CN115820445A - Separated Kosakholderia FB2-3 and strain S thereof 2- Use in degradation - Google Patents
Separated Kosakholderia FB2-3 and strain S thereof 2- Use in degradation Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a separated Kosaxabemyces FB2-3 and a strain thereof in S 2‑ The application in degradation, the preservation number of the strain is CCTCC NO: m2020508, the above Sulfur removal Strain was tested for S in Experimental Shake flasks 2‑ The removal rate of (A) reaches 88.18%. The strain is resuspended in water or 0.01M phosphate buffer, then PEG with the final concentration of 1-2% is added as a protective agent, and the survival rate of the strain is still 78.8% after the strain is stored for 3 months at 25 ℃. On the upper partThe sulfur-removing strain is applied to a biological spray tower H 2 The removal rate of S is about 90%, and the microbial inoculum has low production cost and simple and convenient use.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a separated bacterium Kosakonia oryzae FB2-3 and its strain in S 2- Application in degradation.
Background
The genus Kosakonia is a new genus recently classified from the genus Enterobacter of the family Enterobacteriaceae. Cowanii strains are derived from plants, except for the typical species k.cowanii strains, most of which promote plant growth, and a few Kosakonia species which are phytopathogens. The classification status of some of the members of the genus currently is unknown [1] 。
The current research on the Kosakonia finds that the Kosakonia has the beneficial functions of nitrogen fixation, plant growth promotion, carrier function of enzyme production genes, phosphate dissolution, oil degradation, organic phosphorus pesticide decomposition and the like. But also are pathogenic bacteria of some plants and conditional pathogenic bacteria of human body [2] 。
Liqijie et al isolated and screened an endophytic bacterium Kosakoniaricidincitans with nitrogen fixation ability from corn root system. The strain can dissolve phosphate and fix plant seedling [3] . Mauricio Cruz et al found that the salt-tolerant bacterial phenotype of Kosakoniaricidins increased the root colonization capacity by about 3-fold and showed growth-promoting effects in radish plants [4] . Furthermore, the genus shows excellent tomato growth promoting effect during tomato growth [5]
One strain of radix angelicae sinensis screened by Lidamei et al in Luzhou-flavor liquor brewing processThe Weak acid esterase strain Coxsackia Coxitake is used as an engineering bacterium for producing enzyme to carry out gene editing, so that the enzyme yield of the bacterium is improved [6] 。
In the field of environmental remediation, the genus Coxsacker also has some applications. Ren Jianjun et al successfully isolated a novel oil-degrading bacterium named IUMR B67. The oil degradation rate at 37 deg.C is 95.80%, IUMR B67 is a novel edible oil degrading strain, can hydrolyze oil by lipase activity, and can be used for treating oil and oily food waste [7] . In addition, the Coxsackia separated from the rhizosphere of the paddy rice in the wetland can biodegrade organophosphorus pesticides such as OP insecticide, propanesulide (PF), quinolophos (QP) and the like in the culture medium [8] . The strain has high pesticide tolerance and can synthesize organophosphorus degrading enzyme.
We have now found that the classification of enterobacter species is rapidly changing. To date, one of the blood pathogenic bacteria, k. Radicicinitans, lacks AmpC β -lactamase present on the chromosome of enterobacter species. This is the first report of Kosakonia species on the occurrence of infections in humans by this species [9] 。
The Coxsackia has no report on sulfur removal at present, and as a novel sulfur removal strain, the strain has high removal efficiency and high growth speed, can be applied to large-scale deodorization equipment, has good application value in the aspect of odor treatment, and has good economic benefit.
Reference to the literature
[1] Quality of Li Yuan.A. Classification of bacteria of the genus Kosakonia based on Whole genome sequence systems [ D ]. Zhejiang university, 2016
[2]Bhatti Micah D,KaliaAwdhesh,SahasrabhojanePranoti,KimJiwoong,Greenberg David E,Shelburne Samuel A.Identification and Whole Genome Sequencing of the First Case of Kosakoniaradicincitans Causing a Human Bloodstream Infection.[J].Frontiers in microbiology,2017,8
[3] Separation and identification of Li Joojie, chenjie, sun Shuaixin, chenyunpeng, corn combined azotobacter Kosakonicinsis GXGL-4A and nitrogen fixation characteristic research [ J ] microbiological report 2016,43 (11): 2456-2463
[4]Mauricio Cruz Barrera,Desir é e Jakobs-Schoenwandt,Martha Isabel G ó mez,MatthiasBecker,Anant V.Patel,Silke Ruppel.Salt stress and hydroxyectoine enhance phosphate solubilisation and plant colonisation capacity of Kosakoniaradicincitans[J].Journal of Advanced Research,2019,19
[5]BergerBeatrice,BaldermannSusanne,Ruppel Silke.The plant growth-promoting bacterium Kosakoniaradicincitansimproves fruit yield and quality of Solanum lycopersicum.[J].Journal of the science of food and agriculture,2017,97(14)
[6] The novel phytase gene from Kosakoniaadicidincinnans is efficiently expressed in Pichia pastoris [ J ] by codon optimization, 2016,32 (02): 33-38 in Shanghai agricultural bulletin, 2016,32 (02): 33-38
[7]RenJianjun,WangZhenzhu,NiuDongze,Huhetaoli,HuangXu,FanBo,LiChunyu.Isolation and characterization of the novel oil-degrading strain Kosakoniacowanii IUMR B67 and expression of the degradation enzyme[J].FEMS Microbiology Letters,2020,367(9)
[8]DiptiMayeeDash,W.Jabez Osborne.Rapid biodegradation and biofilm-mediated bioremoval of organophosphorus pesticides using an indigenousKosakoniaoryzaestrain-VITPSCQ3 in a Vertical-flow Packed Bed Biofilm Bioreactor[J].Ecotoxicology and Environmental Safety,2020,192
[9]BhattiMicah,D,Kalia,Awdhesh,Sahasrabhojane,Pranoti,Kim,Jiwoong,Greenberg,David E,Shelburne,Samuel A.Identification and Whole Genome Sequencing of the First Case of Kosakoniaradicincitans Causing a Human Bloodstream Infection.[J].Frontiers in microbiology,2017,8.
Invention of the invention
The invention aims to provide a separated bacterium Kosakonia oryzae, the preservation number of the strain is CCTCC NO: m2020508.
Another object of the present invention is to provide Kosakonia oryzae FB2-3 at S 2- Application in degradation.
In order to achieve the purpose, the invention adopts the following technical measures:
the applicant separates a strain of hydrogen sulfide degrading bacteria from a biological filter filler of a Wuhan biological deodorization treatment system, and the bacteria have good capability of removing hydrogen sulfide and are identified as the Coxsackia bacteria. The strain is preserved in China center for type culture Collection in 09 and 17 months in 2020, wherein the preservation address is Wuhan university in Wuhan, china, and the classification is named as follows: kosakonia oryzae FB2-3 with a deposit number: CCTCC NO: m2020508.
The Kosakusaka (Kosakoniaoryzae) FB2-3 of the present invention was cultured on an LB medium plate for 2 days. The bacterial colony is circular, and the bacterial colony is faint yellow translucent form, and the surface is smooth moist, and the edge is clear, easily picks up.
Preparation of Kosakonia oryzae FB2-3 2- The application of the Kosakonia oryzae FB2-3 in the removal of microbial inoculum comprises directly utilizing the Kosakonia oryzae FB2-3 in S 2- Or preparing the Kosakonia oryzae FB2-3 of the invention into S 2 A degradation preparation.
The Kosakonia oryzae FB2-3 of the present invention can be used for gas phase H 2 S or H dissolved in water 2 And (4) degrading S.
The Kosakonia oryzae FB2-3 strain preservation method, which is to claim 1 strains heavy suspension in water or 0.01M phosphate buffer solution, and then added to the final concentration of 1-2% PEG as a protective agent.
Compared with the prior art, the invention has the following characteristics:
the coxsackiella in the invention has no report of hydrogen sulfide removal function before application date, and has the advantages of rapid growth of thalli, high viable count and strong hydrogen sulfide removal capability; the liquid microbial inoculum has long storage life, high survival rate and stable effect when being applied to a biological spray tower.
The Coxsackie bacteria of the invention has fast growth speed at 30 ℃ and high density of bacteria. The strain has high hydrogen sulfide removing ability, and S is 12h at 30 DEG C 2- The removal rate of (A) reaches 88.18%. The preparation method of the liquid microbial inoculum of the coxsackiella FB2-3 is simple, the cost is low, and the removal effect of the microbial inoculum on the hydrogen sulfide is excellent. When the Kosakholderia fungicide is applied to waste gas and wastewater treatment plants, the effect of high removal rate can be ensured, and meanwhile, the stable removal effect can be maintained, so that the Kosakholderia fungicide has the characteristics of good effect and high efficiency.
Drawings
FIG. 1 is a colony diagram of Kosakholderia FB 2-3.
FIG. 2 is S 2- The standard curve was determined.
FIG. 3 is S of Kosakholderia FB2-3 2- The change chart of the removal rate and the viable count.
FIG. 4 is a graph showing the survival rate of liquid microbial inoculum prepared from the Kosaxakh FB2-3 and different suspending agents.
FIG. 5 is a graph showing the survival rate of bacteria with different protective agent concentrations when the liquid microbial inoculum is prepared from the Kosakesa FB 2-3.
FIG. 6 is a graph showing the change of hydrogen sulfide removal rate in a test conducted with Kosaxabemyces FB 2-3.
Detailed Description
The technical scheme of the invention is a conventional technology if not particularly specified; the reagents or materials, if not specifically mentioned, are commercially available. Three replicates were set up for each experimental group of the present invention unless otherwise specified.
Example 1:
isolation and identification of bacterial FB2-3
(1) Taking 10g of filler in the biological filter in the filler of the biological filter, and adding the filler into a triangular flask containing 90ml of sterile physiological saline and glass beads; and shaking at 30 ℃ and 200rmp for 1-2h until attachments on the filler are basically peeled off and the liquid becomes turbid.
(2) 1ml of the culture obtained in the step (1) is taken to be diluted and coated on an LB solid culture medium, and the grown single colony is continuously streaked and separated to obtain a single strain of bacteria, which is named as FB2-3;
wherein the LB solid medium comprises the following components: 10g of peptone, 5g of yeast extract powder, 10g of sodium chloride, 15g of agar powder, pH7.0-7.2, and the volume is made up to 1L by using distilled water.
Strain identification of strain FB2-3
(1) Morphological characteristics
The colony morphology of the strain FB2-3 on an LB solid culture medium is shown in figure 1, the colony is circular, is light yellow or off-white, has a smooth and wet surface and clear edges, and is easy to pick up.
(2) Gene identification of Strain FB2-3
Extracting the genome DNA of the strain to be detected by using a bacterial DNA extraction kit, and referring to the instruction for the specific operation steps. And (3) amplifying the bacteria by using the extracted bacteria genome DNA as a template, wherein a primer for amplifying 16S rRNA:
27F:5＇-GTTTGATCCTGGCTCAG-3＇
1492R:5＇-TACGGCTACCTTGTTACGACTT-3＇；
the splicing sequence is found to belong to the Kosakh through Blast homologous sequence search, the strain is preserved in China center for type culture Collection in 09.17.2020, the preservation address is Wuhan university in Wuhan, china, and the classification name is as follows: the Kosaknia (Kosakonia oryzae) FB2-3 has a deposit number of: CCTCC NO: M2020508.
Example 2:
growth performance and S of Coxsackia FB2-3 2- Removal test
Culturing FB2-3 in liquid LB seed liquid culture medium at 30 deg.C for 16h to exponential growth end period, collecting seed liquid 5mL (effective bacteria concentration in seed liquid is 4 × 10 before centrifugation) 9 CFU/mL) was centrifuged at 5,000r/min for 4min to remove the supernatant, 5mL of sterile water was used to resuspend the centrifuged cells, and the resuspended cells were inoculated into a sulfur-removal medium at a cell concentration of about 1X 10 7 CFU/mL. Sampling every 1h to determine S 2- Content and viable count.
Wherein: coxsackia FB2-3S 2- The degradation efficiency is measured by adopting methylene blue spectrophotometry S 2- (GB/T 16489-1996)
(1) The principle is as follows: in the presence of ferric ions, S 2- Reacting with p-aminodimethylaniline to generate methylene blue, wherein the color intensity is in direct proportion to the content of sulfide ions, and measuring the absorbance of the solution at the wavelength of 665 nm.
Drawing a standard curve: 2mL of zinc acetate-sodium acetate absorption liquid is respectively added into a 6-piece 25mL colorimetric tube, then 0.00mL, 0.05mL, 0.10mL, 0.20mL, 0.30mL, 0.40mL and 0.50mL of sodium sulfide standard use liquid are respectively added, water is added to a 10mL mark line, then 1mL of N, N-dimethyl-p-phenylenediamine solution is added, and the mixture is uniformly mixed. 0.2mL of ammonium ferric sulfate solution was added, immediately stoppered and shaken well. Standing for 10min, diluting with water to marked line, and shaking. The absorbance was measured at 665nm wavelength with water as reference and blank experiments were performed.
And (3) taking the measured absorbance of each standard solution minus the blank experiment as an ordinate, and taking the content of the sulfur ions in the corresponding standard solution as an abscissa to draw a standard curve (figure 2), so as to obtain a standard curve equation of y =14.822x-0.2617.
(2) Sample measuring method
Taking 1ml of sample solution, centrifuging at 5000r/min for 5min, taking 50-100ul of sample supernatant, and simultaneously taking ck (namely a blank treatment group without ammonia nitrogen). After the absorbance was measured according to the above method, the sulfide ion concentration was calculated according to the following formula.
In the formula: c S 2- —S 2- Content of (1), mg/L;
m-measured from the standard curve S 2- μ g of (d);
v-volume of sample measured, mL.
A sulfur removal culture medium: NH 4 Cl 0.4g，MgCl 2 0.2g，KH 2 PO 4 2g，Na 2 CO 3 0.4g, 1000mL of distilled water, pH7.2-pH7.4, sterilizing at 121 ℃ for 20min, na 2 S·9H 2 Filtering to remove bacteria, adding into sterilized culture medium to obtain S 2- The concentration was 200mg/L. The blank control was a sulfur removal medium without added bacteria.
Wherein S is obtained by screening 2- Degrading bacteria FB2-3 of which 12h is opposite to S 2- The degradation rates of (A) are respectively: 88.18 percent of the total bacteria, and the viable count of the product can reach 2.93 multiplied by 10 9 CFU/mL；S 2- The degradation efficiency was evident (fig. 3), and the specific results are shown in the following table.
Note: the removal rate at 0h is due to the fact that each bottle of culture medium S 2- There was some error in the content, although three replicates and blank controls were set up in this experiment. But after addition of dissolved and sterilized sodium sulfide nonahydrate H 2 S will be volatilized to some extent, and the value measured each time will fluctuate to some extent due to some difference in the sodium sulfide mass of the medium added in portions when the sample is added, so that the sulfur removal rate at 0h is not 0%.
Example 4:
and (3) the storage environment of the FB2-3 liquid microbial inoculum is as follows:
(1) The Colsakella FB2-3 is a non-spore bacterium, so that the Colsakella FB2-3 is prepared into a liquid microbial inoculum to prolong the storage life of the Colsakella FB. As for liquid microbial agents, the pH, osmotic pressure and nutrient substances of the liquid environment of microorganisms have great influence on the survival rate of the microorganisms. The liquid microbial inoculum in the experiment consists of 2 parts, wherein one part is a microbial inoculum resuspension, and the other part is a protective agent.
(2) In experiments where liquid inoculum resuspension was selected: inoculating seed liquid of FB2-3 in a liquid LB culture medium with an inoculation amount of 1%, culturing at 30 ℃,200r/min for 14h, centrifuging, collecting thalli, and washing off culture medium components by using clear water. The cells were resuspended in 0.01mol/L Phosphate Buffer (PB), 0.05mol/L phosphate buffer, 0.10mol/L phosphate buffer, 0.20mol/L phosphate buffer, sterile water, and LB medium diluted 5, LB medium diluted 10, LB medium diluted 20-fold (8 resuspensions), respectively, at equal volumes. After the FB2-3 resuspension solution is preserved in an environment of 25 ℃ for 3 months, viable bacteria of the 8 different resuspension solutions are counted respectively. The resuspension solution with the highest survival rate and relatively economical efficiency is selected.
The survival rate calculation formula is:
the experimental result shows that the survival rate of FB2-3 in 0.01MPB is the highest and reaches 61.90 percent. Considering economic factors such as production cost and the like, water (the survival rate is 60.95%) is selected as a heavy suspension agent of the strain FB2-3 to carry out the next protective agent experiment. (FIG. 4), the specific results are shown in the following table:
| suspending agent | Water (I) | 0.01MPB | 0.1MPB | 0.2MPB | 0.01×LB | 0.1×LB | 0.2×LB |
| Survival rate/%) | 60.95 | 61.90 | 6.67 | 3.81 | 48.67 | 19.52 | 11.91 |
(3) Liquid microbial inoculum protectant selection experiment: collecting the centrifuged thallus from the bacteria liquid, and adding different protective agents (glycerol, polyethylene glycol 6000 (PEG), sorbitol, sucrose and trehalose) based on the heavy suspension (FB 2-3 is re-suspended with sterile water), wherein the final concentration of each protective agent is 1%,2% and 5%. After the bacterial liquid is fully resuspended, all the liquid bacterial agents are stored at 25 ℃, sampling is carried out after 3 months, the number of viable bacteria is determined, and the survival rate of the FB2-3 bacterial strain liquid bacterial agents is calculated.
The experimental results show that the survival rate of FB2-3 in 1-percent PEG is the highest, and the survival rate reaches 72.13 percent, and the specific results are shown in the following table:
example 5:
measurement of removal effect of Coxsackia FB2-3 on hydrogen sulfide in biological spray tower
In the biological spray tower of gas treatment equipment of certain pharmaceutical company in Hubei, the air quantity is about 500m 3 H, uniformly filling 3 layers of PP ball filler in the biological tower, wherein the filling height is 1.8m, and the volume is 0.9m 3 The gas retention time is 20s, the spraying flow of the circulating liquid is about 600L/h, and the spraying is ceaselessly performed. And collecting the field waste gas, and discharging the field waste gas into the biological spray tower. And lifting the nutrient solution containing the Coxsacker liquid microbial inoculum to the top of the biological spraying tower by a circulating water pump to spray downwards, and hanging the filler under the circulating spray for 14 days. And after the film formation is finished, the circulating liquid is directly emptied from the tower. And (3) starting to operate the biological spray tower, discharging the gas into an empty bed, keeping the EBRT for 20s, detecting the concentration of the ammonia gas at the gas inlet and the gas outlet 1 time every day, and operating and measuring for 23 days.
Wherein each liter of the nutrient solution contains: nutrient solution: 10g of glucose, 5g of industrial peptone 4 Cl 2g, vickers 'salt solution 50mL (Vickers' salt solution containing K per liter) 2 HPO 4 5 g，MgSO 4 ·7H 2 O 2.5g，NaCl 2.5g，FeSO 4 ·7H 2 O0.05g, mnSO4 0.05g). (FB 2-3 microbial inoculum concentration is 4X 10 9 CFU/mL, the microbial inoculum is added into the biological tower 10L)
As can be seen from FIG. 6, the removal rate of the bacteria on hydrogen sulfide in the spray tower reaches over 88 percent, and the bacteria have good removal effect on the hydrogen sulfide. The equipment can still carry out high-efficiency treatment on the hydrogen sulfide under the condition of large concentration fluctuation of the hydrogen sulfide.
In conclusion, the Coxsackia FB2-3 in the invention can be applied to the removal of S in water 2- . Can also be used for treating industrial waste gas containing hydrogen sulfide in a biological spray tower, and has stable effect and high removal rate.
Claims (4)
1. Separated Kosakholderia (C.) (Kosakonia oryzae) The preservation number of the strain is CCTCC NO: m2020508.
2. The Coxsackia bacterium of claim 1 in S 2- Application in removal.
3. The use of a bacterium of the genus Kosakhaki as claimed in claim 1 for the preparation of S 2- Application in a degradation agent.
4. The method for preserving the strain according to claim 1, which comprises resuspending the strain according to claim 1 in water or 0.01M phosphate buffer, and adding 1 to 2% PEG as a protective agent.
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| CN116144529A (en) * | 2022-11-01 | 2023-05-23 | 云南大学 | Rice saxophone OOR3-1 strain and application thereof |
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| CN112226384A (en) * | 2020-09-29 | 2021-01-15 | 广州中医药大学(广州中医药研究院) | Pathogenic strain causing patchouli bacterial wilt and application thereof |
| CN112795505A (en) * | 2021-01-07 | 2021-05-14 | 河南省农业科学院植物保护研究所 | Growth-promoting bacterium capable of generating IAA and promoting growth of salvia miltiorrhiza and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112226384A (en) * | 2020-09-29 | 2021-01-15 | 广州中医药大学(广州中医药研究院) | Pathogenic strain causing patchouli bacterial wilt and application thereof |
| CN112795505A (en) * | 2021-01-07 | 2021-05-14 | 河南省农业科学院植物保护研究所 | Growth-promoting bacterium capable of generating IAA and promoting growth of salvia miltiorrhiza and application thereof |
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Cited By (2)
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| CN116144529A (en) * | 2022-11-01 | 2023-05-23 | 云南大学 | Rice saxophone OOR3-1 strain and application thereof |
| CN116144529B (en) * | 2022-11-01 | 2024-02-23 | 云南大学 | Rice saxophone OOR3-1 strain and application thereof |
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