CN1840654A - Chloride ion resistant strain S616 and its screening method - Google Patents

Abstract

The invention relates to a chlorine-resistant microorganism of strain S616, wherein the bacterial strain being Bacillus firmus S616 (CCTCC No: M205146). The bacterial colony shape and main biochemical characters are also described in the specification, the invention can be applied to the highly effective degradation of high concentration organic contaminant.

Description

Chloride ion resistant strain S 616 and screening method thereof

Technical field

The present invention relates to a kind of anti-chlorine microorganism and screening method thereof.

Background technology

The high salinity organic wastewater with difficult degradation thereby has become the worldwide technological puzzle of environmental protection water treatment field as waste water such as oil production, chemical industry, pharmacy.And salt acid system turmeric saponin factory effluent is representative in high density saliferous hardly degraded organic substance waste water.On the one hand, this class waste water complicated component, the organism of difficult degradation is many, and the biodegradability of waste water is poor; On the other hand, the salts contg height in the waste water, chlorine ion concentration is big, and chlorine ion concentration can reach 20000mg/L in its composite waste.At present, salt-containing organic wastewater adopts abiotic method and biological method, abiotic method is owing to its processing costs height, treatment effect are bad, enterprise can't accept, biochemical process is the at first method in the water technology always, the main two sections contact oxidation methods of A-B that adopt, traditional activated sludge process, the SBR method, biological filter, anaerobic filter etc., it is waste water below 3% that but above-mentioned biological process can only be handled saltiness mostly, and high-salt wastewater (saltiness 5%, even 20%) is difficult to handle, therefore need special microorganism.In recent years, reported that abroad relevant seed selection salt tolerant (NaCl) bacterium handles the report of high salt organic waste water, and to this class high-enriched organics content of saponin waste water, high salt (CaCl ₂), contain the complicated waste water of hard-degraded substance (saponin), also belong to the weak field of research so far.

Summary of the invention

The chloride ion resistant strain S 616 and the screening method thereof that the object of the present invention is to provide a strain to handle to contain high-concentration chlorine ion wastewater.

Chloride ion resistant strain S 616 provided by the present invention, be bacillus firmus (Bacillus firmus) S616CCTCCNO.M205146, Chinese typical culture collection center (being called for short CCTCC), preserving number: CCTCC NO.M205146 have been preserved in on December 9th, 2005.

The colonial morphology of a described chlorine-resisting strain S 616: children's bacterium bacterium colony in age is the wheel shape, and oyster white is opaque; Aged bacterium is field gray, the edge is uneven; Physiological characteristic: cell often be to or catenation, peritrichous, gemma is produced in motion, Gram-positive, amphimicrobian, 0.2～0.4 μ m * 1.4～2.1 μ m, the suitableeest growth pH value: 6.5～7.5, growth temperature: 30-40 ℃; Main biochemical character: the catalase positive, oxidase negative, hydrolysed casein, gelatin and starch.

The screening method of one chlorine-resisting strain S 616,

1). zoogleic fragmentation:

Get the anaerobic activated sludge in the saponin waste water treatment system, press anaerobic activated sludge: sterilized water=2g: the proportioning of 100ml, add in the good triangular flask that sterilized water is housed of sterilization in advance, and adding weight is the trisodium phosphate of sterilized water 0.01%, the shaking table vibration, zoogloea is smashed, got mud mixture, standby;

2). the separation of anti-chlorine ion dominant bacteria:

Utilize the above-mentioned mud mixture of aseptic pipette, extract, press mud mixture: isolation medium=0.5ml: the proportioning of 4.5ml, add isolation medium, 30-40 ℃ of anaerobism constant temperature culture 5-7 days treats that the test tube substratum becomes muddy, illustrates that bacterium grows, dull and stereotyped then dilution coating, picking has single bacterium of notable difference on colony characteristics, until the single bacterial strain of acquisition, and preserve on slant medium;

3). the screening of anti-chlorine ion dominant bacteria;

The above-mentioned isolated single bacterium colony of picking is inoculated in respectively in the screening culture medium, 30-40 ℃ of anaerobism constant temperature culture 5-7 days, and the muddy situation of observing bacteria suspension becomes the muddy bacterial strain that is the anti-chlorine ion that filters out; Through screening, obtain the bacterial strain that a strain is numbered strain S 616, i.e. chloride ion resistant strain S 616.

Isolation medium: CH ₃CH ₂COONa 30mmol, CH ₃CH ₂CH ₂COONa 30mmol, CH ₃CHOHCOONa30mmol, yeast 2.0g, MgCl ₂0.1g, NH ₄Cl 1.0g, K ₂HPO4 0.4g, resazurin 0.002g, halfcystine 0.5g, distilled water 1000ml, pH=7.0～7.3.

Screening culture medium: MgSO47H ₂O, 0.2g/L, KH ₂PO ₄0.5g/L, K ₂HPO ₄1.5g/L, NH ₄Cl 0.5/L, CaCl ₂135g/L, the 1ml trace element solution dissolves in 1L saponin waste water (COD concentration is 1000mg/L), pH=7.5,

Trace element solution: FeCl ₃6H ₂O 2g, CoCl ₂2g, MnCl ₂0.5g, CuCl ₂0.03g, ZnCl ₂0.05g, NiCl ₂.6H ₂O 0.05g, EDTA 1g, pH=7.0.

Enlarged culturing base: peptone 10g; Extractum carnis 5g; Sodium-chlor; 5g; Distilled water 1000ml; CaCl ₂18.0g/L.

SC substratum: peptone 10g; Extractum carnis 5g; Sodium-chlor; 5g; Distilled water 1000ml; Agar 20g (Gu); CaCl ₂Add-on be 0-140g.

Slant medium: peptone 10g; Extractum carnis 5g; Sodium-chlor; 5g; Potassium primary phosphate 2g; Ammonium chloride 1g; Agar 20g; Distilled water 1000ml; CaCl ₂18g/L, pH=7.5.

Above substratum is standby after respectively at 121 ℃ of autoclaving 30min.

The treatment process that a described bacillus firmus (Bacillus firmus) S616 CCTCC NO.M205146 is applied to contain high-concentration chlorine ion wastewater is: the single bacterium colony of picking bacillus firmus (Bacillus firmus) S616, in the enlarged culturing base, cultivate 24h, by bacillus firmus (Bacillus firmus) S616 after cultivating: the volume ratio that contains high-concentration chlorine ion wastewater (as mixing saponin waste water) is (1-4): 50 get bacillus firmus (Bacillus firmus) S616 is inoculated in and contains in the high-concentration chlorine ion wastewater (as mixing saponin waste water), 30-40 ℃ of constant temperature culture, the pH value is 7.0-8.0, reacts 48-72 hour.

Separate, screen a strain has the certain growth ability under the high-concentration chlorine ion condition new bacterial strain in the active sludge of the present invention from the saponin waste water treatment system.And its anti-chlorine ability studied, the result shows that under the high-concentration chlorine ion condition, the anti-chlorine ability of chloride ion resistant strain S 616 is good, scope is wide, can be at [Cl ^-] have energy for growth preferably during for 3000-75000mg/L.If it is induced bacterium as adding in the biosystem of the organic waste water of handling high salinity, the microorganism that can overcome general active sludge is subjected to the inhibition of high chloride ion, activity is low, processing efficiency is not high shortcoming.Because in high chloride ion (20000mg/L) environment, thereby being subjected to the effect that chloride permeability presses, the microorganism of traditional technology makes that plasma membrane is impaired, metabolic function descends reduces treatment effect, by contrast, if directly add the high chlorine resistant ion in the biochemical system and have the bacterial strain of good degradation property, just can overcome the shortcoming that above-mentioned traditional technology is handled the high-concentration chlorine ion organic wastewater with difficult degradation thereby.

To contain high salt (CaCl ₂The high concentrated organic wastewater of difficult degradation salt)---saponin waste water is an example, has studied the effect of its degradable organic pollutant.Found that this bacterial strain can effectively remove the ability of COD in the saponin waste water, the COD that can remove in the waste water within 72 hours is about 60-70%.

But the new bacterial strain that the present invention obtains not only can contain existence under the high-concentration chlorine ion environment, but also efficient degradation high concentration organic contaminant.

Description of drawings

Fig. 1-1a is a strain S 616 bacterial strain projection Electronic Speculum picture

Fig. 1-1b is a strain S 616 bacterial strain projection Electronic Speculum picture

Fig. 1-2 is strain S 616 growing state figure on the SC substratum of the different chlorine ion concentrations of liquid

Fig. 1-the 3rd, and the PCR product agarose electrophoresis figure of strain S 616 (1: nuclear DNA, 2: amplified production, M:Marker)

Fig. 1-4a is the following degraded efficiency diagram of strain S 616 under high chloride ion, high COD concentration

Fig. 1-4b is the following degraded efficiency diagram of strain S 616 under high chloride ion, high COD concentration

Fig. 1-4c is the following degraded efficiency diagram of strain S 616 under high chloride ion, high COD concentration

Fig. 1-4d is the following degraded efficiency diagram of strain S 616 under high chloride ion, high COD concentration

Embodiment

One, the screening method of a chlorine-resisting strain S 616:

(1), material is prepared:

1, bacterium source and experiment waste water:

(1) bacterium source: the active sludge in the biochemical system of processing saponin waste water;

(2) experiment waste water (promptly mixing saponin waste water): take from the female Home Co., Ltd in Shiyan City side, Hubei Province diosgenin wastewater; After interior electrolysis and CaO allotment, specific targets are: pH=7.5-8.2, COD=4000-17000mg/L, Cl ^-=8000-30000mg/L, NH ₄-N 140-300mg/L.

2, substratum:

Isolation medium: CH ₃CH ₂COONa 30mmol, CH ₃CH ₂CH ₂COONa 30mmol, CH ₃CHOHCOONa30mmol, yeast 2.0g, MgCl ₂0.1g, NH ₄Cl 1.0g, K ₂HPO4 0.4g, resazurin 0.002g, halfcystine 0.5g, distilled water 1000ml, pH=7.0～7.3.

Screening culture medium: MgSO47H ₂O, 0.2g/L, KH ₂PO ₄0.5g/L, K ₂HPO ₄1.5g/L, NH ₄Cl 0.5/L, CaCl ₂135g/L, the 1ml trace element solution dissolves in 1L saponin waste water (COD concentration is 1000mg/L), pH=7.5,

Trace element solution: FeCl ₃6H ₂O 2g, CoCl ₂2g, MnCl ₂0.5g, CuCl ₂0.03g, ZnCl ₂0.05g, NiCl ₂.6H ₂O 0.05g, EDTA 1g, pH=7.0.

Enlarged culturing base: peptone 10g; Extractum carnis 5g; Sodium-chlor; 5g; Distilled water 1000ml; CaCl ₂18.0g/L.

SC substratum: peptone 10g; Extractum carnis 5g; Sodium-chlor; 5g; Distilled water 1000ml; , agar 20g (Gu); CaCl ₂Add-on be 0-140g; Work as CaCl ₂When add-on is 0g, [Cl in the SC substratum ^-]=3000mg/L.

Slant medium: peptone 10g; Extractum carnis 5g; Sodium-chlor; 5g; Potassium primary phosphate 2g; Ammonium chloride 1g; Agar 20g; Distilled water 1000ml; CaCl ₂18g/L, pH=7.5.

Above substratum is standby after respectively at 121 ℃ of autoclaving 30min.

3, laboratory apparatus and equipment:

State China SHA-B constant temperature oscillator,

SHH150G illumination bio-incubator,

Horizontal pressure steam sterilizer,

WMX-1 type microwave seal is cleared up the COD tacheometer,

The 722S spectrophotometer,

Opticmicroscope,

PH meter,

Bechtop,

Air dry oven,

Ultramicrotome,

The H-7000FA of Hitachi projection microscope,

PTC200 type PCR instrument,

Electrophoresis apparatus and electrophoresis chamber,

Gel ultraviolet visualizer etc.

(2), the separation of bacterial strain and screening:

1). zoogleic fragmentation:

Get the anaerobic activated sludge in the 2g saponin waste water treatment system, add in the triangular flask of the 250ml that the 100ml sterilized water is housed that sterilization is good in advance, and adding weight is the trisodium phosphate of sterilized water 0.01%, the shaking table vibration, zoogloea is smashed, got mud mixture, standby;

2). the separation of anti-chlorine ion dominant bacteria:

Utilize the above-mentioned mud mixture of aseptic pipette, extract 0.5ml, add isolation medium 4.5ml, 30-40 ℃ of (best 35 ℃) anaerobism constant temperature culture 5-7 days, treat that the test tube substratum becomes muddy, illustrate that bacterium grows, dull and stereotyped then dilution coating, picking has single bacterium of notable difference on colony characteristics, until the single bacterial strain of acquisition, and on slant medium, preserve;

3). the screening of anti-chlorine ion dominant bacteria:

The above-mentioned isolated single bacterium colony of picking is inoculated in respectively in the screening culture medium, 30-40 ℃ of (best 35 ℃) anaerobism constant temperature culture 5-7 days, and the muddy situation of observing bacteria suspension becomes the muddy bacterial strain that is the anti-chlorine ion that filters out; Through screening, obtain the bacterial strain that a strain is numbered strain S 616, i.e. chloride ion resistant strain S 616.

Strain S 616 on the liquid SC substratum that adds the different concns chlorion growing state shown in accompanying drawing 1-2.

Two, the microorganism strains of anti-chlorine is identified:

1, to the evaluation of the stronger strain S 616 of chlorine-resistant property:

The stronger strain S 616 of chlorine-resistant property is carried out the evaluation of Physiology and biochemistry and the evaluation of 16S rDNA molecule, determined the kind of chlorine resisting strain from molecular level.

16S rDNA sequential analysis is mainly according to following steps:

1. the extraction of bacterium nuclear DNA:

1) choose single colony inoculation overnight incubation in the enlarged culturing base with autoclaved toothpick, get 1.5ml bacterium liquid in the Eppendorf pipe, 8, the centrifugal 5min of 000rpm room temperature thoroughly removes supernatant.

2) add STE damping fluid 1.5ml washing once, the centrifugal supernatant of abandoning adds 0.6mlTE solution again, resuspended bacterium.

3) add the N,O-Diacetylmuramidase of 30ul10mg/ml, 37 ℃ of water-bath 45min

4) add the SDS of 65 μ l 10%, the Proteinase K 3 μ l of 20mg/ml, 50 ℃ of water-baths 2 hours, clear to solution becomes.

5) add equivalance body phenol chloroform extracting three times, till can't see egg white layer.

6) NaAc (pH5.2) of the 3M of adding 1/10 volume in supernatant liquor.

7) add isopyknic Virahol, deposit D NA.Twine with glass stick and to choose the DNA filament, once with 70% washing with alcohol, natural airing, and DNA is dissolved in the 100 μ l TE solution.

8) adding final concentration is the RNase of 50 μ g/ml, and 37 ℃, 30min removes RNA, and-20 ℃ of preservations are standby.

2. the pcr amplification of 16S rDNA gene

P1: forward primer 5 ' AGAGTTTGATCCTGGCTCAG3 '

P2: reverse primer 5 ' GGTTACCTTGTTACGACTT3 '

In 50 μ L reaction volumes, add l μ L template DNA (0.1 μ g), 0.5 μ L P1 and P2 (final concentration is 0.5 μ M), 1 μ LdNTP (every kind of NTP0.2mM), 0.5 μ LTaq polysaccharase (2U) and 5 μ L, 10 * PCR damping fluid.The pcr amplification condition is: 94 ℃ of pre-sex change 5min; At 94 ℃ of sex change 30s, 61-65 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate 30 times; Last 72 ℃ are extended 10min eventually.

3. the recovery of PCR product

After the PCR product carried out electrophoresis with 1% sepharose, under ultraviolet lamp, cut and contain desire and reclaim segmental gel, put into the 1.5ml centrifuge tube and add 2 times of volume TE, 65 ℃ of water-baths add the extracting of equal-volume water-saturated phenol and get the upper strata water after once centrifugal and use that phenol-chloroform-the primary isoamyl alcohol extracting once again after 10 minutes, reset and add 10mol/L ammonium acetate and 2 times of volume dehydrated alcohol precipitations of 0.1 times of volume in the collection, centrifugal, wash precipitation once with 70% ethanol, be dissolved in an amount of sterilization distilled water after air-dry.4. the complete sequence determination of 16S rDNA and analysis

P-f：CACGACGTTGTAAAACGACAGTTTGATCCTGGCTC；

P-r：GGATAACAATTTCACACAGGAAGGAGGTGATCCAGCC。

Add 1 μ L template DNA (＜0.1 μ g), 30 circulations of pcr amplification (94 ℃ of 1min, 55 ℃ of 30s, 72 ℃ of 2min).Adopt dyestuff terminator termination reaction in the ABI PRISM sequencing kit.Then, on Applied Biosystem 373A dna sequencing instrument, check order.The 16S rDNA sequence that records adopts BLAST software and the comparative analysis of GenBank database, finally determines the kind of this bacterium from molecular level.

2,16S rDNA sequencing:

The present invention adopts the method for 16S rDNA sequencing and analysis is carried out the evaluation of molecular level to bacterium.Nuclear DNA with bacterium is a template, is primer with the universal primer of the pcr amplification of 16S rDNA gene, carries out pcr amplification, obtains the amplified band that length is 1425bp (detecting with 1% agarose gel electrophoresis), as Figure 1-3.After the PCR product is purified, measure its complete sequence.

＜110〉China Geological Univ. Wuhan

＜120〉chloride ion resistant strain S 616 and screening method thereof

<160>1425

<210>1

<211>1425bp

<212>DNA

＜213〉bacillus firmus (Bacillus firmus)

<220>

<221>misc_feature

<222>

<400>1

GCCTATGGTGCGGTCGCGCGAATCTGAGGGAGCTTGGTCCCAAAGATTAGCGGCGGACGG 60

GTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGGAGCTA 120

ATACCGGATAATCCCTTTCCTCACATGAGGAAAGGCTGAAAGACGGTTTCGGCTGTCACT 180

TACAGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGA 240

TGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAAACACGGCCCAAACTCC 300

TACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCG 360

CGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTATCGG 420

AGTAACTGCCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGC 480

AGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGC 540

AGGTGGTTCCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAA 600

CTGGGGAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCAAGTGTAGCGGTGAAATGCGTA 660

GAGATTTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGC 720

GCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGA 780

GTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCG 840

CCTGGGGAGTACGACCGCaAGGTTGAAACTCaAAGGAATTGACGGGGGCCCGCACAAGCG 900

GTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTC 960

TGACAATCCTAGAGATAGGACGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGT 1020

TGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGAT 1080

CTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAA 1140

GGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAAT 1200

GGATGGTACAAAGGGCTGCAAGACCGCGAGGTTTAGCCAATCCCATAAAACCATTCTCAG 1260

TTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCA 1320

GCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGT 1380

TTGTAACACCCGAAGTCGGTGGGGTAACCTTTTGGAGCCAGCCGC 1425

Three, strain S 616 colonial morphology, physiology and biochemical character see Table 1-1; Cellular form is seen Fig. 1-1a, Fig. 1-1b.

Table 1-1:

The colony morphology characteristic of strain S 616 and main physiological characteristic:

Project	Bacterium S616
		Colony morphology characteristic	Children's bacterium bacterium colony in age is the wheel shape, and oyster white is opaque; Aged bacterium is field gray, the edge is uneven.
Physiological characteristic	Cell often be to or catenation, peritrichous, gemma, Gram-positive, amphimicrobian are produced in motion.0.2～0.4 μ m * 1.4～2.1 μ m, the suitableeest growth pH value: 6.5～7.5, growth temperature: 30-40 ℃.

The acid of glucose product+

The glucose aerogenesis-

Wood sugar-

Pectinose-

Fructose-

Sucrose-

Maltose-

N.F,USP MANNITOL+

Seminose-

Oxydase-

Cytochrome C-

Catalase+

Arginine-

Hydrolysed casein+

Gelatin hydrolysate+

Hydrolyzed starch+

Citrate trianion-

NO ₃ ^-Reduction-

Nomenclature in the table: "+": the bacterial strain more than 90% is positive, "-": the bacterial strain more than 90% is negative.

Four, strain S 616 is new bacterial strain:

The 16S rDNA gene order of strain S 616 and the sequence in the international GenBank database are carried out online homology find that relatively the homology of bacterium S616 and a plurality of bacterial strains of Bacillus firmus (bacillus firmus) is up to more than the 98-99%.The Physiology and biochemistry and the Molecular Identification result of comprehensive bacterial strain, can determine that bacterium S616 is bacillus firmus (Bacillus firmus), strain S 616 called after bacillus firmus (Bacillus firmus) S616 (being chloride ion resistant strain S 616), consult pertinent data, still organic wastewater with difficult degradation thereby and to the report of its anti-chlorine capability study under the relevant high-concentration chlorine ion condition of bacteridium.Bacillus firmus (Bacillus firmus) S616 was new bacterial strain, had been preserved in Chinese typical culture collection center (being called for short CCTCC), preserving number: CCTCC NO.M205146 on December 9th, 2005.

The strain S 616 that this experiment separates, filters out has the function of better degrading high concentration chlorion difficult degradation saponin waste water.This has just widened people to the applied research thinking of bacillus firmus (Bacillus firmus) in its function aspects, and provides useful bacterium source and technology for degraded contains the high-concentration chlorine ion saponin waste water, has stronger actual application value.

Five, the application of chloride ion resistant strain S 616:

(1), strain S 616 is to the saponin waste water degradation capability:

Get saponin and produce composite waste, dilution 3-5 multiple, making its COD is 5000mg/L, uses CaCl ₂Regulate [Cl ^-] be 20,000 mg/L, get in the Erlenmeyer flask that waste water 50m1 adds 250ml respectively, add the bacteria suspension 1ml of strain S 616,35 ℃, 140-160rpm, pH value=7.0-7.5, the removal situation of bacterial strain to the COD of waste water calculated in shaking table vibration 3 days, strain S 616 is removed the excellent in efficiency of COD, is 60-70%.The degradation factors experimental result is seen Fig. 1-4a, Fig. 1-4b, Fig. 1-4c, Fig. 1-4d.

Described enlarged culturing base is: peptone 10g; Extractum carnis 55g; Distilled water 1000ml; Agar 20g (Gu); CaCl ₂18.0g/L, pH=7.0-7.5.

(2), the factorial experiments of strain S 616 degraded saponin waste water:

1),, in waste water, adds CaCl to the removal effect of COD in order to investigate bacterial strain under different chlorine ion concentrations ₂9864.2,14980,21061,24780,30080mg/L the adjusting chlorine ion concentration is respectively:.Chloride ion resistant strain S 616 removal effect result such as Fig. 1-4a.

From Fig. 1-4a as can be seen, as [Cl ^-During]=9864.2mg/L, reaction times is 3d, strain S 616 only is 42.96% to the clearance of COD, but rising along with chlorine ion concentration in the waste water, strain S 616 increases the clearance of COD, when chlorine ion concentration in the waste water reaches 20,000 mg/L, strain S 616 can reach 78.89% to the clearance of COD, and strain S 616 increases and changes little to the clearance of COD along with the continuation of chlorine ion concentration in the waste water, when chlorine ion concentration in the waste water during up to 30,000 mg/L, strain S 616 still remains on 70% to the clearance of COD.

2) in order to investigate bacterial strain (6610.4,7906.4,11209.6,13420.0,16196.2mg/L) under different COD concentration,, in waste water, add CaCl to the removal effect of COD ₂Regulate chlorine ion concentration and be respectively 20000mg/L.Chloride ion resistant strain S 616 removal effect result such as Fig. 1-4b.By experiment as can be known: [Cl in waste water ^-]=20000mg/L, when COD concentration reaches 10,000 mg/L, strain S 616 is better to the clearance of COD, can reach 73.2%.

3) from Fig. 1-4c and 1-4d as can be seen, strain S 616 increases afterwards earlier with the increase of pH the clearance of COD and descends, and its highest clearance reaches 70% when pH is 7.5-8.Waste water after the centrifugal treating, when finding that pH is 7-8, the wet thallus amount is 9,10,11 o'clock apparently higher than pH, as seen during in neutrality, helps the growth of bacterial strain at pH; When inoculum size changed between 1-4ml, strain S 616 did not have considerable change to the clearance of COD, was 70% substantially, and when inoculum size was 1ml, its clearance was best.This is the certain nutritive substance of growth needs because of bacterial strain, and when bacteria suspension was 1ml, its material that needs was met easily.

Claims (6)

1. a chlorine-resisting strain S 616 is characterized in that described bacterial strain is bacillus firmus (Bacillus firmus) S616CCTCC N0.M205146.

2. a chlorine-resisting strain S 616 according to claim 1 is characterized in that: the colonial morphology of a described chlorine-resisting strain S 616: children's bacterium bacterium colony in age is the wheel shape, and oyster white is opaque; Aged bacterium is field gray, the edge is uneven; Physiological characteristic: cell often be to or catenation, peritrichous, gemma is produced in motion, Gram-positive, amphimicrobian, 0.2～0.4 μ m * 1.4～2.1 μ m, the suitableeest growth pH value: 6.5～7.5, growth temperature: 30-40 ℃; Main biochemical character: the catalase positive, oxidase negative, hydrolysed casein, gelatin and starch.

3. the screening method of a chlorine-resisting strain S 616 as claimed in claim 1 is characterized in that:

1). zoogleic fragmentation:

Get the anaerobic activated sludge in the saponin waste water treatment system, press anaerobic activated sludge: sterilized water=2g: the proportioning of 100ml, add in the good triangular flask that sterilized water is housed of sterilization in advance, and adding weight is the trisodium phosphate of sterilized water 0.01%, the shaking table vibration, zoogloea is smashed, got mud mixture, standby;

2). the separation of anti-chlorine ion dominant bacteria:

Utilize the above-mentioned mud mixture of aseptic pipette, extract, press mud mixture: isolation medium=0.5ml: the proportioning of 4.5ml, add isolation medium, 30-40 ℃ of anaerobism constant temperature culture 5-7 days treats that the test tube substratum becomes muddy, illustrates that bacterium grows, dull and stereotyped then dilution coating, picking has single bacterium of notable difference on colony characteristics, until the single bacterial strain of acquisition, and preserve on slant medium;

3). the screening of anti-chlorine ion dominant bacteria:

The above-mentioned isolated single bacterium colony of picking is inoculated in respectively in the screening culture medium, 30-40 ℃ of anaerobism constant temperature culture 5-7 days, and the muddy situation of observing bacteria suspension becomes the muddy bacterial strain that is the anti-chlorine ion that filters out; Through screening, obtain the bacterial strain that a strain is numbered strain S 616, i.e. chloride ion resistant strain S 616.

4. the screening method of a chlorine-resisting strain S 616 according to claim 3 is characterized in that: isolation medium: CH ₃CH ₂COONa 30mmol, CH ₃CH ₂CH ₂COONa 30mmol, CH ₃CHOHCOONa 30mmol, yeast 2.0g, MgCl ₂0.1g, NH ₄Cl 1.0g, K ₂HPO ₄0.4g, resazurin 0.002g, halfcystine 0.5g, distilled water 1000ml, pH=7.0～7.3.

5. the screening method of a chlorine-resisting strain S 616 according to claim 3 is characterized in that: screening culture medium: MgSO47H ₂O, 0.2g/L, KH ₂PO ₄0.5g/L, K ₂HPO ₄1.5g/L, NH ₄Cl 0.5/L, CaCl ₂135g/L, the 1ml trace element solution dissolves in the 1L saponin waste water, and COD concentration is 1000mg/L in the saponin waste water, pH=7.5;

Trace element solution: FeCl ₃6H ₂O 2g, CoCl ₂2g, MnCl ₂0.5g, CuCl ₂0.03g, ZnCl ₂0.05g, NiCl ₂.6H ₂O 0.05g, EDTA 1g, pH=7.0.

6. the screening method of a chlorine-resisting strain S 616 according to claim 3 is characterized in that: slant medium: peptone 10g; Extractum carnis 5g; Sodium-chlor; 5g; Potassium primary phosphate 2g; Ammonium chloride 1g; Agar 20g; Distilled water 1000ml; CaCl ₂18g/L, pH=7.5.

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