CN103013850B - Salt-resistant alkali-resistant Cr(VI) reduction microbe and screening method thereof - Google Patents
Salt-resistant alkali-resistant Cr(VI) reduction microbe and screening method thereof Download PDFInfo
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Abstract
The invention discloses a salt-resistant alkali-resistant Cr(VI) reduction microbe and a screening method thereof. The strain is a rod-shaped gram-negative bacteria, salt-resistant and alkali-resistant and high in chromium pollutant tolerated concentration and can reduce high-toxicity Cr(VI) into low-toxicity Cr (III) under an aerobic condition and reduces the Cr(VI) concentration from 130 mg(L<-1>) to 9.1 mg(L<-1>) after being cultured for 114 hours when pH is 8.5 and NaCl concentration is 20 g(L<-1>). The strain provided by the invention has high Cr(VI) reduction efficiency, can be used as a chromium pollution water body and soil bioremediation material and is used for removing the Cr(VI) pollutants contained in water and soil.
Description
Technical field
The present invention relates to a kind of microorganism of repairing for environmental pollution, the Cr (VI) particularly relating to a strain Salt-resistant alkali-resistant reduces bacterial strain and screening method thereof.
Background technology
According to preliminary investigation, the total volume of cargo in storage of current China's chromium slag, more than 6,000,000 tons, is scattered in more than 80 places of 20 Duo Ge provinces and cities.Through rainwater shower, the infiltration of decades, chromium slag muck is deposited place and is heavily polluted, according to relevant expert's estimation, by chromium slag severe contamination, the soil quantity survey that must administer between 4,000,000 tons to 1,000 ten thousand tons, the water pollution caused thus also can not be ignored.Chromium slag contaminated improvement is classified as environmental improvement priority project by " People's Republic of China's national economy and social development 11th Five-Year Plan outline ", explicitly calling for storing up chromium slag and polluted soil carries out the comprehensive regulation, realizing all chromic slag harmlessness of storing up and disposing.Therefore, the repairing and treating work carrying out chromium pollution water and soil is very urgent.
Chromium is main in the environment to be existed with the form of Cr (VI) and Cr (III).Compared with Cr (III), Cr (VI) has the high toxicities such as teratogenesis, carcinogenic, mutagenesis.The organic and inorganic Compound Phase of Cr (III) then easily in environment is combined, and form complicated stable insoluble compound, thus transport property is little, and biological effectiveness is low, and its toxicity is only the thousandth of Cr (VI).Therefore, highly toxic Cr (VI) being reduced to hypotoxic Cr (III) is the basic ideas that Cr (VI) pollutent is repaired.
Compared with the physico-chemical process that physical isolation method, the chemical reduction precipitator method, ion exchange method, reverse osmosis method etc. are traditional, biological restoration has the advantages such as low cost, simple to operate, non-secondary pollution because of it and is subject to paying close attention to widely and paying attention to.In recent years, the chromium of existing different genera also pathogenic microorganism is separated and is reported, as achromobacter Achromobacter sp.Ch-1, microbacterium Microbacterium sp.MP30, anthropi Ochrobactrum sp., golden yellow Arthrobacter Arthrobater aurescens sp., bacillus sp. etc.But not yet there is the report about separating sugared false anthropi Pseudochrobactrum saccharolyticum reduction Cr (VI) so far.Simultaneously, in the microorganism reported, Cr (VI) reduction process of most of bacterial strain is under anaerobic carried out, and salt resistance ability is low, alkaline-resisting limited in one's ability, is thus difficult to the actual repair requirement of high salt content, alkaline environment in satisfied actual contaminated soil and water body.
Therefore, this area in the urgent need to screening obtain new, the Microbial resources that actual polluted-water and soil remediation require can be met, thus provide technical support for the enforcement of pollution of chromium biological restoration.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, provide the Cr (VI) of a strain Salt-resistant alkali-resistant to go back pathogenic microorganism and screening method thereof.This bacterial strain has high tolerance to chromium pollutant, and in the alkaline environment of high salinity, highly toxic Cr (VI) can be reduced to hypotoxic Cr (III).The present invention is that the biological restoration of pollution of chromium provides new Microbial resources.
Object of the present invention is achieved through the following technical solutions: the Cr (VI) of a strain Salt-resistant alkali-resistant goes back pathogenic microorganism, and its 16S rRNA gene has the gene order shown in SEQ ID No.1; The Cr (VI) of this Salt-resistant alkali-resistant goes back pathogenic microorganism and is kept at China Committee for Culture Collection of Microorganisms of the depositary institution common micro-organisms center that Patent Office of the People's Republic of China specifies, and preservation is numbered: CGMCC No.5873.
The Cr (VI) of above-mentioned Salt-resistant alkali-resistant is gone back pathogenic microorganism and is obtained by following steps screening:
(1) enrichment: take 5g chromium-polluted soil (sodium-chlor 5g L in the sterilized LB liquid medium of 50mL
-1, yeast extract 5g L
-1, Tryptones 10g L
-1, pH=7.0 ~ 7.5), 28 DEG C, 160rpm shaking culture;
(2) tame: when soil suspension becomes greyish-green by original yellow, get suspension inoculation in the LB liquid medium containing Cr (VI), 28 DEG C, 160rpm shaking culture, when nutrient solution becomes greyish-green by yellow again, being seeded to another contains in the liquid nutrient medium of higher Cr (VI) concentration, progressively improves Cr (VI) concentration in substratum with this, thus domestication object bacterial strain; Cr (VI) used in domestication process is with the K after filtration sterilization
2cr
2o
7stock solutions adds; Tame Cr (VI) concentration gradient used and be followed successively by 5mM, 8mM, 10mM, 12mM, 15mM;
(3) be separated: when domestication concentration is 15mM Cr (VI), through the cultivation of 5d, nutrient solution becomes greyish-green, using this bacterium liquid as separation mother liquor, gets 1mL and becomes 10 by gradient dilution
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7; Respectively from 10
-5, 10
-6, 10
-7diluent in draw in the solid medium that 50uL coats containing 7mM Cr (VI), be inverted cultivation 2 ~ 3d for 28 DEG C;
(4) purifying: single bacterium colony of picking different shape respectively, carries out streak inoculation in the solid medium containing 7mM Cr (VI); After 28 DEG C of inversion cultivation 2 ~ 3d, picking list bacterium colony lines in Cr-containing medium again; In purifying 3 ~ 4 generation like this, the Cr (VI) obtaining Salt-resistant alkali-resistant goes back pathogenic microorganism.
The invention has the beneficial effects as follows: the present invention screens the Cr (VI) obtaining a kind of new Salt-resistant alkali-resistant and goes back pathogenic microorganism Pseudochrobactrum saccharolyticum LY10, this bacterial strain is Gram-negative bacteria, shaft-like 0.5 ~ 1.0 μm × 1.0 ~ 2.5 μm, under aerobic condition, the high toxicity Cr (VI) in potassium bichromate and potassiumchromate can be reduced to hypotoxic Cr (III); This bacterium has certain salt tolerance and alkali resistance; Be 2 ~ 20g L at substratum sodium chloride concentration
-1, in the scope of pH 7.0 ~ 10.7, all can carry out growth and Cr (VI) reduction well.Cr (VI) reduction efficiency of this bacterial strain is high, can be used for the bioprosthetic material preparing water body or Chromium in Soil pollution, is applied to Cr (VI) pollutent removed in water sweetening of the soil.
Pseudochrobactrum saccharolyticum LY10 bacterial classification is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on March 12nd, 2012, and preserving number is: CGMCCNo.5873; Classification And Nomenclature is for separating sugared false anthropi LY10, and Latin name is: Pseudochrobactrum saccharolyticum LY10.The address at China Committee for Culture Collection of Microorganisms's common micro-organisms center is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and postcode is 100101.
Accompanying drawing explanation
Fig. 1 is the phylogenetic tree of bacterial strain of the present invention;
Fig. 2 is the growing state figure of bacterial strain of the present invention under condition of different pH;
Fig. 3 is that the Cr of bacterial strain of the present invention under condition of different pH (VI) reduces situation histogram;
Fig. 4 is that the Cr of bacterial strain of the present invention under different salinity (VI) reduces situation histogram.
Embodiment
Further illustrate the present invention below in conjunction with embodiment, object of the present invention and effect will become more obvious.
Embodiment 1: the screening of bacterial strain of the present invention be separated
1, enrichment: gather chromium slag muck field, former Red Star chemical plant, Hangzhou contaminated soil, take 5g soil (sodium-chlor 5g L in the sterilized LB liquid medium of 50mL
-1, yeast extract 5g L
-1, Tryptones 10gL
-1, pH=7.0 ~ 7.5), 28 DEG C, 160rpm shaking culture.
2, tame: when soil suspension becomes greyish-green (Cr (III) is in green) by original yellow (Cr (VI) is in yellow), get suspension inoculation and contain in the LB liquid medium of Cr (VI) to what newly configure, 28 DEG C, 160rpm shaking culture, when nutrient solution becomes greyish-green by yellow again, be seeded in the liquid nutrient medium containing higher Cr (VI) concentration, Cr (VI) concentration in substratum is progressively improved with this, thus domestication object bacterial strain.Cr (VI) used in domestication process is with the K after filtration sterilization
2cr
2o
7stock solutions adds.Tame Cr (VI) concentration gradient used and be followed successively by 5mM, 8mM, 10mM, 12mM, 15mM.
3, be separated: when domestication concentration is 15mM Cr (VI), through the cultivation of 5d, nutrient solution becomes greyish-green, using this bacterium liquid as separation mother liquor, gets 1mL and becomes 10 by gradient dilution
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7.Respectively from 10
-5, 10
-6, 10
-7diluent in draw in the solid medium that 50uL coats containing 7mM Cr (VI), be inverted cultivation 2 ~ 3d for 28 DEG C.
4, purifying: single bacterium colony of picking different shape respectively, carries out streak inoculation in the solid medium containing 7mM Cr (VI).After 28 DEG C of inversion cultivation 2 ~ 3d, picking list bacterium colony lines in Cr-containing medium again.In purifying 3 ~ 4 generation like this, obtain the bacterial strain with high density Cr (VI) tolerance, called after LY10.
Embodiment 2: the Physiology and biochemistry of bacterial strain and 16S Molecular Identification
1, physiological and biochemical property
After bacterial strain LY10 grows 24h on the LB solid plate not containing chromium, bacterium colony is opaque circle, and smooth surface is moistening, is creamy white.Rule bacterial strain LY10 in the solid medium containing 7mM Cr (VI), after cultivating 72h, bacterium colony is faint yellow because being enriched a certain amount of chromium.This bacterial strain is Gram-negative bacteria, shaft-like, atrichia, and size is 0.5 ~ 1.0 × 1.0 ~ 2.5um.The Testing and appraisal of this bacterial strain entrusts Institute of Microorganism, Academia Sinica to carry out (2011 No. 243rd, micro-searching).The oxidase test result of bacterium is positive, and catalase test result is positive.Its BIOLOG qualification result is as table 1.
Table 1.LY10 is to the Utilization ability of 95 kinds of carbon substrates on BIOLOG GN plate
| Test subject | Result | Test subject | Result | Test subject | Result |
| Water | - | D-melibiose | - | Quinic acid | - |
| Cyclodextrin | - | Beta-methyl-D-Glucose glycosides | - | D-saccharic acid | - |
| Dextrin | - | D-Psicose | - | Sebacic acid | - |
| Glycogen | - | D-raffinose | - | Succsinic acid | ＋ |
| Polysorbate40 | - | L-rhamnosyl | - | Dibromo-succinic acid | ＋ |
| Tween 80 | ＋ | D-glucitol | - | Succinamic acid | - |
| N-ethanoyl-D GalN | - | Sucrose | - | Glucuronamide | - |
| N-acetyl-D-glucose amine | ＋ | D-trehalose | - | ALANINE amine | ＋ |
| Ribitol | - | Turanose | - | D-alanine | ＋ |
| L-arabinose | ＋ | Xylitol | - | ALANINE | ＋ |
| D-R | - | Methyl-prop ketone acid | ＋ | L-alanyl-glycine | ＋ |
| D-cellobiose | - | Monomethyl succsinic acid | ＋ | Altheine acid | ＋ |
| Erythritol | - | Acetic acid | ＋ | ASPARTIC ACID | ＋ |
| D-Fructose | ＋ | Cis-equisetic acid | - | Pidolidone | ＋ |
| L-fructose | - | Citric acid | - | Glycyl-ASPARTIC ACID | ＋ |
| D-semi-lactosi | - | Formic acid | - | Glycyl-L-glutamic acid | ＋ |
| Gentiobiose | - | D-lactobionolactone | - | Serine | ＋ |
| Alpha-D-glucose | ＋ | D-galacturonic acid | - | L-threonine | ＋ |
| M-inositol | - | Maltonic acid | - | D, VBT | - |
| α-D-lactose | - | D-Glucose amino acid | - | γ-aminobutyric acid | ＋ |
| Lactulose | - | D-Glucose aldehydic acid | - | Urocanic acid | ＋ |
| Maltose | - | Alpha-hydroxybutyric acid | ＋ | Inosine | ＋ |
| PEARLITOL 25C | - | Beta-hydroxy-butanoic acid | - | Uridine | ＋ |
| D-MANNOSE | - | Gamma-hydroxybutyric acid | - | Thymidine | - |
| P-hydroxyl phenylacetic acid | - | L-Histidine | ＋ | Phenylethylamine | - |
| Methylene-succinic acid | - | OH-L-proline | ＋ | Butanediamine | - |
| α-one butyric acid | ＋ | L-Leu | - | 2-monoethanolamine | - |
| α-ketoglutaric acid | ＋ | L-Orn | ＋ | 2,3-butanediol | - |
| α-one valeric acid | - | L-Phe | - | Glycerol | - |
| D, Pfansteihl | ＋ | L-PROLINE | ＋ | D, L-alpha-phosphate glycerine | - |
| Propanedioic acid | - | L-Glutimic acid | - | Cori's eater Cori | - |
| Propionic acid | ＋ | D-Ser | - | G6P | - |
2,16S rRNA gene and phylogenetic tree analysis
UNIQ-10 pillar bacterial genomes DNA extraction agent box is adopted to extract bacteria total DNA.PCR primer used is universal primer: forward primer BSF8/20 sequence is as shown in SEQ ID NO.2: 5 '-AGAGTTTGATCCTGGCTCAG 1 '; Reverse primer BSR1541/20 sequence is as shown in SEQ IDNO.3: 5 ' one AAGGAGGTGATCCAGCCGCA 1 '.PCR primer order-checking is completed by Shanghai Ying Jun Bioisystech Co., Ltd, and gained 16S rRNA gene order is as shown in SEQ ID NO.1.This sequence compared in ncbi database, phylogenetic tree construction as shown in Figure 1.Comparison result shows, and this bacterial strain belongs to Pseudochrobactrum saccharolyticum kind, therefore called after Pseudochrobactrum saccharolyticum LY10.Bacterial strain has been submitted patented procedure microorganism and has been preserved on March 12nd, 2012 to China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number is CGMCC No.5873.
Embodiment 3.: the alkali resistance of bacterial strain LY10 and salt tolerance detect
1, pH is on the impact of Cr (VI) reducing power of bacterial strain LY10
Bacterial strain P.saccharolyticum LY10 is cultured to increased logarithmic phase, is seeded to containing 130mg L according to the inoculum size of volume ratio 1%
-1cr (VI), pH are respectively in the LB liquid medium of 5.5,7.0,8.3,9.2,10.7,28 DEG C, shaking culture under 160rpm condition.Respectively at Cr (VI) concentration in different time sections sampling and measuring microbial biomass OD600 and nutrient solution.
Experimental result shows, P.saccharolyticum LY10 has certain resistance to alkali ability and wider appropriate pH growth scope, under neutral-alkaline environment, (pH7.0 to 10.7) all can grow and carry out Cr (VI) reduction preferably, shown in result Fig. 2, Fig. 3.When pH is 8.3, this bacterium has maximum growth amount and the highest Cr (VI) reducing power, and after cultivating 84h, Cr (VI) reduction ratio is 60.57%.This bacterium is that its application in different pH, particularly alkaline environment provides guarantee to the eurytropy of pH.
2, salinity is on the impact of Cr (VI) reducing power of bacterial strain LY10
By the bacterial strain being cultured to increased logarithmic phase according to the inoculum size of volume ratio 1% be seeded to different salinity (NaCl concentration is respectively 0,1,2,5,10,20,40,60g L
-1), initial Cr (VI) concentration is 130mg L
-1lB substratum in, 28 DEG C, sample after cultivating 114h under 160rpm condition, get after the centrifugal 10min of 10000rpm supernatant measure Cr (VI) content.
Experimental result shows that bacterial strain P.saccharolyticum LY10 has higher salt resistance ability, is 1 ~ 20g L in NaCl concentration
-1condition under all can grow well and carry out Cr (VI) reduction, and along with the increase of NaCl concentration, Cr (VI) reduction ratio increases gradually.When NaCl concentration is 20g L
-1time, bacterial strain, can by Cr (VI) concentration from 130mg L after cultivating 114h
-1be down to 9.1mg L
-1, reduction ratio is up to 95.2%, and result as shown in Figure 4.The salt-tolerant trait of this bacterium is that its application in the chromium slag contaminated soil of high salt content creates condition.
<110> Hangzhou Gao Bo Environmental Protection Technology Co., Ltd
The Cr (VI) of <120> mono-strain Salt-resistant alkali-resistant goes back pathogenic microorganism and screening method thereof
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1361
<212> DNA
<213> Pseudochrobactrum saccharolyticum
<400> 1
tggtcgcctg cctccttgcg gttagcacag cgccttcggg taaaaccaac tcccatggtg 60
tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg cggcattctg atccgcgatt 120
actagcgatt ccaacttcat gcactcgagt tgcagagtgc aatccgaact gagatggctt 180
ttggagatta gctcgacctc gcggtctcgc tgcccactgt caccaccatt gtagcacgtg 240
tgtagcccag cccgtaaggg ccatgaggac ttgacgtcat ccccaccttc ctccagctta 300
tcactggcag tccctttaga gtgcccaact aaatgatggc aactaaaggc gagggttgcg 360
ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacagcc atgcagcacc 420
tgtgtcctac gccccgaaag gcccaaagtg tctccactaa ggttcatagg catgtcaaga 480
gctggtaagg ttctgcgcgt tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc 540
ccccgtcaat tcctttgagt tttaatcttg cgaccgtact ccccaggcgg aatgtttaat 600
gcgttagctg cgccaccgaa gtgtaaacac cccgacggct aacattcatc gtttacggcg 660
tggactacca gggtatctaa tcctgtttgc tccccacgct ttcgcacctc agcgtcagta 720
atggaccagt aagccgcctt cgccactggt gttcctgcga atatctacga atttcacctc 780
tacactcgca attccactta cctcttccat actcaagact tccagtatca aaggcagttc 840
cggggttgag ccccgggatt tcacccctga cttaaaagtc cgcctacgtg cgctttacgc 900
ccagtaaatc cgaacaacgc tagccccctt cgtattaccg cggctgctgg cacgaagtta 960
gccggggctt cttctccggt taccgtcatt atcttcaccg gtgaaagagc tttacaaccc 1020
tagggccttc atcactcacg cggcatggct ggatcaggct tgcgcccatt gtccaatatt 1080
ccccactgct gcctcccgta ggagtctggg ccgtgtctca gtcccagtgt ggctgatcat 1140
cctctcagac cagctatgga tcgtcgcctt ggtaggcctt taccctacca actagctaat 1200
ccaacatggg ctcatcattc tccgataaat ctttccccaa aagggcgtat acggtattag 1260
cacaagtttc cctgagttat tccgtagaga acggtagatt cccatgcatt actcacccgt 1320
ctgccactgc ctccgaagag accgttcgac ttgcatgtgt a 1361
<210> 2
<211> 20
<212> DNA
<213> engineer
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 20
<212> DNA
<213> engineer
<400> 3
aaggaggtga tccagccgca 20
Claims (1)
1. the Cr (VI) of a strain Salt-resistant alkali-resistant goes back pathogenic microorganism, it is characterized in that, its 16S rRNA gene has the gene order shown in SEQ ID No.1; The Cr (VI) of this Salt-resistant alkali-resistant goes back pathogenic microorganism and is kept at China Committee for Culture Collection of Microorganisms of the depositary institution common micro-organisms center that Patent Office of the People's Republic of China specifies, and preservation is numbered: CGMCC No.5873.
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| CN107138521B (en) * | 2017-05-08 | 2020-08-07 | 北京航空航天大学 | Microbial remediation method for cadmium-polluted bottom mud |
| CN109593667A (en) * | 2017-09-30 | 2019-04-09 | 天津科技大学 | The identification of Cr (VI) reducing bacteria and its cultural method |
| CN108034619A (en) * | 2018-01-26 | 2018-05-15 | 南华大学 | A kind of preparation method of galvanotaxis microorganism formulation and its application in uranium-containing waste water is handled |
| CN110117545B (en) * | 2018-02-07 | 2021-12-21 | 南京农业大学 | Ectomycorrhizal fungi with Cr (VI) tolerance and reducing capability and application thereof |
| CN108728103A (en) * | 2018-04-17 | 2018-11-02 | 仁维国际股份有限公司 | The extracting method of geobiont element |
| CN109135754A (en) * | 2018-09-04 | 2019-01-04 | 孙爱友 | A kind of microorganism in situ cultural method for agricultural soil |
| CN111235055B (en) * | 2020-01-16 | 2021-09-24 | 浙江亿丰海洋生物制品有限公司 | Heronawinia heronans and application thereof in reduction of hexavalent chromium |
| CN111924980A (en) * | 2020-08-05 | 2020-11-13 | 盐城工学院 | Application of lysine bacillus fusiformis in removing hexavalent chromium in eutrophic wastewater |
| CN114657089B (en) * | 2022-03-02 | 2023-07-21 | 有研资源环境技术研究院(北京)有限公司 | Chromium-reducing bacillus and method for repairing chromium-polluted soil by using same |
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