CN114317302B - Black and odorous water body remediation microbial inoculum and application thereof - Google Patents
Black and odorous water body remediation microbial inoculum and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of microbial agents, and particularly relates to a black and odorous water body remediation microbial agent and application thereof. The specific technical scheme is as follows: a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae) is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 09 days 2021, with the preservation numbers as follows: CGMCC No.24061. The invention can effectively restore the black and odorous water body, reduce the concentration of pollutants such as COD, ammonia nitrogen and the like in the water, improve DO, ORP and transparency of the water body, and restore the severe black and odorous water body to be in a state without black and odorous.
Description
Technical Field
The invention relates to the technical field of microbial agents, and particularly relates to a black and odorous water body remediation microbial agent and application thereof.
Background
With the acceleration of the urbanization process and the rapid development of economy, more and more production wastewater and domestic wastewater are discharged into rivers, and the rivers are polluted to different degrees, wherein the phenomenon of black and odorous rivers is more severe. The black and odorous water body becomes a ubiquitous problem in recent decades, and a lot of harm exists, so that the urban image is influenced, the water body ecological system is also deteriorated, and the health of surrounding residents is influenced. Therefore, the research on the black and odorous water body remediation technology is urgent.
The black and odorous water body remediation technology can be divided into chemical remediation, physical remediation and biological remediation. Wherein the chemical remediation has the risk of secondary pollution caused by introducing pollutants into the water body; physical remediation cost is high, the water body ecosystem is easily damaged, and black odor is easy to recur; bioremediation has the advantages of environmental friendliness, low cost, simple technology, easy maintenance and the like, and is a hotspot in the research fields of water environment remediation and black and odorous water treatment at present.
The microbial inoculum is a common bioremediation method, the decomposition function of the microorganisms is strengthened, and the purpose of degrading the pollutants is achieved by utilizing the absorption and transformation of the microorganisms on the pollutants. At present, in the repair of black and odorous water, a complex microbial inoculum construction method is unclear, a preparation process is complex, the variety of composition bacteria is various, the application cost is high, and the problem that the growth condition of the microbial inoculum cannot be quantitatively detected after the microbial inoculum is added into the environment is ubiquitous, and needs to be solved urgently.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a black and odorous water body remediation microbial inoculum which can effectively remediate black and odorous water bodies.
In order to realize the purpose, the invention is realized by the following technical scheme:
the invention discloses a Saccharomyces cerevisiae (Saccharomyces cerevisiae) which is preserved in China general microbiological culture Collection center at 12 months and 09 days 2021, wherein the preservation numbers are as follows: CGMCC No.24061.
Correspondingly, the 16S rDNA sequence of the saccharomyces cerevisiae is shown as SEQ ID NO:2, respectively.
Correspondingly, the repairing microbial inoculum comprises the saccharomyces cerevisiae.
Preferably, the bacillus cereus, the klebsiella and the streptomyces are also included.
Preferably, the bacillus cereus is deposited in the general microbiological culture collection center of the committee for preservation management of chinese microbiological culture in 2021, 12 months and 09 days, and the deposit number is: CGMCC No.24062; the streptomyces is preserved in China general microbiological culture Collection center on 09.12.2021, with the preservation numbers as follows: CGMCC No.24063.
Preferably, the viable bacteria amount of the bacillus cereus, the klebsiella, the saccharomyces cerevisiae and the streptomyces is 2.1 multiplied by 10 respectively 9 cfu/mL、8×10 8 cfu/mL、6.2×10 8 cfu/mL、6.4×10 9 cfu/mL。
Preferably, the construction mode of the remediation microbial inoculum is as follows: and mixing the independently fermented streptomyces with the bacterial liquid obtained by mixing and fermenting the bacillus cereus, the klebsiella and the saccharomyces cerevisiae.
Preferably, the ratio of the bacillus cereus to the klebsiella to the saccharomyces cerevisiae to be mixed and fermented is 0.31:0.18:0.51, the mass ratio of the streptomyces fermented alone to the bacterial liquid after mixed fermentation is 1.
Correspondingly, the remediation microbial inoculum is applied to treatment of the black and odorous water body.
Preferably, the adding mode of the remediation microbial inoculum in the black and odorous water body is as follows: adding a bacterial liquid into the black and odorous water body in a spraying mode, aerating for 24 hours, and stopping; adding the mixture again after 3d, aerating for 24h, and then stopping adding; and adding the mixture again after 3d, and aerating for 24h.
Preferably, the adding amount of the repairing microbial inoculum for one time is 0.1-1% according to the volume ratio.
The invention has the following beneficial effects:
1. the repairing microbial inoculum provided by the invention consists of four strains, namely bacillus cereus, klebsiella, saccharomyces cerevisiae and streptomyces; through the optimization of the compound proportion and the optimization of degradation conditions, the compound microbial inoculum can effectively restore the black and odorous water body, reduce the concentration of pollutants such as COD (chemical oxygen demand), ammonia nitrogen and the like in water, improve DO (dissolved oxygen demand), ORP (oxidation-reduction potential) and transparency of the water body, and restore the severe black and odorous water body to be in a state without black and odorous water; moreover, the microbial inoculum has simple composition, simple fermentation method and small addition amount, and can effectively reduce the repair cost of the black and odorous water body.
2. The repairing microbial inoculum disclosed by the invention can quantitatively detect the planting situation of the microbial inoculum added into the environment by a fluorescence quantitative PCR detection method; on one hand, the microbial inoculum can be periodically supplemented according to the existence condition of the microbial inoculum, on the other hand, the growth characteristics of the microbial inoculum can be deeply researched, and the composite microbial inoculum is further optimized; the result shows that the strain is still planted in the environment after being added for 67 days, and the environmental stability is good.
Drawings
FIG. 1 shows that 20 strains of bacteria can treat COD and NH in black and odorous water body 3 -five day removal of N;
FIG. 2 shows DO and ORP values of 20 strains after the black and odorous water body is repaired;
FIG. 3 shows five-day removal rates of 40 groups of composite bacterial agents on COD and NH3-N in the black and odorous water body;
FIG. 4 shows DO and ORP values of the black and odorous water body repaired by the 40 groups of composite bacterial agents;
FIG. 5 is a scanning electron microscope image of FN4 (left) and complex microbial inoculum (right);
FIG. 6 shows the COD removal rate and NH content when the amount of the complex microbial inoculum is 0.1% 3 -N removal rate response results;
FIG. 7 shows the colony morphology of strains C111, B16, J13, FN 4;
FIG. 8 shows the fluorescent quantitative PCR standard curve for strains C111, B16, J13 and FN 4;
FIG. 9 shows the results of quantitative detection of complex microbial inoculum in overlying water;
FIG. 10 shows the result of quantitative detection of complex microbial inoculum in surface sediments.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art.
The reagents and culture media involved in the invention are as follows:
1. LB medium (enriched medium): 10g of peptone, 10g of NaCl, 5g of yeast powder, 1000mL of water, 7.0-7.2 of pH value, and sterilizing for 20min at 121 ℃. The solid LB medium is prepared by adding 20g of agar, and the semisolid LB medium is prepared by adding 7.5g of agar.
2. Black and odorous water body slant culture medium: 1000mL of black and odorous water and 20g of agar.
3. Primary screening medium (black and odorous substrate sludge medium): 100g of black and odorous bottom mud, 20g of agar and water are added to the mixture until the total volume is 1000ml.
4. Artificially simulating a black and odorous water body: beef extract 0.35g, naCl0.16g, KCl0.03g 2 0.022g，MgSO 4 ·7H 2 O 0.022g，K 2 HPO 4 0.84g，KH 2 PO 4 0.11g, 0.05mL of lactic acid, 0.0073g of sulfanilic acid, 0.0073g of ferric citrate, 0.06g of urea, 0.36g of peptone, 50mL of natural black-odor water, and carrying out anaerobic standing at 30 ℃ for 5d.
1. Single-bacterium screening for black and odorous water body remediation
1. The source of the single bacterium.
The method comprises the steps of purifying and separating indigenous strains 59 from black and odorous water bodies in Wuzhou city and Shijie 370255 (the black and odorous water in the Ten Ling river, donghu park, chuan Duhua pond, the black and odorous section of the Jinjiang river and the division pond in the Kejie) and black and odorous bottom mud in the four places (the black and odorous bottom mud in the Ten Ling river, the bottom mud in the double sand bridge, the bottom mud in the double flow black and odorous river, and the black and odorous bottom mud in Shijie 37025.
2. And (5) domesticating the strains.
Inoculating all strains into corresponding liquid culture media from a slant, and culturing for 2-3 days at a constant temperature of 30 ℃ and 140r/min in a shaking box to obtain seed liquid; preparing a culture medium (black smelly water: LB culture medium =3: 7, v/v), inoculating the seed solution for passage for one time, and culturing for 2-3 days at 140r/min and 30 ℃ in a constant-temperature shaking box; preparing a culture medium (black smelly water: LB culture medium =5, v/v), carrying out secondary passage, and culturing for 2-3 days at a constant temperature shaking box of 30 ℃ and 140 r/min; preparing a culture medium (black smelly water: LB culture medium = 7) and carrying out three passages, and culturing for 2-3 days at 140r/min in a constant-temperature shaking box at 30 ℃; preparing a culture medium (black smelly water: LB culture medium = 9) for four passages, and culturing for 2-3 days at 140r/min and 30 ℃ in a constant-temperature shaking box; and finally, storing the strain in a black and odorous water slant culture medium.
3. And (5) performing qualitative primary screening.
The microorganism capable of being planted in the black and odorous bottom mud is selected to prepare the black and odorous water body remediation microbial inoculum, so that the added microbial inoculum can be ensured to stably exist in the black and odorous water body for a long time. Therefore, the domesticated strains were further screened with a sterilized black and odorous substrate. And (3) carrying out plate streaking on the domesticated strains, culturing for five days at the temperature of 30 ℃ in a constant-temperature incubator, observing and recording the growth conditions of the strains on a plate, and selecting 20 strains with good growth on a primary screening culture medium, wherein the colony forms of the 20 strains are shown in the following table 1.
TABLE 1 morphological characteristics of the 20 strains
| Strain numbering | Colony morphology |
| B16 | Milky white colony, round shape, large colony, wet and sticky surface |
| Y1279 | The colony is transparent, has raised middle part, and is dispersed all around, irregular shape, and sticky |
| C11 | Milky white, round, smooth, convex, moist surface |
| J16 | Milky white, round, smooth, convex, moist surface |
| Y1250 | White round, convex in the middle, smooth surface, dry edge |
| J13 | White colony, irregular, smooth and moist surface and convex middle |
| J25 | Irregular shape, relatively transparent, moist surface and convex middle |
| FN4 | The diameter of the bacterial colony is about 2mm, the bacterial colony is round, dry, convex in the middle, white, and transparent in the edge |
| FN7 | The bacterial colony is circular, has a raised middle part and is white, and the edge is transparent and concave |
| Y1122 | White colony, irregular edge, wet surface |
| Q17 | White circle, diameter of about 1.5mm, raised middle part and wet surface |
| Y1117 | White round, moist surface, flat and without humps |
| Y115 | Pale yellow round colony, raised middle part, and wet surface |
| Q11 | Round point shape, colony diameter of about 1mm, and surface drying |
| C111 | White round, about 3mm in diameter, with wet surface and fluffy edge |
| Y1115 | White round, diameter of about 2mm, wet surface, smooth edge |
| Y106 | Light yellow round, wet edge, concentric upward convex top, white and dry convex part |
| CX4 | Yellow colony, irregular round shape, wet surface and raised middle part |
| C13 | White colony, irregular edge, dry surface, flat and no ridge |
| Q24 | White colony with yellowish edge, transparency, middle swelling, milky white surface and wet surface |
4. And (6) quantitatively re-screening.
The strain is inoculated in 150mL LB culture medium and cultured for 48h at 140r/min in a constant temperature shaking box at 30 ℃. In a sterile environment, 3mL of culture solution is sucked by a pipette, centrifuged for 5min at 5000r/min, the supernatant is poured off, and the precipitate is washed three times by sterile normal saline and then added into 100mL of artificial simulated black and odorous water. And measuring COD, ammonia nitrogen, DO and ORP of the black and odorous water body after five days, and evaluating the repairing effect of the black and odorous water body according to a water quality index formula. COD removal rate, NH after five days 3 the-N removal rate is shown in figure 1, and the ORP value and DO value of the water body are shown in figure 2. The degradation test results show that the strains FN4, Y1122 and Q24 have the highest removal rate on the black and odorous water COD for five days, namely 63.9%, 57.9% and 56.8% respectively; strains Y115, C111 and B16 for black odorous water NH 3 The removal rate is the highest in five days of-N, and is respectively 13.04%, 12.4% and 11.7%; the strains J16, C11 and J13 can improve the dissolved oxygen of the black and odorous water body most obviously; the strains FN4, FN7 and B16 have the most obvious improvement on the ORP value of the black and odorous water body.
5. And (4) measuring antagonism.
Antagonism among 20 strains was determined by oxford cup method. The double-layer culture substrate layer is an LB solid culture medium, the upper layer is an LB semisolid culture medium with agar content of 0.75% and bacterial liquid content of 10%, and the selection mode of the added bacterial liquid is as follows: when the antagonism of the two bacteria is measured, the bacterial liquid of one of the bacteria is selected and added into LB semisolid culture medium. And after the bottom layer culture medium is solidified, inserting the oxford cup, pouring the upper layer culture medium containing substrate bacterium liquid into a culture dish, taking out the oxford cup after solidification, adding 100 mu L of reactant bacterium liquid into each hole, culturing at 30 ℃ for 24 hours, and observing and recording the diameter of the inhibition zone. The results are shown in table 2 below.
TABLE 2 determination of zone of inhibition of antagonistic action between strains (mm)
Note: the diameter of the Oxford cup is 8mm, and the diameter of the inhibition zone is more than 8mm, which shows that the growth inhibition effect is achieved between the two strains.
2. Construction of black and odorous water body remediation microbial inoculum
1. And (4) preparing seed liquid.
Fermenting 20 strains of bacteria with LB culture medium to late logarithmic phase, diluting with sterile water, and measuring OD of bacteria solution 600 Adjusting the OD value of the bacterial liquid to 0.2 to be used as a seed liquid, wherein the OD value of the bacterial liquid is adjusted by adjusting the concentration of the bacterial liquid.
2. And (5) mixing and fermenting.
COD and NH after treating black and odorous water body with each strain 3 And the removal rate of N, the repair effect of DO and ORP in the water body are four levels, the first three strains with the optimal levels are selected as three factors according to the degradation test, and a four-factor three-level full-factor composite test is designed to obtain 81 groups of microbial communities. Colonies containing Y115-FN4, B16-FN7, and C11-Y115 were removed by antagonism to finally leave 40 groups of composite bacteria designated EM1 to EM40, and the corresponding constituent strains are shown in Table 3.
Table 3 40 groups of complex microbial inoculum and constituent strains thereof
And (3) mixing the single bacterium seed solutions according to a volume ratio of 1:1:1: the ratio of 1 is inoculated into an LB culture medium, and cultured for 2-3 days at the constant temperature of 30 ℃ in a shaking box at 140r/min to obtain 40 groups of composite microbial inoculum. In a sterile environment, 3mL of culture solution is sucked by a pipette, centrifuged for 5min at 5000r/min, the supernatant is poured off, and the precipitate is washed three times by sterile normal saline and then added into 100mL of artificial simulated black and odorous water. And measuring COD, ammonia nitrogen, DO and ORP of the black and odorous water body after five days, and evaluating the repairing effect of the black and odorous water body according to a water quality index formula. COD removal rate, NH after five days 3 The removal rate of-N is shown inFIG. 3 shows ORP and DO values of the water body as shown in FIG. 4. Comprehensively considering four main indexes, the composite microbial inoculum EM31 (FN 4, C111, B16 and J13) has obvious effect on repairing the black and odorous water body, the five-day removal rate of COD in the black and odorous water body by the community is 77%, the removal rate of ammonia nitrogen is 44%, the removal rate is 13.5% higher than that of single-bacterium COD, and the removal rate of ammonia nitrogen is 31%.
3. And (5) optimizing the compound microbial inoculum.
3.1 ratio optimization
Different inoculation ratios can lead to different interaction degrees among the strains, so that the optimization of the inoculation ratio among the strains is beneficial to improving the treatment effect of the composite microbial inoculum on the black and odorous water body. The material mixing design mainly researches the relation between different proportions of each experimental factor and reaction variables, obtains a regression equation of the test index and the percentage of each component in the material mixing through material mixing tests of different percentages, and gives a statistical conclusion through the regression equation. The proportion of the composite microbial inoculum is determined by the mixed material design, the relation between the test index and the proportion of each bacterium can be obtained through a small amount of tests, and the mixing effect can be more accurately analyzed and predicted.
Mixing materials are designed by adopting minitab software, four strains are inoculated into 150mL LB culture medium according to the proportion in the test group for mixed fermentation, and the mixture is cultured for 48 hours at the constant temperature of 30 ℃ and 140r/min in a shaking box. In a sterile environment, 3mL of culture solution is sucked by a pipette, centrifuged for 5min at 5000r/min, the supernatant is poured off, and the precipitate is washed three times by sterile normal saline and then added into 100mL of artificial simulated black and odorous water. Measuring COD and ammonia nitrogen in the black and odorous water body after five days, and determining the removal rate of COD (COD EF%) and the removal rate of ammonia Nitrogen (NH) 3 -N EF%) as response value. Mixing design 15 experimental group settings and response value measurements are shown in table 4.
Table 4 compounding design test groups and response values
The model fitting optimization result with the COD removal rate as a response value is FN4: c111: b16: j13=0: 0.31:0.18:0.51, effect of ammonia nitrogen removalThe strain ratio and ammonia nitrogen removal rate have no corresponding relation. Considering that FN4 has a nitrate reduction effect and FN4 has a spherical structure, the method is favorable for constructing a high-efficiency microbial community with a space structure, promotes the transfer of substances such as metabolic intermediates, cofactors, vitamins and the like generated among strains, ensures the growth of the strains, enhances the effective transfer of metabolites and signal molecules among different cells, and improves the pressure resistance of the community to the environment by combining the physiological and biochemical characteristics of the strains, so that the high-efficiency microbial community composite microbial inoculum is constructed by adopting a mode of 'single fermentation FN4 mycelium pellet + mixed fermentation (C111: B16: J13)'. C111: b16: the volume ratio of the J13 mixed fermentation is 0.31:0.18:0.51.FN4 mycelial pellet to mixed fermented (C111: B16: J13) ratio of 1:1 (mass ratio), wherein the viable bacteria amount of Bacillus cereus, klebsiella, saccharomyces cerevisiae and Streptomyces is 2.1 × 10 9 cfu/mL、8×10 8 cfu/mL、6.2×10 8 cfu/mL、 6.4×10 9 cfu/mL. The mycelium pellets before and after fermentation were subjected to electron microscope scanning (FIG. 5), and it was preliminarily determined from the microbial morphology that three additional strains were supported on the FN4 mycelium.
3.2 degradation Condition optimization
And optimizing three factors of pH, temperature and microbial inoculum addition when the composite microbial inoculum degrades the black and odorous water body by taking the COD removal rate and the ammonia nitrogen removal rate as response values. Adopting minitab to carry out response surface design, carrying out mixed microbial inoculum fermentation according to the optimized result of 3.1, sucking 3mL of culture solution by a liquid transfer device in a sterile environment, centrifuging for 5min at 5000r/min, pouring off the supernatant, washing the precipitate for three times by using sterile normal saline, and adding the precipitate into 100mL of artificial simulated black and odorous water. Measuring COD and ammonia nitrogen in the black and odorous water body after five days, and determining the removal rate of COD (COD EF%) and the removal rate of ammonia Nitrogen (NH) 3 -N EF%) as response value. The response surface of 17 test groups and test results are shown in table 5, wherein the adding amount of the microbial inoculum is determined according to the volume of the black and odorous water body.
TABLE 5 response surface test groups and response values
Model fitting was performed on the results of the removal of COD and the removal of ammonia nitrogen (i.e., data in table 5), and the results obtained were that when the amount of addition was 0.1%, the pH was 6.187, and the temperature was 20.964 ℃, the removal of COD and ammonia nitrogen had the maximum expected values, the optimal degradation conditions and expected values are shown in table 6, and the response surface three-dimensional response graph is shown in fig. 6.
TABLE 6 response surface optimization results
| pH | Temperature/. Degree.C | Addition amount/% | COD EF％ | NH 3 -N EF％ | Expected value |
| 6.187 | 20.964 | 0.100 | 78.995 | 52.396 | 0.640 |
3.3 optimization result verification
And verifying that the compound microbial inoculum and the optimized compound microbial inoculum carry out degradation tests on the artificially simulated black and odorous water according to the optimization results, wherein the results are shown in table 7. The results in table 7 show that the COD removal rate of the compound microbial inoculum before optimization is 73.3%, the ammonia nitrogen removal rate is 40%, the COD removal rate of the compound microbial inoculum after optimization is 83.16%, and the ammonia nitrogen removal rate is 53.23%.
TABLE 7 Complex microbial inoculum optimization results
| Index (es) | COD(mg/L) | Ammonia nitrogen (mg/L) | DO(mg/L) | ORP(mV) | Transparency (cm) | Degree of black odor |
| Black and odorous water body | 172.5 | 16 | 0.1 | -217 | 14.4 | Severe black odor |
| Complex microbial inoculum before optimization | 46 | 9.6 | 5.11 | 220 | >30 | Mild black odor |
| Optimized composite microbial inoculum | 29.05 | 7.48 | 6.27 | 287 | >30 | No black odor |
3. Identification of strains
1. And (5) identifying physiological and biochemical characteristics.
Inoculating C111, B16, J13, and FN4 onto LB culture medium, culturing in 30 deg.C incubator, and observing colony morphology after 24 hr, wherein the colony morphology is shown in FIG. 7. The sugar fermentation experiments of the strains can measure the utilization capacity of the strains to different carbon sources, and the flocculation capacity of the strains plays an important role in removing organic pollutants in sewage. The four strains are subjected to flocculation activity determination, glucose, lactose and sucrose fermentation experiments and nitrate reduction.
The detection methods are as follows:
(1) And (3) testing the flocculation activity: 5g of kaolin is weighed, 1L of deionized water is added, a magnetic stirrer is adopted to rotate at a high speed for 5min, and the pH value is adjusted to 7.0 by NaOH solution. 20mL of kaolin suspension is weighed into a 25mL colorimetric cylinder, and 1mL of CaCl is added 2 The solution (10 g/L) was added with 5mL of fermentation supernatant, shaken gently up and down, and allowed to stand for 5min to measure the absorbance at 550 nm. Deionized water was used as a blank control.
The calculation formula for flocculation activity = (absorbance at 550nm for blank-absorbance at 550nm for sample set)/absorbance at 550nm for blank 100%.
(2) Glucose fermentation experiment: preparing a sugar fermentation liquid culture medium (peptone 1%, naCl 0.5%, 1.6% bromocresol purple ethanol solution 1%, glucose solution 1%, and the balance of water), putting an inverted Du's small tube into the culture medium, respectively inoculating the four strains into the culture medium in an aseptic environment, and culturing in a constant-temperature incubator at 37 ℃ for 48 hours, wherein the culture medium turns yellow to be acid-producing positive, bubbles in the Du's small tube are gas-producing positive, and the other strains are negative.
(3) Lactose fermentation experiments: preparing a sugar fermentation liquid culture medium (peptone 1%, naCl 0.5%, 1.6% bromocresol purple ethanol solution 1%, lactose solution 1%, and the balance water), putting an inverted Du's small tube into the culture medium, respectively inoculating the four strains of bacteria into the culture medium in an aseptic environment, and culturing for 48 hours in a constant-temperature incubator at 37 ℃, wherein the culture medium turns yellow to be acid-producing positive, bubbles in the Du's small tube are gas-producing positive, and the other strains are negative.
(4) Sucrose fermentation experiment: preparing a sugar fermentation liquid culture medium (peptone 1%, naCl 0.5%, 1.6% bromocresol purple ethanol solution 1%, sucrose solution 1%, and the balance water), putting an inverted Du's small tube into the culture medium, respectively inoculating the four strains of bacteria into the culture medium in an aseptic environment, and culturing for 48 hours in a constant-temperature incubator at 37 ℃, wherein the culture medium turns yellow to be acid-producing positive, bubbles in the Du's small tube are gas-producing positive, and the other strains are negative.
(5) Nitrate reduction experiments: preparation of nitrate Medium (KNO) 3 0.1 percent, 0.5 percent of sodium citrate, 0.1 percent of dipotassium hydrogen phosphate, 0.02 percent of magnesium sulfate heptahydrate, 0.1 percent of dipotassium hydrogen phosphate trihydrate and the balance of water), putting the inverted small Du's tube into a culture medium, respectively inoculating four strains of bacteria into the culture medium in an aseptic environment, putting the culture medium in a constant-temperature incubator at 37 ℃ for culturing for 1-4 days, equivalently mixing a detection reagent liquid A (0.8g and 5mol/L acetic acid 100mL of p-aminobenzenesulfonic acid) and an acetic acid liquid (0.5g and 5mol/L acetic acid 100mL of alpha-naphthylamine) respectively (0.5 mL of each), adding the mixture into the culture medium, and judging that the culture medium is red and is positive in nitrate reduction, otherwise, the culture medium is negative.
The results are shown in Table 8, wherein "-" indicates negative and "+" indicates positive.
TABLE 8 identification results of physiological and biochemical indicators
2. And (5) molecular identification.
(1) C111: extracting purified DNA of the C111 bacteria by using an industrial bacteria DNA extraction kit, selecting a 16S rRNA bacteria universal primer for PCR amplification by using the DNA as a template, performing electrophoresis purification and recovery, and sequencing a PCR product by using the industrial bacteria DNA extraction kit to obtain a sequence with the length of 840bp, wherein the specific sequence is shown as SEQ ID NO:1 is shown.
The determined sequence was compared for homology using BLAST with the sequences already registered in the GenBank/EMBL/DDBJ database, the most recent strain for C111 homology was Bacillus cereus (Bacillus cereus), the sequence coverage was 98%, and the similarity was 99%. The strain C111 is named as Bacillus cereus ZD-C, and is preserved in China general microbiological culture Collection center (CGMCC) in 12 months and 09 days in 2021, and the preservation number is as follows: CGMCC No.24062, the preservation address is: xilu No. 1 Hospital No. 3, beijing, chaoyang, beicheng.
(2) B16: extracting purified B16 bacteria DNA by using an industrial bacteria DNA extraction kit, selecting a 16S rRNA bacteria universal primer for PCR amplification by using the DNA as a template, performing electrophoresis purification and recovery, and sequencing a PCR product by using the industrial bacteria DNA extraction kit to obtain a sequence with the length of 1190 bp.
The determined sequences were compared for homology using BLAST with the sequences already registered in the GenBank/EMBL/DDBJ database, the most recent strain with B16 homology was Klebsiella (Klebsiella sp.), the sequence coverage was 96%, and the similarity was 99%. Meanwhile, the sequence detected by searching and finding the strain B16 from a microbial resource library of the institute of Oesophagostomum of Chinese academy of sciences is the same as the sequence of the Klebsiella bacterium disclosed in the BFX-01 strain of the high-yield bioflocculant and the obtained bioflocculant with the Chinese patent application number of 201711116739.9. Therefore, the Klebsiella required by the present invention can be obtained by the single bacteria purification screening disclosed in the present invention, by screening in the manner disclosed in the above prior patent, or by direct purchase.
(3) J13: extracting the purified DNA of the J13 strain by adopting an artificial fungus DNA extraction kit, selecting a 16S rRNA fungus universal primer for PCR amplification by taking the DNA as a template, performing electrophoresis purification and recovery, and sequencing a PCR product by engineering to obtain a sequence with the length of 300bp, wherein the specific sequence is shown as SEQ ID NO:2, respectively.
The determined sequence was compared for homology using BLAST with the sequences already registered in the GenBank/EMBL/DDBJ database, the most recent strain of homology for J13 was Saccharomyces cerevisiae (Saccharomyces cerevisiae), the sequence coverage was 98%, and the similarity was 99%. The strain J13 is named as saccharomyces cerevisiae ZD-J, is preserved in China general microbiological culture Collection center (CGMCC) at 09 months 12 and 2021, and has the preservation number as follows: CGMCC No.24061, the preservation address is: xilu No. 1 Hospital No. 3, beijing, chaoyang, north.
(4) FN4: extracting the purified DNA of FN4 bacteria by using an artificial fungus DNA extraction kit, selecting a 16S rRNA actinomycetes universal primer for PCR amplification by using the DNA as a template, carrying out electrophoretic purification and recovery, and sequencing a PCR product by organism to obtain a sequence with the length of 378bp, wherein the specific sequence is shown as SEQ ID NO:3, respectively.
The determined sequence was compared for homology using BLAST with the sequences already registered in the GenBank/EMBL/DDBJ database, the most recent strain for homology to J13 being Streptomyces sp, with 99% sequence coverage and 99% similarity. The bacterial strain FN4 is named as streptomycete ZD-F, is preserved in China general microbiological culture Collection center (CGMCC) at 09 month 12 in 2021, and has the preservation number as follows: CGMCC No.24063, the preservation address is: xilu No. 1 Hospital No. 3, beijing, chaoyang, north Chen.
Thus, the inventor constructs a black and odorous water body remediation microbial inoculum, and the constituent bacteria of the black and odorous water body remediation microbial inoculum are C111 (Bacillus cereus bacillus), B16 (Klebsiella sp. Klebsiella), J13 (Saccharomyces cerevisiae) and FN4 (streptomyces sp. Streptomyces).
4. Quantitative detection method of black and odorous water body remediation microbial inoculum
1. And (3) designing a primer.
The SEQ sequences of the strains were input into GeneBank, and the primer sequences with the highest score were selected by synthesizing the GC content of the primers and the annealing temperature, and the primer sequences of the four strains are shown in Table 9.
TABLE 9 specific primers corresponding to the four strains
2. Optimizing and establishing a fluorescent quantitative PCR reaction program and a reaction system.
By the pair of Mg 2+ And selecting concentration, primer concentration and annealing temperature, and determining a reaction system and a reaction program by taking the lowest Ct value and the highest RFU as screening indexes.
(1) The PCR reaction program is: (1) pre-denaturation at 95 ℃ for 3min, (2) fluorescence collection at 95 ℃ for 10sec, annealing temperature (B16 ℃; C111 58 ℃; J13 59 ℃; FN4 69 ℃) for 30sec, and fluorescence signal collection and amplification are performed for 40 cycles after each cycle is finished, and (3) dissolution curve analysis is performed, wherein the temperature of 55 ℃ is gradually increased to 95 ℃, and fluorescence is collected at intervals of 0.2 ℃.
(2) The PCR reaction system is as follows: the total volume of the system is 20 mu L, and the system comprises: 2xSYBR Gerrnabstract PCR Mix 10 uL, template 1 uL, primer final concentration B16 0.3 uM, C111 0.2 uM, J13 0.3 uM, FN 4.7 mM, ddH 2 Make up to 20. Mu.L of O.
3. And (4) preparing a standard curve.
(1) And (4) obtaining a target gene fragment.
Respectively culturing the strains C111, B16, J13 and FN4 overnight, extracting total DNA in each culture solution, determining the purity and concentration of DNA by ultraviolet spectrophotometer and gel electrophoresis, and performing fluorescent quantitative PCR reaction on DNA of each strain by using the above primers and reaction program.
And identifying the PCR product through agarose gel electrophoresis, cutting a target gene band under an ultraviolet lamp, and purifying and recovering the PCR product by using a gel recovery kit to obtain the purified target gene fragment of each strain.
(2) And (5) preparing a standard substance.
Measuring the value of the target gene DNA A260 of each strain by an ultraviolet spectrophotometer, and calculating the DNA concentration of each sample, wherein the calculation formula is as follows: DNA concentration (μ g/mL) = a260 × dilution factor × 50 μ g/mL.
Calculating the molecular copy number according to the relative molecular mass and the Avgalois constant, wherein the calculation formula of the DNA copy number is as follows: copies/. Mu.L = (6.02X 10) 23 copies/mol) × (DNA concentration g/mL. Times.10 -9 ) /(number of bases. Times.660 g/mol), the concentration of the standard was adjusted to 10 9 copies/uL, then 10-fold serial dilutions of the PCR products to obtain a concentration of 10 9 ，10 8 ,10 7 ,10 6 ,10 5 ,10 4 ,10 3 ,10 2 1mL of copies/uL of DNA solution per sample.
(3) And (5) establishing a standard curve.
And (3) taking the standard products obtained in the step (2) as templates for real-time fluorescence quantitative PCR reaction for amplification, and drawing corresponding threshold cycle numbers by logarithmic values of target gene copy numbers of different dilution gradients to prepare a standard curve. The fluorescence quantitative PCR standard curve corresponding to C111, B16, J13 and FN4 is shown in FIG. 8. According to the standard curve, the growth condition of the microbial inoculum after the microbial inoculum is added into the natural black and odorous water body can be quantitatively detected.
The invention is further illustrated by the following specific examples.
Example 1
1. Preparing bacterial liquid: c111, B16 and J13 are inoculated into an LB culture medium according to the optimized proportion of 3.1, the mixture is cultured for 24 hours at the constant temperature of 30 ℃ and 140r/min in a shaking box, FN4 is independently inoculated into the LB culture medium at the constant temperature of 30 ℃ and 140r/min in the shaking box, the mixture is cultured for 24 hours at the constant temperature of 30 ℃ and 140r/min in the shaking box, the two fermentation liquors are mixed, and the mixture is cultured for 10 hours at the constant temperature of 30 ℃ and 140r/min in the shaking box, so that the compound microbial inoculum is obtained. The viable bacteria amount of Bacillus cereus, klebsiella, saccharomyces cerevisiae, and Streptomyces in the bacterial solution is 2.1 × 10 9 cfu/mL、8× 10 8 cfu/mL、6.2×10 8 cfu/mL、6.4×10 9 cfu/mL。
2. The repairing method comprises the following steps: the natural black and odorous water body environment is relatively complex, and in order to enable the bacterial strains to be planted in the natural environment, under the condition that the volume of the black and odorous water body is not changed, the single inoculation amount is 10 times larger than the 3.3 optimization result verification (the experimental object is artificial simulated black and odorous water, and the addition amount is 0.1%). The number of times of adding the bactericide was changed to 3 times. When the water temperature is 20-25 ℃, the test group adds 1% of compound microbial inoculum bacterial liquid into the black and odorous water body in a spraying mode, the water body is aerated for 24 hours in the day of spraying, and then the water is stopped; spraying 1% of composite microbial inoculum liquid again after 3d, aerating the water body in the current day for 24h, and stopping spraying; spraying 3 times of bacteria solution. The total repair time was 67d. The control group was not supplemented with the bacterial solution. And measuring COD, ammonia nitrogen, DO, ORP and transparency of the water bodies of the test group and the control group before and after the restoration.
Wherein, the chemical oxygen demand COD is determined by a Lovibond COD determinator according to a HJ/T399-2007 standard rapid digestion spectrophotometry; ammonia nitrogen is measured according to HJ 535-2009 nNa reagent spectrophotometry; dissolved oxygen DO was measured in situ with a portable measuring instrument according to the electrode method described in methods for monitoring and analyzing water and wastewater (fourth edition); the oxidation-reduction potential ORP was measured in situ by the electrochemical method described in Water and wastewater monitoring and analysis methods (fourth edition); the transparency was measured by a transparency meter according to the method described in methods for monitoring and analyzing Water and wastewater (fourth edition); and judging the grade of the black and odorous water according to the grading standard of the pollution degree of the urban black and odorous water in the urban black and odorous water treatment working guideline.
The results are shown in Table 10. As can be seen from the table, under natural conditions, most of COD in the black and odorous water body can be degraded, but the problems of high ammonia nitrogen concentration and low dissolved oxygen concentration still exist, the removal rate of ammonia nitrogen in the black and odorous water body can be greatly improved by adding the composite microbial inoculum, the dissolved oxygen concentration is increased, and the severe black and odorous water body can be recovered to be in a black and odorous state. The 3.3 optimization results prove that the removal rate of the composite microbial inoculum to ammonia nitrogen in the black and odorous water body simulation experiment is 53.23% at most, while in the actual black and odorous water body pilot-scale repair experiment, the removal rate of the composite microbial inoculum to ammonia nitrogen in the black and odorous water body is 95.39%, presumably because the addition of the composite microbial inoculum interacts with indigenous microorganisms in the environment, the growth of certain indigenous strains is stimulated, and the removal rate of ammonia nitrogen is increased.
TABLE 10 repairing effect of bacterial agent on actual black and odorous water body
3. And (4) quantitatively detecting the complex microbial inoculum.
Extracting total DNA of the overburden water and the surface sediment of a test group and a control group before and after repair by adopting a biological soil DNA extraction kit, respectively carrying out fluorescence quantitative PCR amplification according to a primer and a reaction system shown in a table 8 to obtain a CT value, and calculating the target gene copy number of strains C111, B16, J13 and FN4 according to a standard curve shown in a figure 8. The gene copy number changes of four strains in the overlying water and the surface sediments when the complex microbial inoculum is added into the black and odorous water body are shown in figures 9 and 10.
The results show that: before the microbial inoculum is added (in a period D0), target genes are not detected in overlying water and sediments, after the microbial inoculum is added (in a period D12), the copy numbers of the target genes of four strains in the overlying water are higher, the copy number logarithm is respectively 5.11, 4.16, 4.62 and 6.57, at the moment, the copy numbers of the target genes of the four strains in surface sediments are lower, the copy number logarithm is respectively 0.33, 0, 0.54 and 1.14, after the microbial inoculum is added, and when an experiment is finished (in a period D67), the copy numbers of the four strains in the overlying water are reduced, and the copy numbers of the strains in the surface sediments are obviously increased except for C111. Therefore, the growth characteristics of the compound microbial inoculum can be seen, namely three strains (B16, J13 and FN 4) in the compound microbial inoculum can be planted in the black and odorous water body and black and odorous substrate sludge, wherein the planting effect of the FN4 is the best, and the other strain C111 can only be planted in the black and odorous water body. By quantitatively detecting the existence condition of the complex microbial inoculum in the environment, the microbial inoculum can be supplemented periodically according to the existence condition of the microbial inoculum on one hand, and the growth characteristics of the microbial inoculum can be deeply researched to further optimize the complex microbial inoculum on the other hand.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention are intended to fall within the scope of the present invention defined by the claims.
Sequence listing
<110> institute of biological research of Chengdu of Chinese academy of sciences
<120> black and odorous water body remediation microbial inoculum and application thereof
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<213> Bacillus cereus ZD-C (Bacillus cereus)
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actgagacac ggcccagact yctacgggag gcagcagtag ggawyyykcc gcaatggacg 60
aaagtctgac ggagcaacgc cgcgtgagtg atgaaggctt tcgggtcgta aaactctgtt 120
gttagggaag aacaagtgct agttgaataa gctggcacct tgacggtacc taaccagaaa 180
gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttatccgga 240
attattgggc gtaaagcgcg cgcaggtggt ttcttaagtc tgatgtgaaa gcccacggct 300
caaccgtgga gggtcattgg aaactgggag acttgagtgc agaagaggaa agtggaattc 360
catgtgtagc ggtgaaatgc gtagagatat ggaggaacac cagtggcgaa ggcgactttc 420
tggtctgtaa ctgacactga ggcgcgaaag cgtggggagc aaacaggatt agataccctg 480
gtagtccacg ccgtaaacga tgagtgctaa gtgttagagg gtttccgccc tttagtgctg 540
aagttaacgc attaagcact ccgcctgggg agtacggccg caaggctgaa actcaaagga 600
attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa 660
ccttaccagg tcttgacatc ctctgaaaac cctagagata gggcttctcc ttcgggagca 720
gagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 780
gcaacgagcg caacccttga tcttagttgc catcattaag ttgggcactc taaggtgact 840
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agatttctgt gcttttgtta taggacaatt aaaaccgttt caatacaaca cactgtggag 180
ttttcatatc tttgcaactt tttctttggg cattcgagca atcggggccc agaggtaaca 240
aacacaaaca attttattta ttcattaaat ttttgtcaaa aacaaraatt ttcgtaactg 300
<210> 3
<211> 378
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<213> Streptomyces ZD-F (Streptomyces sp.)
<400> 3
gcagccgcgg taatacgtag ggcgcgagcg ttgtccggaa ttattgggcg taaagagctc 60
gtaggcggct tgtcacgtcg gttgtgaaag cccggggctt aaccccgggt ctgcagtcga 120
tacgggcagg ctagagttcg gtaggggaga tcggaattcc tggtgtagcg gtgaaatgcg 180
cagatatcag gaggaacacc ggtggcgaag gcggatctct gggccgatac tgacgctgag 240
gagcgaaagc gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacggt 300
gggcactagg tgtgggcgac attccacgtc gtccgtgccg cagctaacgc attaagtgcc 360
ccgcctgggg agtacgga 378
Claims (10)
1. A strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae) is characterized in that: the strain is preserved in China general microbiological culture Collection center at 09.12.2021, with the preservation number as follows: CGMCC No.24061.
2. A remedial agent comprising the saccharomyces cerevisiae of claim 1.
3. The remediation microbial inoculum of claim 2, wherein: also included are Bacillus cereus, klebsiella and Streptomyces.
4. The remedial bacterial agent according to claim 3, wherein: the bacillus cereus is preserved in the China general microbiological culture Collection center on 09.12.2021, with the preservation numbers as follows: CGMCC No.24062; the streptomyces is preserved in China general microbiological culture Collection center at 09.12.2021, with the preservation numbers as follows: CGMCC No.24063.
5. The remediation microbial inoculum of claim 3, wherein: the viable bacteria amount of the bacillus cereus, the Klebsiella, the saccharomyces cerevisiae and the streptomyces is 2.1 multiplied by 10 respectively 9 cfu/mL、8×10 8 cfu/mL、6.2×10 8 cfu/mL、6.4×10 9 cfu/mL。
6. The remediation microbial inoculum of claim 3, wherein: the construction mode of the remediation microbial inoculum is as follows: and mixing the independently fermented streptomyces with the bacterial liquid obtained by mixing and fermenting the bacillus cereus, the klebsiella and the saccharomyces cerevisiae.
7. The remediation microbial inoculum of claim 6, wherein: the volume ratio of the bacillus cereus to the klebsiella to the saccharomyces cerevisiae in mixed fermentation is 0.31:0.18:0.51, the mass ratio of the streptomyces fermented independently to the bacterial liquid after mixed fermentation is 1.
8. The use of the remediation microbial inoculum according to any one of claims 3 to 7 in black and odorous water body treatment.
9. Use according to claim 8, characterized in that: the adding mode of the remediation microbial inoculum in the black and odorous water body is as follows: adding a bacterium solution into the black and odorous water body in a spraying mode, aerating for 24 hours, and stopping; adding the mixture again after 3d, aerating for 24h, and then stopping; and adding the mixture again after 3 days, and aerating for 24 hours.
10. Use according to claim 9, characterized in that: according to the volume ratio, the adding amount of the repairing microbial inoculum for one time is 0.1-1%.
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