and odorous water according to claim 1, which is characterized in that: the sulfur bacteria adopts aerobic energy-conversion nutrient bacteria without producing pigment, the pichia pastoris adopts a milder purification mode to purify target protein, ammonium sulfate precipitation is adopted, and then the protein is further purified by methods of ion exchange, gel filtration chromatography and hydrophobic chromatography.

6. The compound biological agent for purifying black and odorous water according to claim 1, which is characterized in that: the bacillus strain is Bacillus cereus, and is prepared by separating Bacillus cereus from various foods at 28-37 deg.C, including meat, dairy products, vegetables, fish, potato, paste, soy sauce, pudding, parched rice and various desserts, and extracting and culturing.

7. The compound biological agent for purifying black and odorous water according to claim 1, which is characterized in that: the culture solution adopts complete inorganic salt culture solution and comprises 1g of calcium nitrate, 0.25g of monopotassium phosphate, 0.25g of magnesium sulfate, 0.25g of ferric chloride, 0.25g of potassium chloride and 1L of distilled water.

8. The compound biological agent for purifying black and odorous water according to claim 1, which is characterized in that: the em bacteria liquid adopts photosynthetic bacteria, lactic acid bacteria, actinomycetes and saccharomycetes as components.

9. The compound biological agent for purifying black and odorous water according to claim 8, wherein the compound biological agent comprises: the photosynthetic bacteria comprise chlorophyll a, water serving as a hydrogen donor and an electron donor, can convert light energy into chemical energy through photosynthesis, assimilate CO2 as organic substances, are plant-derived lactic acid bacteria, are spore bacteria, and secrete yeasts by plants.

10. The preparation method of the composite biological agent for purifying black and odorous water body according to claim 1, comprising the following steps:

the method comprises the following steps: respectively connecting fresh inclined planes of parent strains T4412 and TT2 to a test tube filled with a liquid complete culture medium, and carrying out shake culture at 36 ℃ for 14 h;

step two: transferring 1ml of bacterial liquid from T4412 and TT2 into a 250ml conical flask filled with 20ml of liquid complete culture medium, and performing shaking culture at 36 ℃ for 3h to enable the cell growth to enter the prophase of logarithm;

step three: adding 25u/ml penicillin into T4412 and TT2 respectively to make the final concentration 0.3u/ml, and continuing shaking culture for 2 h;

step four: taking 10ml of bacterial liquid from T4412 and TT2 respectively, centrifuging for 10min at 4000r/min, discarding supernatant, suspending the thalli in phosphate buffer, centrifuging, repeatedly washing twice, suspending the thalli in 10ml of SMM, wherein each ml of SMM contains 108-109 viable bacteria;

step five: 0.5ml of bacterial liquid is taken from T4412 and TT2 respectively, diluted by normal saline, and 1ml of 10-5, 10-6 and 10-7 respectively is taken, poured into a complete culture medium, cultured for 24 hours at 36 ℃ and counted;

step six: respectively taking 5ml of bacterial suspension of each parent strain, adding 5ml of lysozyme solution, wherein the concentration of lysozyme is 100ug/ml, uniformly mixing, carrying out heat preservation treatment in a water bath at 36 ℃ for 30min, sampling at regular time, observing the formation condition of protoplasts through microscopic examination, centrifuging for 10min at 4000r/min when more than 95% of cells become spherical protoplasts, removing supernatant, washing with hypertonic buffer solution to remove enzyme, and suspending the protoplasts in 5ml of hypertonic buffer solution to obtain bacillus subtilis;

step seven: plates 1/5-1/3 were coated and the coated areas were cross-hatched using an inoculating loop to form single colonies.