CN112481153B - Rhodoblastus sphagnicola coupled composite microbial agent and application thereof - Google Patents
Rhodoblastus sphagnicola coupled composite microbial agent and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses a Rhodoblastus sphagnicola coupling composite microbial inoculum and application thereof in the aspect of degrading starch wastewater, according to the water quality characteristics of the starch wastewater, rhodoblastus sphagnicola PNSB-MHW and bacillus atrophaeus ecological niche differentiation and metabolism complementation characteristics are utilized, liquid seeds are prepared by optimizing the proportion of the Rhodoblastus sphagnicola PNSB-MHW and bacillus atrophaeus ecological niche differentiation and metabolism complementation characteristics, the finally obtained composite microbial inoculum is inoculated into the starch wastewater according to the mass volume ratio of 15%, COD (chemical oxygen demand), ammonia nitrogen, total phosphorus, mercaptan, hydrogen sulfide and the like of the starch wastewater within 144 hours are obviously reduced, the degradation efficiency is more than 98%, and the degraded wastewater realizes the bacterial strain self-flocculation function, so that the steps of fussy acid-base regulation, flocculation precipitation and the like in wastewater treatment are simplified. The Rhodoblastus sphagnicola PNSB-MHW coupled composite microbial agent has wide application value in the technical field of microbial strain application by utilizing the degradation characteristic of the composite microbial agent.
Description
Technical Field
The invention relates to the technical field of microorganisms and application thereof, in particular to a novel Rhodoblastus sphagnicola coupled composite microbial agent and the technical field of application thereof in degrading starch wastewater.
Background
The open production process or the semi-closed production process is adopted in China, so that the numerical values of COD, BOD5, SS and the like in potato starch wastewater in China are higher. The production wastewater of corn starch is discharged, so that the cost of the starch is high, the water consumption of the starch is high, the corn loss is large, and serious environmental pollution is caused due to the putrefaction of protein, fat and other organic matters in the wastewater and the residual sulfurous acid.
The purpose of the starch wastewater treatment is to remove pollutants in the wastewater, so that various indexes of the treated wastewater reach the discharge standard. The existing starch wastewater treatment method comprises a physical method; a chemical process; a physicochemical method; a biochemical method; physical biological methods; enzymatic methods. The biological treatment method of the waste water provides proper conditions, and organic pollutants in a dissolved or colloid state in the waste water are degraded and converted into useful substances by utilizing the metabolism of microorganisms, so that the waste water is struggled. Compared with the physical and chemical treatment methods of wastewater, such as adsorption and coagulation, the methods only transfer organic matters from the wastewater, and also need to consider the subsequent treatment, so that the treatment of both the principal and the secondary aspects is not achieved. The biological treatment method is a more thorough way of degrading organic matters, so the biological treatment method is more and more important and is also a main way of treating wastewater. Another type of starch wastewater treatment method uses a lot of photosynthetic bacteria, namely a bacterial generic name which can carry out photosynthesis under anaerobic conditions but does not release oxygen, namely photosynthetic bacteria which are mainly rhodopseudomonas and degrade organic matters by utilizing photosynthesis, and experimental researches of the seventies of century, japanese patent application No. Lin Zhengtai and the like reveal important roles of the photosynthetic bacteria, and people begin to use the photosynthetic bacteria to treat high-concentration organic wastewater in large quantities. The photosynthetic bacteria sewage treatment method has simple process, low treatment cost, stable operation and no secondary pollution, and is one of the preferred strains for biological treatment of organic wastewater. At present, although the photosynthetic bacteria sewage treatment method is applied to various organic wastewater treatments, only a few of the methods enter industrialized operation, and a large amount of work still stays in the experimental research stage. In addition, the photosynthetic bacteria wastewater treatment has the problems of poor stability of a reaction system, treatment limit of treated water, incapability of directly degrading macromolecular substances, difficult bacterial settlement and the like, so that the application of the photosynthetic bacteria in the wastewater is still to be further studied.
Bacillus atrophaeus (Bacillus atrophaeus) is a non-pathogenic, bacillus subtilis variant that forms soluble black colonies on carbohydrate-containing media, primarily isolated from soil. The method has wide application in the fields of agriculture, industry, scientific research, medical treatment, sanitation and the like, and has no report on the application of the method in starch wastewater degradation.
Disclosure of Invention
Aiming at the problems that in the prior art, photosynthetic bacteria have poor stability of a reaction system, treatment limit of starch wastewater treatment, incapability of directly degrading macromolecular substances, difficult bacterial settlement and the like in the aspect of starch wastewater degradation, no application of coupling of photosynthetic bacteria and bacillus atrophaeus in the aspect of starch wastewater degradation is reported in the literature. The invention aims to provide a novel bacterium Rhodoblastus sphagnicola coupled composite microbial inoculum and application thereof in degrading starch wastewater. According to the invention, rhodoblastus sphagnicola PNSB-MHW which is an acidophilic photosynthetic bacterium and is separated from acid wastewater sludge of quincuncial monosodium glutamate sewage disposal ditch in five Xinjiang, is compounded with bacillus atrophaeus (Bacillus atrophaeus) BA-BC which is separated from saline alkali soil in the Xinjiang, and according to the characteristics of differentiation and metabolism complementation of photosynthetic bacteria and bacillus atrophaeus ecological niches, rhodoblastus sphagnicola PNSB-MHW and bacillus atrophaeus (Bacillus atrophaeus BA-BC) are mixed according to a volume ratio of 10:1 to obtain Rhodoblastus sphagnicola coupled composite microbial inoculum, the composite microbial inoculum is inoculated to starch wastewater according to an inoculum size of 15% in mass-volume ratio, the degradation efficiency of the starch wastewater COD, ammonia nitrogen, total phosphorus, mercaptan, hydrogen sulfide and the like is obviously reduced to be more than 98%, the degraded wastewater can realize the self flocculation function of the strain, the steps of fussy acid-base adjustment, flocculation precipitation and the like in wastewater treatment are simplified, and the composite microbial inoculum has wide value for expanding the application field of microbial strains.
The invention particularly provides a novel bacterium Rhodoblastus sphagnicola coupling composite microbial inoculum, which is a coupling Rhodoblastus sphagnicola composite microbial inoculum formed by compounding Rhodoblastus sphagnicola PNSB-MHW and bacillus atrophaeus (Bacillus atrophaeus) according to a proportion.
In the invention, the strain Rhodoblastus sphagnicola PNSB-MHW is screened and separated from a water sample for sewage of quincuncial monosodium glutamate acidic wastewater in Wujia Jiujia City in Xinjiang, and molecular level identification of a strain system and physiological and biochemical system test verification of the strain are well known in the art, and the obtained acidophilic photosynthetic bacteria belongs to a typical new strain in the category, and the strain Rhodoblastus sphagnicola PNSB-MHW is preserved in China general microbiological culture Collection center, with the preservation number: CGMCC No.20882, preservation date: 10 months and 13 days 2020.
The gene sequence of the Rhodoblastus sphagnicola PNSB-MHW CGMCC No.20882 strain is shown as SEQ ID NO. 1.
The bacillus atrophaeus (Bacillus atrophaeus) BA-BC in the coupling composite microbial inoculum is verified by molecular level identification of a strain system and physiological and biochemical system experiments of strains which are well known in the art, and the obtained bacillus atrophaeus (Bacillus atrophaeus) BA-BC belongs to bacillus atrophaeus (Bacillus atrophaeus) which is common in the art, and the bacillus atrophaeus (Bacillus atrophaeus) strain culture medium and the culture method which are disclosed by the common technical personnel can be purchased through public channels and are suitable for the implementation of the invention.
In the invention, the number average of viable bacteria of Rhodoblastus sphagnicola PNSB-MHW and bacillus atrophaeus (Bacillus atrophaeus) in the coupling composite microbial inoculum is more than or equal to 10 9 cfu/mL, rhodoblastus sphagnicola PNSB-MHW and bacillus atrophaeus (Bacillus atrophaeus) are compounded according to a volume ratio of 10:1.
In the invention, rhodoblastus sphagnicola PNSB-MHW enrichment medium: KH (KH) 2 PO 4 0.04g/L,NH 4 Cl 0.1g/L,MgCl 2 ﹒6H 2 O 0.01g/L,CaCl 2 ﹒2H 2 O 0.05g/L,CH 3 Coona1.0g/L, trace element mother liquor 1ml, vitamin complex mother liquor 1ml, ph=5.0.
In the present invention, rhodoblastus sphagnicola PNSB-MHW isolation medium (RCVBN) (g/L): KH (KH) 2 PO 4 0.5g/L,K 2 HPO 4 0.3g/L，NH 4 Cl 1.0g/L,MgCl 2 ﹒6H 2 O 0.2g/L,CaCl 2 ﹒2H 2 O 0.5g/L,CH 3 COONa 1.0g/L,CH 3 CH 2 COONa 0.5g/L, yeast powder 0.1g/L, agar 7.0g/L, trace element mother liquor 1ml, pH=5.0.
Meanwhile, the invention provides a preparation method of the novel bacterium Rhodoblastus sphagnicola coupled composite microbial inoculum, which specifically comprises the following steps:
(1) Inoculating the preserved strain Rhodoblastus sphagnicola PNSB-MHW CGMCC No.20882 into a liquid modified Nisson & Dundas culture medium, standing and culturing in an illumination incubator at 30 ℃ and an illumination intensity of 1000-2000lx, shaking 2 times per day, and culturing for 3-5 days to a stationary phase, wherein the concentration of thalli contains 10g of dry thalli per liter; inoculating selected Bacillus atrophaeus (Bacillus atrophaeus) into liquid self-grinding culture medium, and culturing at 30deg.C and rotation speed of 120rad/min in shaking table for 3-5d to reach stationary phase, wherein the thallus concentration is 15g dry bacteria per liter.
(2) Taking the two bacterial liquids cultured in the step (1), respectively carrying out centrifugal separation at room temperature, discarding supernatant, precipitating, fully suspending with sterile water, centrifuging again, repeating the step for 3-5 times, and suspending the precipitate with sterile water to obtain the liquid seeds of the two bacterial strains.
(3) Under the aseptic condition, the Rhodoblastus sphagnicola PNSB-MHW obtained in the step (2) and the bacillus atrophaeus (Bacillus atrophaeus) liquid seeds are respectively compounded according to the volume ratio to prepare the Rhodoblastus sphagnicola coupling compound microbial inoculum.
Further, the invention provides application of the novel bacterium Rhodoblastus sphagnicola coupled composite microbial inoculum in degrading starch wastewater.
Specifically, the invention provides application of a novel bacterium Rhodoblastus sphagnicola coupled composite microbial inoculum in degrading starch wastewater. Rhodoblastus sphagnicola the coupling composite microbial inoculum is inoculated in starch wastewater according to the inoculum size of 15 percent of the mass volume ratio, the temperature is 30 ℃, the anaerobic culture is carried out for 72 hours after the illumination, the anaerobic illumination, the darkness and the aerobiotic alternate culture are carried out for 72 hours, the standing is carried out for 24 hours, and the wastewater degradation process is finished after the bacterial body is completely self-flocculated and precipitated.
By implementing the technical scheme provided by the invention, the following beneficial effects can be achieved:
(1) The invention provides a strain Rhodoblastus sphagnicola PNSB-MHW, which is proved by molecular level identification of a strain system and physiological and biochemical system test verification of the strain which are well known and accepted in the field, and the obtained acidophilic photosynthetic bacteria with the strain number PNSB-MHW belongs to a typical new strain, and further needs to be preserved according to legal requirements.
(2) In the invention, a Rhodoblastus sphagnicola PNSB-MHW and bacillus atrophaeus (Bacillus atrophaeus) coupling reaction system is stable in a new bacterium Rhodoblastus sphagnicola coupling composite microbial inoculum: the two strains have very strong adaptability, can adapt to water environment with pH of 4.0-8.0 and salinity of 0.1% -3%, and the strain Rhodoblastus sphagnicola PNSB-MHW CGMCC No.20882 can perform No-illumination aerobic respiration, thus ensuring the wide applicability of the two strains in cooperation use. In addition, the acid-resistant and alkali-producing properties of Rhodoblastus sphagnicola PNSB-MHW CGMCC No.20882 degrade the organic acid micromolecule substances in the acid starch wastewater to raise the pH value of the wastewater, and the added bacillus atrophaeus can secrete a large amount of amylase and other enzyme substances to degrade the macromolecular substances in the starch, and simultaneously the produced organic acid and micromolecule substances reduce the environmental pH value and stimulate the bacterial strain PNSBThe activity of MHW, rhodoblastus sphagnicola PNSB-MHW CGMCC No.20882 viable count in the composite microbial inoculum>10 10 cfu/mL, number of viable bacteria of Bacillus atrophaeus (Bacillus atrophaeus)>10 9 cfu/mL can automatically adjust the pH of the water body, so that the bacterial degradation wastewater is always in a stable state.
(3) The novel bacterium Rhodoblastus sphagnicola coupled composite microbial inoculum provided by the invention is applied to degradation of starch wastewater, and the number of Rhodoblastus sphagnicola PNSB-MHW CGMCC No.20882 viable bacteria in the degraded wastewater is more than 10 8 cfu/mL, experiments prove that the synergistic coupling of the two bacteria is good, and the activity of Rhodoblastus sphagnicola PNSB-MHW CGMCC No.20882 is enhanced by the bacillus atrophicus (Bacillus atrophaeus), so that the sewage is deodorized by degrading pollutants such as COD, ammonia nitrogen, nitrate nitrogen, total phosphorus and the like in the starch sewage with high efficiency and degrading odor generated by the sewage, volatile odor organic acid, mercaptan and the like, and data support is provided for the application of the sewage in the aspect of biological bacterial manure.
(4) The novel bacterium Rhodoblastus sphagnicola coupled composite microbial inoculum provided by the invention is applied to degradation of starch wastewater, the degraded starch wastewater has no odor, and after standing for a certain time, the self-flocculation phenomenon occurs, bacterial sludge is settled to the bottom of a container according to the settling rate of 3cm/d, and the turbidity (suspended matters) of supernatant fluid is less than 100mg/L. The settled bacterial mud can be recycled, thereby further reducing the wastewater treatment cost.
(5) The novel bacterium Rhodoblastus sphagnicola coupled composite microbial agent provided by the invention is applied to degrading starch wastewater, the Rhodoblastus sphagnicola PNSB-MHW CGMCC No.20882 coupled bacillus atrophaeus (Bacillus atrophaeus) composite microbial agent provided by the invention can degrade macromolecular substances difficult to degrade in the starch wastewater, and metabolic products can activate other microbial activities in a natural system, and the novel bacterium Rhodoblastus sphagnicola coupled composite microbial agent provided by the invention provides data support for expanding the application field of microbial strains.
Drawings
FIG. 1 is a phylogenetic tree of Rhodoblastus sphagnicola PNSB-MHW constructed based on the 16S rDNA gene.
FIG. 2 is a phylogenetic tree of Bacillus atrophaeus (Bacillus atrophaeus) BA-BC constructed based on the 16S rDNA gene.
FIG. 3 shows the absorbance peak of the cytochrome of Rhodoblastus sphagnicola PNSB-MHW viable bacteria.
FIG. 4 is a graph showing the effect of the initial pH of different media on the number of viable Rhodoblastus sphagnicola PNSB-MHW.
FIG. 5 is a graph showing the effect of different salt concentrations on the number of viable Rhodoblastus sphagnicola PNSB-MHW.
FIG. 6 is a graph showing the effect of different temperatures on the number of viable Rhodoblastus sphagnicola PNSB-MHW.
FIG. 7 is a graph showing the effect of different illumination intensities on the number of viable Rhodoblastus sphagnicola PNSB-MHW.
FIG. 8 is a graph of Rhodoblastus sphagnicola PNSB-MHW growth curves and pH changes of fermentation broths thereof.
FIG. 9 is a graph showing the growth curve of Bacillus atrophaeus and the pH change of the fermentation broth.
FIG. 10 shows the growth curve of Rhodoblastus sphagnicola PNSB-MHW coupled Bacillus atrophaeus complex and the pH change curve of the fermentation broth.
FIG. 11 is an optimized graph of culture conditions of a Rhodoblastus sphagnicola PNSB-MHW coupled bacillus atrophaeus composite, wherein A is a comparison of COD degradation efficiency of composite bacterial agents with different viable count ratios; b is the pH comparison of fermentation culture mediums of the composite microbial inoculum with different viable count proportions; C. comparing flocculation sedimentation rates of the composite microbial agents with different viable count ratios; and D, when the ratio of the Rhodobastus sphagnicola PNSB-MHW to the number of viable bacillus atrophaeus is 10:1, the composite microbial inoculum has a COD degradation curve and a pH change curve.
FIG. 12 is a graph showing the effect of different Rhodoblastus sphagnicola coupled composite microbial inoculum sizes and treatment modes on COD degradation efficiency of starch wastewater.
FIG. 13 shows the effect of different sodium acetate addition amounts Rhodoblastus sphagnicola on starch wastewater degradation by coupling the composite microbial agents.
FIG. 14 is a graph of Rhodoblastus sphagnicola coupled complex microbial agents versus starch wastewater contaminant degradation.
Detailed Description
The present invention will be described with reference to the following examples, but the present invention is not limited to the examples. All raw materials and auxiliary materials selected in the invention, as well as selected methods of culturing the strains, are well known in the art, and all percentages referred to in the invention are weight percentages unless otherwise indicated.
Rhodoblastus sphagnicola PNSB-MHW enrichment medium: KH (KH) 2 PO 4 0.04g/L,NH 4 Cl0.1g/L,MgCl 2 ﹒6H 2 O 0.01g/L,CaCl 2 ﹒2H 2 O 0.05g/L,CH 3 COONa1.0g/L, trace element mother liquor 1ml, vitamin complex mother liquor 1ml, ph=5.0.
Rhodoblastus sphagnicola PNSB-MHW isolation Medium (RCVBN) (g/L): KH (KH) 2 PO 4 0.5g/L,K 2 HPO 4 0.3g/L，NH 4 Cl 1.0g/L,MgCl 2 ﹒6H 2 O 0.2g/L,CaCl 2 ﹒2H 2 O 0.5g/L,CH 3 COONa 1.0g/L,CH 3 CH 2 COONa 0.5g/L, yeast powder 0.1g/L, agar 7.0g/L, trace element mother liquor 1ml, pH=5.0.
Example 1: isolation and characterization of Rhodoblastus sphagnicola PNSB-MHW and Bacillus atrophaeus (Bacillus atrophaeus) BA-BC
Identification of Rhodoblastus sphagnicola PNSB-MHW
(1) Rhodoblastus sphagnicola PNSB-MHW separation and purification
The strain is separated from a water sample of a sewage drain ditch of quincuncial monosodium glutamate acid waste water in five Xinjiang ditches, the pH value of the waste water is 4.5, and sediment is collected. Directional enrichment is carried out by using an enrichment culture method. Culturing at 25deg.C for 7d under 2000lx illumination intensity, and sucking thallus to separate strain after the color of culture medium changes to red. After the diluted thalli are coated on an RCVBN culture medium, an anaerobic culture tank is used, carbon dioxide is introduced into the top layer of the tank body to empty air, then the tank body is sealed and placed in an illumination culture box for constant-temperature culture at 25 ℃, the illumination intensity is 2000lx, and colony separation is carried out according to the occurrence time, the size and the colony morphology of colonies on a flat plate.
(2) Rhodoblastus sphagnicola PNSB-MHW Classification and identification
Rhodoblastus sphagnicola PNSB-MHW (hereinafter referred to simply as "strain PNSB-MHW") 16Sr DNA sequence determination and analysis, the total DNA of the extracted strain PNSB-MHW is used as a template for PCR amplification, and the primers are bacterial 16S rDNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1429R (5'-CTACGGCTACCTTGTTACGA-3'). The PCR reaction system was 25. Mu.L: 1.0. Mu.L of DNA template, 10. Mu. Mol/L of 27F/1429R each 1.0. Mu.L, 10mmol/L of dNTPs 1.5. Mu.L, 10 XPCR Buffer (2.5 mmol/L MgCl) 2 ) 2.5. Mu.L, 2.5U/. Mu.L TaqDNA polymerase 0.5. Mu.L, and sterilized ultrapure water was supplemented to 25. Mu.L. The reaction conditions are as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 45s, annealing at 58℃for 45s, elongation at 72℃for 1.5min,35 cycles; extending at 72deg.C for 7min, and storing at 4deg.C. After the amplified product is detected by 10g/L agarose gel electrophoresis, the PCR product is subjected to clone sequencing by Shanghai biological engineering Co., ltd, and the sequencing result is shown as SEQ ID No. 1.
The sequence result was subjected to BLAST search at the National Center for Biological Information (NCBI), a model strain with high similarity was selected as a reference strain, and a 16S rDNA phylogenetic tree was constructed using the Neighbor-joining method (Neighbor-joining) in MEGA5.0 software, and a Bootstrap value (Bootstrap) of 1000. As a result, referring to FIG. 1, the strains PNSB-MHW and Rhodoblastus sphagnicola form a branch, the 16S rDNA gene sequence has higher similarity with the model strain Rhodoblastus sphagnicola (AM 040096), the homology with Rhodoblastus sphagnicola (AM 040096) strain is highest, the similarity reaches 98.32%, and the comprehensive judgment of the similarity and the homology of the strains proves that the obtained acidophilic photosynthetic bacteria belongs to a typical new strain in the category of PNSB-MHW through the molecular level identification of the strain systems and the physiological and biochemical system test of the combined strains which are well known in the art.
Based on the above biological characteristics, the strain PNSB-MHW was identified as Rhodoblastus sphagnicola. The strain has been deposited in the Budapest treaty International deposit unit of microorganisms: china general microbiological culture Collection center (CGMCC). Preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing. The preservation date is 10 months and 13 days in 2020, and the preservation number is CGMCC No.20882.
Isolation and identification of Bacillus atrophaeus (Bacillus atrophaeus) BA-BC
Bacillus atrophaeus (Bacillus atrophaeus) BA-BC was isolated from a salt farm soil of Cuminum cyminum Bai Xicun, atsu district, xinjiang. Collecting saline soil with the thickness of 0-10cm on the surface layer of a salt field, using an LB culture medium, adding 5% sodium chloride, uniformly coating the diluted solution with different concentrations on a solid plate containing salt LB by adopting a dilution gradient method, culturing at the constant temperature of 30 ℃ for 24 hours, picking single colony according to different aggregation forms, streaking and purifying, then carrying out 16SrRNA gene sequencing, and constructing a 16S rDNA gene phylogenetic tree by utilizing an adjacent method (Neighbor-joining) in MEGA5.0 software, wherein the result is shown in the attached figure 2. The results show that the strain has 99.45% similarity with bacillus atrophaeus (Bacillus atrophaeus), and the analysis result of phylogenetic tree also shows that the strain belongs to bacillus atrophaeus clusters, and bacillus atrophaeus (Bacillus atrophaeus) BA-BC belongs to common bacillus atrophaeus (Bacillus atrophaeus).
Example 2: physiology assay of Rhodoblastus sphagnicola PNSB-MHW
(1) Bacterial strain PNSB-MHW living bacterium cytochrome light absorption wavelength
As shown in figure 3, 4 characteristic peaks appear in the range of 500-900nm, and the peaks are respectively positioned at 480nm, 510nm, 590nm and 810nm, which belong to the light absorption peak positions of the chlorophyll a of the typical purple non-sulfur photosynthetic bacteria, but are clearly different from the common bacteria in the same genus range of the acidophilic photosynthetic bacteria, and belong to the characteristics of new bacteria in the same genus of the acidophilic photosynthetic bacteria.
(2) Growth of the strain PNSB-MHW at different initial pH conditions
The growth condition of the strain PNSB-MHW under different initial pH conditions is shown in the accompanying figure 4, the optimal growth pH of the strain is 5.0-5.5, the growth of the strain is reduced along with the increase of the pH, the activities of the strain PNSB-MHW are completely inhibited at the pH of 3.5 and 9.0, the strain can grow at the pH of 4.0 and 8.5, but the activity is only 30% of the highest activity, and the activity of the strain is 60% at the pH of 7.5, so the strain belongs to acidophilic bacteria, generally likes to be in an acidic environment, has a certain tolerance to alkali, has clear distinction from the common acidophilic photosynthetic bacteria in the same genus range, and belongs to the characteristics of new strains in the acidophilic photosynthetic bacteria in the same genus.
(3) Growth of the strain PNSB-MHW under conditions of different salt concentrations
The growth condition of the strain PNSB-MHW under different salt concentration conditions is shown in the accompanying figure 5, the viable count of the strain is higher under the condition of 0.5 to 3 percent of sodium chloride concentration, the bacterial growth activity is highest under the condition of 1 to 1.5 percent of salt concentration, the bacterial growth is obviously affected when the salt concentration is higher than 3.5 percent, the activity is reduced by 70 percent, the activity is basically completely inhibited after the salt concentration is higher than 4 percent, the bacterial does not grow, but the strain has clear salt tolerance difference with the common strain in the same genus range of acidophilic photosynthetic bacteria, and the strain belongs to the characteristics of a new strain in the same genus of acidophilic photosynthetic bacteria.
(4) Growth of the strain PNSB-MHW at different culture temperatures
The growth condition of the strain PNSB-MHW under different culture temperature conditions is shown in the accompanying figure 6, and the result shows that the strain has higher activity at the optimum growth temperature of 30 ℃ and 20-25 ℃, the active growth of the strain is inhibited at the temperature of over 40 ℃, and the strain does not grow basically at the temperature of 45 ℃, but has clear growth characteristic difference from the common strain in the same genus range of acidophilic photosynthetic bacteria, and belongs to the characteristic of a new strain in the same genus of acidophilic photosynthetic bacteria.
(5) Growth of the strain PNSB-MHW under different illumination intensities
The effect of illumination intensity on the growth of the strain PNSB-MHW is shown in a figure 7, and the result shows that the strain has higher growth activity under the light intensity of 500-6000 lx, wherein the strain has highest growth performance under the light intensity of 1000-2000lx, and the growth performance is higher under the light intensity of 500lx, so that the strain can be potentially applied to degradation of high-chroma water pollutants which are difficult to transmit light. When the illumination intensity exceeds 10000lx, the activity is inhibited, which shows that the cytochrome reaches light saturation and the growth is inhibited, but the bacterial strain in the same genus range with the common acidophilic photosynthetic bacteria has a clear difference on the illumination intensity, and belongs to the characteristic of a new bacterial strain in the same genus with the acidophilic photosynthetic bacteria.
Example 3: preparation of Rhodoblastus sphagnicola coupled composite microbial inoculum
The embodiment further provides a preparation method of the Rhodoblastus sphagnicola coupling composite microbial inoculum based on the embodiment 1-2, which specifically comprises the following steps:
(1) Inoculating the preserved strain Rhodoblastus sphagnicola PNSB-MHW CGMCC No.20882 into a liquid modified Nisson & Dundas culture medium, standing and culturing in an illumination incubator at 30 ℃ and an illumination intensity of 1000-2000lx, shaking 2 times per day, and culturing for 3-5 days to a stationary phase, wherein the concentration of the thallus is 10g dry thallus per liter; inoculating selected Bacillus atrophaeus (Bacillus atrophaeus) into liquid self-grinding culture medium, and culturing at 30deg.C and rotation speed of 120rad/min in shaking table for 3-5d to reach stationary phase, wherein the thallus concentration is 15g dry bacteria per liter.
(2) Taking the two bacterial liquids cultured in the step (1), respectively carrying out centrifugal separation at room temperature, discarding supernatant, precipitating, fully suspending with sterile water, centrifuging again, repeating the step for 3-5 times, and suspending the precipitate with sterile water to obtain the liquid seeds of the two bacterial strains.
(3) Under the aseptic condition, the Rhodoblastus sphagnicola PNSB-MHW obtained in the step (2) and the bacillus atrophaeus (Bacillus atrophaeus) liquid seeds are respectively compounded according to the volume ratio to prepare the Rhodoblastus sphagnicola coupling compound microbial inoculum.
Example 4: preparation of Rhodoblastus sphagnicola coupled composite microbial inoculum
This example examined culture conditions of the strain PNSB-MHW, bacillus atrophaeus culture conditions, and a combination of both on the basis of example 3.
(1) Culture of Rhodoblastus sphagnicola PNSB-MHW
The strain PNSB-MHW was cultured on modified Nisson & Dundas medium at 30deg.C under light of 1000-2000lux for 2 times daily shaking for 3-5d to stationary phase, pH value of the fermentation broth was detected, and growth condition of Rhodoblastus sphagnicola PNSB-MHW was examined, and specific data are shown in FIG. 3.
As can be seen from the data of FIG. 3, rhodobastus sphagnicola PNSB-MHW enters a growth index period from the 2 nd of initial inoculation, the 8 th reaches a platform period, the bacterial body is kept still for preventing and controlling the 12d viable count from obviously decreasing, and the viable count is 6.86x10 after the culture is finished 10 cfu/ml. In addition, the pH value of the fermentation medium can be seen that Rhodoblastus sphagnicola PNSB-MHW can survive under the condition of initial pH=4.0, and the pH value after fermentation is finished is between 8.0 and 9.0, which shows that Rhodoblastus sphagnicola PNSB-MHW can have wide acid-base adaptability and can produce alkali in the growth process, but has clear distinction on the characteristics of producing alkali from the common strains in the same genus range of acidophilic photosynthetic bacteria, and belongs to the characteristics of new strains in the same genus of acidophilic photosynthetic bacteria.
(2) Cultivation of Bacillus atrophaeus (Bacillus atrophaeus)
The isolated and purified Bacillus atrophaeus (Bacillus atrophaeus) was cultured on a self-grinding medium at 30℃for 3-5d to stationary phase in a shaking table at 1200 rpm. The growth status of bacillus atrophaeus (Bacillus atrophaeus) and the pH change of the fermentation broth were examined, and the specific results are shown in FIG. 4.
As can be seen from the data of FIG. 4, bacillus atrophaeus (Bacillus atrophaeus) can grow in large quantity from the 2 nd day to the exponential phase, and the 5 th day to the logarithmic phase, and the viable count does not decrease within 15 days with the increase of the storage time, and the viable count is 9.11×10 after the cultivation 12 cfu/ml. Bacillus atrophaeus (Bacillus atrophaeus) can reduce pH of fermentation broth by using culture medium containing starch, glucose, etc. to generate acids, and the pH value is reduced with the increase of culture time, and is stabilized at the level after the pH value is reduced to about 4.0 at day 5. Thus, bacillus atrophaeus (Bacillus atrophaeus) is able to withstand low pH environments, which can utilize sugars to produce acid.
(3) Preparation of Rhodoblastus sphagnicola coupled composite microbial inoculum
Based on experiments 1-2, the experiment uses a bacillus atrophaeus (Bacillus atrophaeus) culture medium, the pH of the culture medium is reduced to 4.5 by adding sterilized lactobacillus fermentation broth, and Rhodoblastus sphagnicola PNSB-MHW and bacillus atrophaeus (Bacillus atrophaeus) are connected into the culture medium according to a volume ratio of 1:1. And the number of viable bacteria and the pH change of the fermentation broth of each bacterial cell are measured by taking only the PNSB-MHW strain and only the bacillus atrophaeus strain as control groups, and the specific results are shown in figure 10.
As can be seen from the data of FIG. 10, bacillus atrophaeus (Bacillus atrophaeus) did not grow at the initial culture pH=4.5, and the cell concentration did not change substantially, but after Rhodoblastus sphagnicola PNSB-MHW was compounded, the Bacillus atrophaeus cell concentration showed an upward trend, and the OD value of the cells cultured for 16d was 2 times that of the cells cultured alone. The photosynthetic bacteria show the same change curve, the OD value of Rhodoblastus sphagnicola PNSB-MHW bacteria increases along with the increase of the culture time, and the concentration of Rhodoblastus sphagnicola PNSB-MHW bacteria after being compounded with the bacillus atrophaeus shows a straight line rising trend, and the number of viable bacteria is about 4 times that of the single culture. Meanwhile, monitoring the dynamic change of the pH of the culture medium, wherein the pH of the bacillus atrophaeus is maintained in an initial state without change, the pH of Rhodoblastus sphagnicola PNSB-MHW is increased along with the increase of the culture time and finally to about 7.5, the pH of the coupled complex bacteria is increased along with the increase of the time before 8 days of culture, but the pH starts to drop sharply on the 9 th day, reaches the lowest 5.0 when the pH reaches 11 days, then shows a straight line rising trend, and finally the pH is about 9.0. It is shown that the combination of two bacteria can promote each other to grow, mainly by removing the repressor affecting the growth of both bacteria. Rhodoblastus sphagnicola PNSB-MHW can utilize bacillus to decompose organic acid generated by sugar starch to generate alkaline substances, so that pH inhibition of bacillus is relieved, and after the inhibition of bacillus is relieved, continuous growth of the bacillus is promoted, decomposed organic matters continue to produce acid, and deficiency of photosynthetic bacteria growth nutrient substances is relieved, so that photosynthetic bacteria are stimulated to grow to produce alkali, pH is regulated, and growth of bacillus atrophaeus is inhibited due to limited substrate, and simultaneously, growth of Rhodoblastus sphagnicola PNSB-MHW thallus is also inhibited. After fermentation, the pH value of the culture medium is about 8.5, the viable count of the two bacteria is several times higher than that of the bacteria when the two bacteria are cultured independently, and the self-flocculation precipitation phenomenon occurs in bacillus atrophaeus (Bacillus atrophaeus) and Rhodoblastus sphagnicola PNSB-MHW.
Example 5: optimized culture of Rhodoblastus sphagnicola PNSB-MHW CGMCC No.20882 coupled bacteria composite microbial inoculum
In order to verify the actual degradation effect of the Rhodoblastus sphagnicola PNSB-MHW CGMCC No.20882 coupling composite microbial inoculum on starch wastewater, the starch wastewater is used as a culture medium, the influence of the optimal compounding proportion, inoculation amount and culture mode (light anaerobic, dark aerobic and light anaerobic-dark aerobic) of Rhodoblastus sphagnicola PNSB-MHW CGMCC No.20882 and bacillus atrophaeus (Bacillus atrophaeus) on the COD degradation rate and pH of the starch wastewater is explored, and the flocculation condition of the degraded wastewater is compared.
(1) Influence of different Rhodoblastus sphagnicola PNSB-MHW (Bacillus atrophaeus) (Bacillus atrophaeus) compound ratios on starch wastewater degradation efficiency
The rest conditions are unchanged, and the experiment examines the influence of different Rhodoblastus sphagnicola PNSB-MHW CGMCC No.20882 and bacillus atrophaeus (Bacillus atrophaeus) composite proportion on the degradation efficiency of starch wastewater. As shown in the figure 11, according to the data in the figure 11, when the volume ratio of Rhodoblastus sphagnicola PNSB-MHW (PSB) to bacillus atrophaeus (Bacillus atrophaeus) (BA) is 10:1, the highest degradation efficiency on the COD of starch wastewater is 98%, the pH at the end of fermentation is about 8.0, and the pH change in the fermentation process is lower, so that the pH can be maintained between 7 and 8 all the time; further, the flocculation condition of the degraded wastewater was measured, and the flocculation sedimentation rate was 5cm/d when the volume ratio of Rhodoblastus sphagnicola PNSB-MHW (PSB) to Bacillus atrophaeus (Bacillus atrophaeus) (BA) was 10:1.
(2) Test of COD degradation efficiency of starch wastewater by inoculum size
Based on the determination of the ratio of the viable bacteria in the test (1), the effect of the inoculation amount and the inoculation mode on the degradation efficiency of the starch wastewater is examined, and the result is shown in the figure 12. As can be seen from the data of fig. 12, the COD degradation efficiency shows an increasing trend with the inoculum size, wherein the starch wastewater degradation rate is more than 90% when the inoculum size is more than 15%, and the inoculum size is 15% based on the practical application of cost consideration; the treatment patterns were further compared at 15% inoculation conditions and the results are shown in figure 12. The highest COD degradation efficiency under the light anaerobic-dark aerobic condition is 95 percent, and then the dark aerobic treatment is carried out. Because photosynthetic bacteria can grow and degrade COD and produce alkali at the same time under the anaerobic illumination condition, and bacillus atrophaeus belongs to aerobic bacteria which can only work under the condition of oxygen, the set illumination anaerobic-dark aerobic condition is that firstly illumination anaerobic 48h,Rhodoblastus sphagnicola PNSB-MHW is carried out to grow and produce alkali, and then anaerobic illumination is carried out: the reaction is carried out in the dark for oxygen=12h:12h, and the COD degradation efficiency is 95.21% under the condition.
(3) Test of the influence of intermediate metabolite on starch degradation Rate
Acetic acid is an intermediate metabolite of many anaerobic reactions and is also an important substrate for stimulating the growth of photosynthetic bacteria. Therefore, the effect of adding a certain amount of sodium acetate to starch wastewater on COD degradation efficiency was further examined, and the result is shown in FIG. 13. Compared with the control group, the sodium acetate with different concentrations can improve the degradation rate of the COD of the starch wastewater, but the improvement range is not large, and the sodium acetate with the concentration of 1g/L can improve the degradation rate of the COD of the wastewater to about 98 percent.
Example 6: application of Rhodoblastus sphagnicola coupled composite microbial inoculum in starch wastewater degradation
Based on the embodiment 2-3, the optimal compounding ratio of Rhodoblastus sphagnicola PNSB-MHW and bacillus atrophaeus (Bacillus atrophaeus) in the Rhodoblastus sphagnicola coupled compound microbial inoculum is verified, the influence of Rhodoblastus sphagnicola PNSB-MHW coupled compound microbial inoculum on indexes such as ammonia nitrogen, nitrate nitrogen, total phosphorus, mercaptan, hydrogen sulfide and the like in starch wastewater along with the extension of degradation time is examined, and the specific result is shown in figure 14.
As can be seen from the data of FIG. 14, under the optimal condition, the Rhodoblastus sphagnicola PNSB-MHW coupled composite microbial inoculum has the degradation rate of ammonia nitrogen, nitrate nitrogen and total phosphorus in the water body of 6d>99%, the degradation efficiency of mercaptan and hydrogen sulfide of the starch wastewater malodorous source is over 98%, and the water quality reaches the national sewage discharge standard. After the wastewater treatment is finished, the number of two coupled composite bacteria in the wastewater is measured,the viable count of Rhodoblastus sphagnicola PNSB-MHW can reach 1.19X10 8 cfu/ml, the number of viable bacteria of the bacillus atrophaeus (Bacillus atrophaeus) can reach 6.21 multiplied by 10 8 cfu/ml. Rhodoblastus sphagnicola PNSB-MHW and bacillus atrophaeus (Bacillus atrophaeus) are compounded according to the volume ratio of 10:1, the inoculation amount is 15% of the total volume of starch wastewater, anaerobic illumination (1000-2000 lx) culture is carried out for 0-72h, and then anaerobic illumination is carried out: the method is characterized in that the method comprises the steps of continuously culturing for 72 hours in dark aerobic condition (12 hours: 12 hours), adding 1g/L sodium acetate into sewage before culturing, so that the COD degradation rate of starch waste water can reach 98.21%, the degradation efficiency of ammonia nitrogen, nitrate nitrogen, total phosphorus, mercaptan, hydrogen sulfide and other pollutants in a water body can reach more than 98%, meanwhile, after fermentation is finished, the pH value of a fermentation liquor is about 8.0, under the pH condition, thalli begin to self flocculate, suspended particles and high-chroma particulate matters in the sewage can be precipitated (8 cm/hour), and the waste water can be discharged without additional treatment, and can be used as water for irrigation, flushing and recycling of greening raw water.
The above examples are only illustrative of the invention and are not intended to be limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While remaining within the scope of the invention, obvious variations or modifications thereof are contemplated.
Sequence listing
<110> institute of microbiological applications of the academy of agricultural sciences of Xinjiang (Xinjiang-Asian Biotechnology research and development center of China)
<120> Rhodoblastus sphagnicola coupled composite microbial inoculum and application thereof
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<170> SIPOSequenceListing 1.0
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<211> 1418
<212> DNA
<213> Rhodoblastus sphagnicola(Rhodoblastus sphagnicola)
<400> 1
atcatggctc agaacgaacg ctggcggcag gcctaacaca tgcaagtcga accgatgtag 60
caatacatca gtggcagacg ggtgaggaac acgtgggaac ataccctttg gttcggaata 120
actcagggaa acttgagcta ataccggata agtcggcaac gagaaagatt catcgccgaa 180
ggaatggccc gcgtccgatt agctagttgg tgaggtaacg gctcaccaag gcgacgatcg 240
gtagctggtc tgagaggatg atcagccaca ttgggactga gacacggccc aaactcctac 300
gggaggcagc agtggggaat attggacaat gggcgcaagc ctgatccagc catgccgcgt 360
gagtgatgac ggccttaggg ttgtaaagct ctttcaccca cgacgataat gacggtagtg 420
ggagaagaag ccccggctaa cttcgtgcca gcagccgcgg taatacgaag ggggctagcg 480
ttgttcggat ttactgggcg taaagcgcac gtaggcggat ctttaagtca ggggtgaaat 540
gccggagctc aacttcggaa ctgccttaga tactggagat ctagagatcg agagacgtga 600
gtggaactgc gagtgtagac gtgaaattcg tagatattcc caagaacacc agtggcgaag 660
gcggctcaca ggctcgatac tgacgctgag gtgcgaaagc gtggggagca aacaggatta 720
gataccctgg tagtccacgc cgtaaacgat ggatgctagc cgttaggcag cttgctgctt 780
agtggcgcag ctaacgcttt aagcatcccg cctggggagt acggtcgcaa gattaaaact 840
caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtataatt cgatgcaacg 900
ggcagaacct taccagcttt tgacatgtcc tggacggttt ccagagatgg attcctctct 960
tcggagccag gaacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg 1020
ttaagtcccg caacgagcgc aacccacgtc cttagttgcc aacatttagt tgggcactct 1080
agggagactg ccggtgataa ggggagagga aggtgtggat gatgtcaagt gctcatggcc 1140
cttacacgct cggctacaca cgtgctacaa tggcggtgac aatgggatgc gaaggggcga 1200
cccttagcaa atctcaaaaa tccgtctcag ttcggattgt tctctgcaac tcgagagcat 1260
gaaggtggaa tcgctagtaa tcgcagatca gaacgctgcg gtgaatacgt tcccgggcct 1320
tgtacacacc gcccgtcaca ccatgggagt tggctttacc cgaaggcgtt tcgctaaccg 1380
caaggaggca gacgaccacg gtagggtcag cgactggg 1418
Claims (3)
1. New fungusRhodoblastus sphagnicolaThe coupling composite microbial inoculum is characterized by comprisingRhodoblastus sphagnicolaPNSB-MHW and Bacillus atrophaeusBacillus atrophaeus) The preparation method comprises the steps of compounding according to a volume ratio of 10:1; the saidRhodoblastus sphagnicolaPNSB-MHW was deposited in China general microbiological culture Collection center, accession number: CGMCC No.20882, date of preservation: 10 months 13 days 2020; in the coupling composite microbial inoculumRhodoblastus sphagnicolaPNSB-MHW and Bacillus atrophaeusBacillus atrophaeus) The number average of viable bacteria is more than or equal to 10 9 cfu/mL。
2. The method as claimed in claim 1Rhodoblastus sphagnicolaThe coupling composite microbial inoculum is characterized in thatRhodoblastus sphagnicolaThe gene sequence of PNSB-MHW is shown in SEQ ID NO. 1.
3. A method as claimed in claim 1Rhodoblastus sphagnicolaThe application of the coupling composite microbial inoculum in degrading starch wastewater is characterized in that,Rhodoblastus sphagnicolainoculating the coupled composite microbial inoculum into starch wastewater according to the inoculum size of 15% of the mass volume ratio, carrying out anaerobic light-dark aerobic alternate culture for 72h after carrying out light anaerobic culture for 72h at the temperature of 30 ℃, and standing for 24h until the degradation process of the wastewater is finished after the bacterial cells are completely self-flocculated and precipitated.
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