CN114703090B - Moderately halophilic nitrobenzene degrading bacterium and application thereof - Google Patents
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Abstract
The invention discloses a moderately halophilic nitrobenzene degrading bacterium and application thereof, belonging to the technical field of biological treatment of organic pollutants. The invention uses high salinity exposed activated sludge as a bacterial source, uses glucose and nitrobenzene as a carbon source and uses a high salt (8.5% -9.5% w/v NaCl) inorganic salt culture medium as a screening culture medium, and adopts a streaking method to separate and purify so as to obtain the moderate halophilic bacillus capable of degrading nitrobenzene. The molecular biological identification shows that Bacillus is named Bacillus pumilus NJUST and the preservation number is CCTCC NO: M2022199. The moderately halophilic bacillus of the invention can be cultivated and grown in a high-salt environment (8.5% -9.5% w/v NaCl), so that the efficient degradation of nitrobenzene is synchronously realized, and the Extracellular Polymer (EPS) plays an important role in the degradation process. Bacillus pumilus NJUST51 has high salinity adaptability and organic matter degradation capability, is suitable for biological treatment of nitrobenzene-containing chemical wastewater with high salt concentration, and extracellular polymers of the nitrobenzene-containing chemical wastewater play a non-trivial role in the degradation process.
Description
Technical Field
The invention belongs to the technical field of biological treatment of environmental organic pollutants, and particularly relates to moderate halophilic nitrobenzene degrading bacteria and application thereof.
Background
In the past decades of industrial production activities, such as medicines, textiles, dyes, pesticides, etc., large amounts of high salt concentration industrial waste water are produced, with salt concentration contents varying up to 1% -20%. The main substances of salts in the high-salt wastewater are generally considered to be Na + 、Ca 2+ 、Mg 2+ 、Cl - 、SO 4 2- Often, the main inhibitory effect on microorganisms is cationic and its total content is generally higher than 1.5% (calculated as NaCl). Nitrobenzene is often used as an aromatic organic compound in the above industrial activities, and the production process is often accompanied by the discharge of a large amount of salt-containing organic wastewater. Because of the special chemical structure and relatively stable chemical property of nitrobenzene, conventional degradation methods such as photocatalytic degradation and physical decomposition are difficult to realize, and biological methods are widely focused as economic and environment-friendly treatment methods. However, the high salt concentration can lead to cell dehydration and rupture and inhibit the activity of enzyme, so that the traditional industrial wastewater biological treatment process faces great challenges, and meanwhile, the high salt wastewater is directly discharged, so that great pressure is brought to the environment and resource waste is caused.
Through searching, the application with the Chinese patent application number of CN202110737115.9 discloses a salt-tolerant bacillus (Bacillus subtilis strain), the salt tolerance range of which is 3-5%, 50mg/L ammonia nitrogen can be completely degraded within 48-72 h, and the salt-tolerant bacillus has good nitrification, but cannot adapt to high-salt-concentration nitrobenzene-containing chemical wastewater. The application with the Chinese patent application number of CN201610283793.1 discloses a biological agent containing bacillus subtilis, which can quickly form dominant species in printing and dyeing wastewater, degrade aniline substances, strengthen COD treatment effect in the wastewater, but not strengthen biodegradation of high-salt chemical wastewater.
Because the physical method and the chemical method of the high-salt nitrobenzene-containing wastewater have higher economic treatment cost, and the salt stress can lead to the weakening of the microbial activity, so that the biological method is difficult to carry out, the separation of a salt-resistant high-efficiency nitrobenzene degradation strain is necessary.
Disclosure of Invention
1. Problems to be solved
Aiming at the problems of weak biological activity and poor treatment effect caused by high salinity in the current biological treatment process of wastewater containing pollutant nitrobenzene, the invention provides a bacillus strain capable of degrading nitrobenzene, which has stronger adaptability and tolerance to living environment and can treat nitrobenzene in wastewater under the condition of high salt concentration.
2. Technical proposal
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the invention takes high salinity exposed activated sludge as a bacterial source, takes glucose and nitrobenzene as a carbon source and takes an inorganic salt culture medium containing high salt (9%w/vNaCl) as a screening culture medium, and performs purification and separation of bacterial strains to obtain a Bacillus strain which can degrade nitrobenzene and is moderately halophilic, the Bacillus strain is named Bacillus pumilus NJUST51, and the Bacillus strain is preserved in China Center for Type Culture Collection (CCTCC) at the year 2022 and 03 month 04, and has a preservation address of China university of Wuhan, and the preservation number is M2022199 in CCTCC NO.
Preferably, the 16sRNA gene sequence of the moderately halophilic bacillus strain is shown in SEQ ID NO. 1.
The invention also provides a culture method of the bacillus strain, which comprises the steps of inoculating Bacillus pumilus NJUST51 into an inorganic salt culture medium with the pH value of 6.5-7.5, and culturing at the temperature of 30-35 ℃.
Preferably, the composition of the inorganic salt culture medium is as follows: 1.268g L -1 K 2 HPO 4 ，3.48g L -1 KH 2 PO 4 ，4g L -1 (NH 4 ) 2 SO 4 ，1.43g L -1 MgSO 4 ·7H 2 O，0.5g L -1 Glucose, microelement solution SL-4 (10 mLL -1 ) Microelement SL-6 (10)0mL L -1 )；
Microelement SL-4: EDTA (0.5 g L) -1 )，FeSO 4 ·7H 2 O(0.2g L -1 )；
Microelement SL-6: znSO (ZnSO) 4 ·7H 2 O(0.01g L -1 )，MnCl 2 ·4H 2 O(0.03g L -1 )，H 3 BO 4 (0.3g L -1 )，CoCl 2 ·6H 2 O(0.2g L -1 )，CuCl 2 ·2H 2 O(0.01g L -1 )，NiCl 2 ·6H 2 O(0.02g L -1 )，Na 2 MoO 4 ·2H 2 O(0.03g L -1 )。
Preferably, the culture medium contains nitrobenzene and/or sodium chloride, and nitrobenzene and NaCl are convenient for calculating the logarithmic phase time of cell growth.
Preferably, the nitrobenzene concentration in the inorganic salt culture medium is 96.8-102.3 mg L -1 。
Preferably, the concentration of sodium chloride in the inorganic salt medium is 8.5% -9.5% (w/v).
The invention also provides application of the bacillus strain in high-salinity nitrobenzene-containing wastewater.
Preferably, the nitrobenzene-containing wastewater is high salinity nitrobenzene-containing wastewater, wherein the high salinity means that the NaCl concentration exceeds 1% (w/v).
Preferably, the above application comprises: inoculating Bacillus pumilus NJUST seed liquid into nitrobenzene wastewater with high salt concentration, wherein the culture temperature is 30-35 ℃ and the culture pH is 6.5-7.5.
Preferably, the inoculation amount of the Bacillus pumilus NJUST seed liquid is 5-10%, and the OD of the water sample after inoculation 600 Between 1.0 and 1.4.
Preferably, the above application comprises: the bacillus strain is made into a microbial inoculum and added into the wastewater.
The invention also provides a microbial inoculum for degrading nitrobenzene in high-salinity wastewater, which contains the screened bacillus strain Bacillus pumilus NJUST.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) The Bacillus pumilus NJUST provided by the invention has stronger adaptability and tolerance to living environment, and can treat nitrobenzene in wastewater under the condition of high salt concentration.
(2) According to the application of Bacillus pumilus NJUST in high-salinity nitrobenzene-containing wastewater, nitrobenzene can be degraded by taking glucose as a carbon source in a high-salt environment, the nitrobenzene removal rate reaches 94.6+/-1.3% in 48h at the salt concentration of 3.5%, and the Extracellular Polymer (EPS) secreted by the strain has a positive effect on degradation due to the fact that the extracellular polymer can prevent NaCl from diffusing well to protect cells, and compared with the effect that the nitrobenzene removal rate of a 0% salt concentration group is only 75.6+/-1.7% under the same condition, the strain can achieve a relatively ideal removal effect in biologically treating the high-salinity nitrobenzene-containing wastewater.
Drawings
FIG. 1 is a colony chart of the strain Bacillus pumilus NJUST provided by the invention grown in a solid medium plate.
Fig. 2 is a scanning electron microscope image of the strain Bacillus pumilus NJUST provided by the present invention.
FIG. 3 shows OD of strain Bacillus pumilus NJUST of the present invention in nitrobenzene-containing wastewater of different salt concentrations 600 A variation graph.
Fig. 4 is a graph showing the degradation effect of nitrobenzene in nitrobenzene-containing wastewater with different salt concentrations by the strain Bacillus pumilus NJUST provided by the invention.
FIG. 5 is a graph showing the effect of EPS removal or paranitrobenzene degradation of the strain Bacillus pumilus NJUST in nitrobenzene-containing wastewater with different salt concentrations.
Detailed Description
The invention is further described below in connection with specific embodiments.
The terms such as "upper", "lower", "left", "right", "middle" and the like are also used in the present specification for convenience of description, and are not intended to limit the scope of the present invention, but rather to change or adjust the relative relationship thereof, and are also considered to be within the scope of the present invention without substantial change of technical content.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
As used herein, the term "about" is used to provide the flexibility and inaccuracy associated with a given term, metric or value. The degree of flexibility of a particular variable can be readily determined by one skilled in the art.
As used herein, the term "is intended to be synonymous with" one or more of ". For example, "at least one of A, B and C" expressly includes a only, B only, C only, and respective combinations thereof.
Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limits of 1 to about 4.5, but also include individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be construed to include all such values and ranges. Moreover, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
Any steps recited in any method or process claims may be performed in any order and are not limited to the order set forth in the claims.
Example 1
The present embodiment provides for screening, separating and identifying Bacillus pumilus NJUST, specifically comprising the steps of:
(1) Isolation of strains
10mL of activated sludge is taken from an existing activated sludge reactor for treating high-salt nitrobenzene-containing wastewater in a laboratory, added into 100mL of saline with corresponding salt concentration, stirred uniformly and stood for two hours. Adding 1mL of supernatant into 50mL of inorganic salt culture medium, performing enrichment culture in a constant temperature shaking incubator for three days (180 rpm), continuously enriching for three times, and diluting the culture solution with sterile water gradient to 10 -4 ～10 -10 Multiple times. Inorganic salt agar solid culture medium plates are prepared, 20 mu L of diluted culture solution is respectively coated on the inorganic salt agar solid culture medium, and the culture medium plates are placed in a biochemical incubator for three days at 30 ℃. Single bacterial colony with obvious difference on the culture dish is selected, purified culture is carried out by adopting a plate streak separation method, and after five times of continuous purification, single bacterial strain is obtained and inclined surface preservation is carried out. Preparing an inorganic salt liquid culture medium containing nitrobenzene and high NaCl (8.5-9.5%w/v), filling the liquid culture medium into a triangular flask, sterilizing at a high temperature of 121 ℃, inoculating, separating and purifying to obtain a pure strain, culturing in a constant-temperature shaking incubator at 180 rpm and 35 ℃, and monitoring the concentration change of the nitrobenzene in the culturing process. Selecting a strain which can effectively remove nitrobenzene in a culture medium, named NJUST51, and performing inclined plane preservation and low-temperature preservation at-80 ℃.
The composition of the LB medium was as follows: tryptone (10 g L) -1 ) Yeast extract (5 g L) -1 ) Sodium chloride (90 g L) -1 )。
The composition of the inorganic salt medium (MSM) is as follows: 1.268g L -1 K 2 HPO 4 ，3.48g L -1 KH 2 PO 4 ，4g L -1 (NH 4 ) 2 SO 4 ，1.43g L -1 MgSO 4 ·7H 2 O，0.5g L -1 Glucose, microelement solution SL-4 (10 mLL -1 ) Microelement SL-6 (100 m)L L -1 )；
Microelement SL-4: EDTA (0.5 g L) -1 )，FeSO 4 ·7H 2 O(0.2g L -1 )；
Microelement SL-6: znSO (ZnSO) 4 ·7H 2 O(0.01g L -1 )，MnCl 2 ·4H 2 O(0.03g L -1 )，H 3 BO 4 (0.3g L -1 )，CoCl 2 ·6H 2 O(0.2g L -1 )，CuCl 2 ·2H 2 O(0.01g L -1 )，NiCl 2 ·6H 2 O(0.02g L -1 )，Na 2 MoO 4 ·2H 2 O(0.03g L -1 )。
The amounts of nitrobenzene and NaCl were added as required.
Adding 20g L based on liquid culture medium -1 After being autoclaved for 20 minutes at 121 ℃ in a sterilizing pot, the agar powder is poured into a sterile culture dish to be cooled to room temperature, and then the inorganic salt agar solid culture medium is obtained.
(2) Identification of strains
The strain NJUST51 is subjected to morphological, physiological and biochemical tests. Determining the 16SrRNA gene sequence of the strain, comparing the homology of the 16SrRNA gene sequence of the strain with gene sequences in a GenBank database, and analyzing the result to determine the species of the strain from the molecular biology level.
Morphological features: NJUST51 is yellow, cells are rod-shaped, the surface is smooth and opaque, the edges are neat and glossy, and the cells are diffusible and turbid in a liquid culture medium. FIGS. 1 and 2 are a solid medium plate colony and a scanning electron microscope, respectively, of bacteria NJUST51.
Molecular biology identification: the nuclear DNA of the strain NJUST51 is used as a template, and the universal primer for bacterial amplification is used for PCR amplification to determine the gene sequence of the strain NJUST51. The 16S rRNA gene sequence of the strain is submitted to a GenBank database for homology comparison, and the result shows that the sequence similarity of NJUST51 and Bacillus pumilus BFB30 reaches 100%.
From morphological, physiological and biochemical tests and molecular biological analyses of NJUST51, NJUST51 was identified as Bacillus genus and named Bacillus pumilus NJUST.
Example 2
This example provides the performance of strain Bacillus pumilus NJUST to degrade nitrobenzene at different salt concentrations.
Bacillus pumilus NJUST51 is inoculated into LB culture medium, 180 rpm shaking culture is carried out at 35 ℃, NJUST51 strain enrichment is carried out, after the strain enters the logarithmic phase (about 48 hours), the obtained bacterial liquid is centrifuged by a centrifuge for 10 minutes (6000 rpm), and deposited bacterial bodies are obtained.
Preparation of the composition containing 100mg L -1 Nitrobenzene and NaCl (0%, 1%, 3.5 w/v) in an inorganic salt broth were used as simulated wastewater. Adding the deposited thallus into simulated wastewater with corresponding salt concentration after sterilization treatment, re-suspending, separating the core at 6000 rpm for 10 minutes, washing for three times, and controlling the OD thereof 600 Culturing at 1.2+ -0.2, shaking at 30deg.C and 180 rpm, and monitoring OD 600 Variation and nitrobenzene concentration.
As shown in FIGS. 3 and 4, the results of the experiment show that the OD was high at a high salt concentration (3.5% NaCl) 600 The trend of increasing and decreasing occurs, while the lower salt concentration (0%, 1% NaCl) OD 600 The value is always decreasing. And the nitrobenzene removal rate can reach 75.6+/-1.7% in 48h at the salt concentration of 0%, and the nitrobenzene removal rate can reach 94.6+/-1.3% in 48h at the salt concentration of 3.5%.
This example illustrates that the isolated Bacillus pumilus NJUST51 can be grown in a high salt environment while enabling efficient degradation of nitrobenzene.
Example 3
This example provides the effect of the extracellular polymeric substance of strain Bacillus pumilus NJUST on its degradation.
Bacillus pumilus NJUST 51A culture medium containing sterilized LB medium with different salt concentrations (0% 1%3.5% w/v NaCl) was inoculated for expansion, and as shown in example 2, a post log phase washing treatment was performed to obtain deposited cells.
Preparing NaCl solution with low salt concentration (0.9% w/v) and high salt concentration (3.5% w/v), culturing the thallus in 0% and 1% LB culture mediumRe-suspending in 0.9% physiological saline, suspending the strain in 3.5% LB culture medium in 3.5% saline solution, washing for three times, suspending again, heating in 45deg.C water bath for 1 hr, centrifuging again (6000 rpm) for 20 min, discarding supernatant to obtain deposited strain, re-suspending with sterilized inorganic salt liquid culture medium with corresponding salt concentration (0% 1.5% w/v NaCl), centrifuging again, washing repeatedly for three times, suspending strain in sterile liquid inorganic salt culture medium to obtain seed solution, inoculating into sterilized simulated wastewater (controlling OD) 600 1.2.+ -. 0.2), and then put into a constant temperature shaking table at a rotation speed of 180 rpm at 30 ℃. Different salt concentrations can stimulate the generation of different amounts of EPS, and the cell obtained by washing with the saline water with the corresponding salt concentration has higher integrity, is not easy to crack due to water loss, has higher bacterial activity and is more thoroughly removed.
The nitrobenzene is respectively degraded by using the bacterial liquid with EPS removed and the bacterial liquid without EPS removed, the experimental result is shown in fig. 5, compared with the degradation effect of the nitrobenzene without EPS, the degradation effect of the nitrobenzene with EPS removed is reduced, after degradation is 48, the nitrobenzene removal rate is 67.7+/-2.1% when the salt concentration is 0%, and is 64.6+/-1.3% when the salt concentration is 3.5%, because the diffusion speed of NaCl in EPS is proportional to the distance in the diffusion direction, the high salt concentration stimulates the secretion of EPS, the EPS layer is thickened, and NaCl can be better prevented from diffusing into cells, so that the degradation effect of the nitrobenzene can be greatly influenced by the EPS removal.
The example shows that the separated Bacillus pumilus NJUST can degrade nitrobenzene-containing wastewater under the high-salt condition to achieve a better nitrobenzene removal effect, and the extracellular polymer plays a vital role in the degradation process.
Sequence listing
<110> university of Nanjing's science
<120> a strain of moderately halophilic nitrobenzene degrading bacteria and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
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<211> 1453
<212> DNA
<213> Bacillus pumilus NJUST51(Bacillus pumilus NJUST51)
<400> 1
atggctcagg acgaacgctg gcggcgtgcc taatacatgc aagtcgagcg gacagaaggg 60
agcttgctcc cggatgttag cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa 120
gactgggata actccgggaa accggagcta ataccggata gttccttgaa ccgcatggtt 180
caaggatgaa agacggtttc ggctgtcact tacagatgga cccgcggcgc attagctagt 240
tggtgaggta acggctcacc aaggcgacga tgcgtagccg acctgagagg gtgatcggcc 300
acactgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccgc 360
aatggacgaa agtctgacgg agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa 420
gctctgttgt tagggaagaa caagtgcaag agtaactgct tgcaccttga cggtacctaa 480
ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 540
gtccggaatt attgggcgta aagggctcgc aggcggtttc ttaagtctga tgtgaaagcc 600
cccggctcaa ccggggaggg tcattggaaa ctgggaaact tgagtgcaga agaggagagt 660
ggaattccac gtgtagcggt gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc 720
gactctctgg tctgtaactg acgctgagga gcgaaagcgt ggggagcgaa caggattaga 780
taccctggta gtccacgccg taaacgatga gtgctaagtg ttagggggtt tccgcccctt 840
agtgctgcag ctaacgcatt aagcactccg cctggggagt acggtcgcaa gactgaaact 900
caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 960
cgaagaacct taccaggtct tgacatcctc tgacaaccct agagataggg ctttcccttc 1020
ggggacagag tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt 1080
aagtcccgca acgagcgcaa cccttgatct tagttgccag catttagttg ggcactctaa 1140
ggtgactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt 1200
atgacctggg ctacacacgt gctacaatgg acagaacaaa gggctgcgag accgcaaggt 1260
ttagccaatc ccacaaatct gttctcagtt cggatcgcag tctgcaactc gactgcgtga 1320
agctggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg 1380
tacacaccgc ccgtcacacc acgagagttt gcaacacccg aagtcggtga ggtaaccttt 1440
atggagccag ccg 1453
Claims (8)
1. Bacillus pumilusBacillus pumilusCharacterized in that the Bacillus pumilus is named asBacillus pumilusNJUST51, the strain is preserved in China Center for Type Culture Collection (CCTCC) NO: M2022199.
2. The method for culturing Bacillus pumilus according to claim 1, whereinBacillus pumilusThe NJUST51 is inoculated into an inorganic salt culture medium with the pH value of 6.5-7.5, and the culture temperature is 30-35 ℃.
3. The method for culturing Bacillus pumilus according to claim 2, wherein the inorganic salt medium contains nitrobenzene and high-concentration sodium chloride, and the concentration of sodium chloride is 8.5% -9.5% (w/v).
4. The method for culturing Bacillus pumilus according to claim 3, wherein the concentration of nitrobenzene in the inorganic salt medium is 96.8-102.3 mg L -1 。
5. The use of bacillus pumilus according to claim 1 for degrading nitrobenzene in high salinity nitrobenzene-containing wastewater, wherein the high salinity is NaCl concentration exceeding 1% (w/v).
6. The use of bacillus pumilus according to claim 5 in high salinity nitrobenzene-containing wastewater, wherein the bacillus pumilus is formulated into seed solution or added into the wastewater after being formulated into a microbial inoculum.
7. The application of bacillus pumilus in high-salinity nitrobenzene-containing wastewater according to claim 6, wherein the wastewater treatment temperature is 30-35 ℃ and/or the pH is 6.5-7.5.
8. A microbial inoculum for degrading nitrobenzene, characterized in that it contains the Bacillus pumilus strain according to claim 1Bacillus pumilus NJUST51。
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| 酒精耐受性短小芽孢杆菌在硝基苯降解中的应用;张向东;杨谦;;化工进展(第S1期);摘要 * |
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| CN114703090A (en) | 2022-07-05 |
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