CN113373076B - Heavy metal tolerant nitrile degrading bacterium and application thereof in production of organic acid - Google Patents
Heavy metal tolerant nitrile degrading bacterium and application thereof in production of organic acid Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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Abstract
The invention belongs to the technical field of biology, and particularly relates to application of a heavy metal resistant nitrile degradation and organic acid production bacterium. The invention provides a nitrile compound biodegradation method, and obtains a bacterium capable of producing mandelic acid, acrylic acid and nicotinic acid. Combined with morphological, physiological and biochemical characteristics and 16S rDNA sequencing analysis, it was identified as leubacter (Leucobacter sp.) DLY 11. The strain is preserved in the China general microbiological culture Collection center. The bacterium can degrade mandelonitrile, acrylonitrile, 3-cyanopyridine, adiponitrile and n-valeronitrile in a culture medium with nitrile compounds as the only carbon source. The production efficiency of acrylic acid is about 159mg-381mg per hour per gram of dry cells, and the production efficiency of mandelic acid can reach 453mg per hour per gram of dry cells. The invention has important application prospect in the fields of relevant environmental pollution treatment, chemical engineering and medicine and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a heavy metal tolerant nitrile degrading bacterium and application thereof in production of organic acid.
Background
Nitrile compounds are highly toxic organic compounds containing a-CN (cyano) group, have toxic effects on many organisms, generally have strong pungent odor and are difficult to degrade. How to effectively treat the substances to prevent toxic and harmful discharge and reduce environmental pressure is always a trouble of nitrile-containing sewage enterprises. Chemical processes for the treatment of nitrile compounds generally require extreme environments such as high temperatures, strong acids or bases, and may also produce toxic and harmful by-products during the reaction, and the high requirements for processing equipment and processing techniques have greatly limited their use. Compared with the nitrile compound treated by a chemical method, the microbial method has the advantages of mild reaction, economy, practicability, low energy consumption, strong pertinence and the like. Therefore, the strain capable of degrading nitrile compounds is obtained and applied, which is beneficial to stabilizing the treatment effect of a microbiological method and improving the degradation efficiency of microorganisms on harmful substances in sewage. The nitrile compound degradation conversion bacteria have very important application value in the aspect of producing organic acid with high added value. For example: the mandelic acid produced by using the mandelonitrile is an important intermediate for synthesizing amino acid, penicillin, antitumor drugs, coenzyme A and other drugs; nicotinic acid produced by using 3-cyanopyridine can be used for synthesizing various medicines, such as nicotemide, inositol nicotinate and the like.
Disclosure of Invention
The present invention aims at providing a nitrile compound-degrading bacterium aiming at practical problems and needs in production practice.
The invention also aims to provide the application of the bacterium in producing organic acid.
The nitrile compound degrading strain provided by the invention is a gram stain reaction positive bacterium, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 6 th and 17 th of 2020 (address: China, Beijing, West Lu No. 1 institute of China academy of sciences No. 3 of the morning area of the morning sun, Taiwan, zip code: 100101). The classification was named as Leuconobacter (Leuconobacter sp.) DLY 11. The preservation number is CGMCC No: 1.16127.
the main biological characteristics of the nitrile compound degrading bacteria are as follows: no flagellum, rod-like, no motility, growth pH of 4.0-7.0, growth temperature of 15-37 deg.C, growth concentration of NaCl of 0-5% (w/v), and tolerance of heavy metal Cr 6+ (2g/L) and Ni 2+ (3g/L)。
The nitrile compound degrading bacteria colony appears milky yellow on the surface of a TSA culture medium (Qingdao Haibo organism, China), and the surface of the bacterial colony is smooth and uniform in color.
The invention provides degradation of the nitrile compound degrading bacteria: mandelonitrile, acrylonitrile, 3-cyanopyridine, adiponitrile, and n-valeronitrile.
The invention also provides a method for producing organic acid by the nitrile compound degrading bacteria, which comprises the following steps:
activating thalli: single colonies were picked from the surface of TSA solid medium and inoculated into activated medium, followed by shaking culture in a constant temperature shaker (30 ℃ C., 150rmp) for 48 hours, and the pellet was collected.
Preparing a seed solution: inoculating the leucine DLY11 to a basic culture medium containing 10mM acrylonitrile (as a unique nitrogen source) for degrading nitrile compounds according to the inoculation amount of 10 percent, carrying out shake culture for 48 hours, and collecting thallus precipitates;
production of organic acids from nitriles: adding seed solution into a basal culture medium containing 20mM nitrile (acrylonitrile, 3-cyanopyridine, adiponitrile, n-valeronitrile), and performing shaking culture at 30 ℃ and 150rpm, wherein 5mL of the culture medium is sampled each time; adding 20mM nitrile into the culture system after each sampling; the method for producing mandelic acid comprises the following steps: after the activation of the cells, the cells were inoculated into the activation medium of example 2 in an inoculum size of 3%, shake-cultured for 48 hours using a shaker, and the cell precipitates were collected; respectively adding bacterial suspension, a nitrile degradation basal medium and mandelonitrile with the concentrations of 10mM, 30mM and 50mM, namely 1.33g/L, 3.99g/L and 6.65 g/L; the yield of organic acids such as mandelic acid, acrylic acid, nicotinic acid and the like and the degradation effect of nitrile compounds are detected by gas/liquid chromatography and a phenol-sodium hypochlorite method.
The invention has great application prospect in the fields of relevant environmental pollution treatment, chemical industry, biomedicine and the like.
Drawings
FIG. 1 is a photograph of the morphology of strain DLY 11.
FIG. 2 is a phylogenetic tree.
FIG. 3 is a graph showing the dry weight of bacterial cells and absorbance.
FIG. 4 is a chromatogram of a gas chromatography detection of acrylonitrile/acrylic acid.
FIG. 5 is a chromatogram of liquid chromatography detection of mandelonitrile/mandelic acid.
FIG. 6 is a graph showing the degradation curves of mandelonitrile at different concentrations and at different times.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 isolation, identification and preservation of Leuconostoc DLY11
II, separation
About 1ml of an activated sludge sample (obtained from a petrochemical oil refining and chemical wastewater treatment system) was sequentially diluted with sterile distilled water to 10-fold, 20-fold, 50-fold, 100-fold and 1000-fold volumes. And coating 100 mu L of the diluted sample on the surface of a solid culture medium by using a coating plate method, culturing at 30 ℃, and observing the growth condition of surface colonies every day. Colonies with different colors and morphologies are picked, purified and cultured by a three-region streaking method until single colonies with the same morphologies, sizes and other characteristics grow on the surface of the culture medium. Single colonies were obtained by purification. The colonies appeared milky yellow on the surface of a TSA culture medium (Qingdao Haibo organism, China), and the surfaces of the colonies were smooth and uniform in color.
II, identification
The strain obtained by purification was inoculated on a TSA plate, cultured at 30 ℃ for 24 hours, and then the morphology, size, and the like of the cells were observed using a transmission electron microscope (see FIG. 1 for a photograph of the strain DLY 11). Single colonies were picked from the surface of the TSA plate cultured for 24 hours, and gram-stained according to a standard method, and the gram-stained bacteria were observed using an inverted fluorescence microscope. A semi-solid culture medium TSB (Qingdao Haibobao, China) is prepared, and the motility and aerobic condition of bacteria are observed by adopting a puncture inoculation method.
Based on liquid medium TSB: preparing culture medium systems with different NaCl concentrations (NaCl gradient of 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0g/100mL), placing the inoculated culture medium systems in a shaking table at 30 ℃ for shaking culture for 3-5d, and detecting the growth condition of the bacteria at OD600 by using a UV-2450 spectrophotometer to determine the optimal salinity growth range; preparing liquid culture media with different pH values (pH gradient set to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0), inoculating, placing in a shaking table at 30 ℃ for shaking culture for 3-5d, and taking an non-inoculated culture medium with the same culture conditions as a blank control to determine the optimal growth pH range; setting the temperature gradient at 4 deg.C, 20 deg.C, 28 deg.C, 30 deg.C, 35 deg.C, 37 deg.C, 40 deg.C, 45 deg.C, and 50 deg.C to determine the optimumThe growth temperature range is optimized; set Pb 2+ 、Ni 2+ 、Cr 6+ 、Cu 2+ 、Co 2+ Five heavy metal ions (concentration gradient of 0.5, 1.0, 1.5, 2.0, 3.0 and 4.0 g/L). On the basis of a liquid carbon source culture medium, different carbohydrates with different concentrations are added to be used as a unique carbon source, and the assimilation of bacteria is detected. According to the method in the handbook of identifying common bacteria systems, the nitrate reducing capability, nitrite reducing capability and denitrification capability of bacteria as well as the existence of lipase and catalase are detected. Zeae DSM 103238 T And L. dentificans DSM 25936 T As a reference strain. The results are shown in tables 2 and 3 and 4.
TABLE 2
TABLE 3
TABLE 4
The bacterial genomic DNA extraction kit is used for extracting the genomic DNA of the strain DLY11, and 16S rDNA amplification is carried out by adopting universal primers 27F and 1492R. The amplified product was analyzed for product bands by agarose gel electrophoresis to obtain target bands and purified using Tiangen DNA purification kit DP 209. The purified product was ligated into the vector pMD19-T and the vector of interest was transformed into E.coli DH 5. alpha. using the pMD19-T cloning kit. The recombinant strain is sent to Qingdao Okagaku Biotechnology Limited company for sequencing to obtain a 16S rDNA sequence 1393bp (see a sequence diagram 1). The sequence of 16S rDNA of each relevant gene was retrieved using GenBank database and phylogenetic trees of the strains were constructed using MEGA version 7.0. Phylogenetic analysis of bacteria was performed using the nearest neighbor ligation (NJ). Based on 1000 replicates, the topology of the phylogenetic tree was determined using Bootstrap analysis. See fig. 2.
The above identification results show that the strain DLY11 belongs to the phylum Actinobacillaceae (Microbacteriaceae), the genus Leuconobacter (Leuconobacter).
III, preservation
Leuconobacter (Leuconobacter sp.) DLY11 was deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 17.6.2020 (address: China, Beijing, Yokogao Yang district, West Chen, Xilu No. 3, China academy of sciences, Japan; postal code: 100101). The preservation number is CGMCC No: 1.16127.
example 2 degradation of Likelacterium DLY11 nitrile and production of organic acid
Na 2 HPO 4 /NaH 2 PO 4 The formula of the buffer solution is as follows: disodium hydrogen phosphate 14.2g, sodium dihydrogen phosphate 12.2g, dissolved in 800mL ddH 2 O, using ddH 2 And adding O to the volume of 1L, and adjusting the pH value to 7.0.
The formula of the activation medium is as follows: tryptone Soy Broth Medium (TSB)30g, in ddH 2 Dissolving O, diluting to 1L, and adjusting pH to 7.0.
Basic culture medium for nitrile degradation: disodium hydrogen phosphate 2.0g, potassium dihydrogen phosphate 1.0g, yeast powder 0.5g, magnesium sulfate 0.2g, ferrous sulfate 0.03g, dissolved in 800mL Na 2 HPO 4 /NaH 2 PO 4 Buffer, then add glycerol 3.25mL and mix well, then add Na 2 HPO 4 /NaH 2 PO 4 Buffer volume was adjusted to 1L.
Firstly, preparing seed liquid
Inoculating the leucine DLY11 into an activation culture medium, and performing shaking culture at 30 ℃ and 150rpm to obtain a seed solution. OD of seed liquid 600nm The value is 0.6-0.8.
Secondly, preparing a cell dry weight standard curve
1. And (3) inoculating the seed solution prepared in the step one to an activation culture medium according to the inoculation amount of 10%, and then carrying out shaking culture at 30 ℃ and 150rpm for 48 hours.
2. After completion of step 1, 40mL of sample was taken and OD was adjusted with activation medium 600nm Value, obtaining OD 600nm The values of the respective bacterial liquids were different.
3. And (3) taking the bacterial liquid obtained in the step (2), centrifuging at 8000rpm for 15min, and collecting bacterial precipitates.
4. And (3) taking the thallus precipitate obtained in the step (3), fully washing the thallus precipitate by using normal saline, drying the thallus precipitate at the temperature of 60 ℃, and measuring the dry weight of the thallus precipitate.
5. Preparation of OD 600nm Standard curve of values versus biomass (dry cell weight) (see fig. 3). The abscissa is OD 600 The value on the ordinate is the cell biomass (in mg cell dry weight per mL mgdcw. mL-1). The standard curve equation is that y is 2.864 x-0.015; r 2 ＝0.9998。
Thirdly, making a bacterial growth curve and detecting the degradation rate of acrylonitrile
1. Inoculating the seed solution prepared in the first step to a basic culture medium containing 10mM acrylonitrile (as a unique nitrogen source) for degrading nitrile compounds, performing shaking culture at 30 ℃ and 150rpm, and detecting OD every 12h 600nm The value is obtained. The results show that OD was obtained after 48 hours of shaking culture 600nm The value reaches a maximum value.
2. Taking the culture system of the step 1 after shaking culture for 48h, centrifuging at 8000rpm for 15min, collecting thallus precipitate, and washing for 2-3 times (the washing method is Na 2 HPO 4 /NaH 2 PO 4 Suspending the cells in buffer, centrifuging at 8000rpm for 15min, collecting the cells), and adding Na 2 HPO 4 /NaH 2 PO 4 Suspending the cells in a buffer to obtain OD 600nm Bacterial suspension with value of 0.43.
3. 100mL of a basic medium for degrading a nitrile compound containing 20mM acrylonitrile was added with 5mL of the bacterial suspension prepared in step 2, followed by shaking culture at 150rpm at 30 ℃ for 104 hours. Sampling every 2h for 0-8h, sampling every 12h for 9-32h, and sampling every 24h for 33-104 h; 5mL of samples are taken each time; after each sampling 20mM of nitrile was added to the culture system.
4. Preparation of bacterial growth curves
Taking 3mL of the sample from step 3, and determining the OD 600nm Calculating each time point of the shaking culture according to the standard curve of the step twoBiomass per liter of culture system (biomass on a dry cell weight basis).
During the cultivation, the biomass of the bacteria is increased and then decreased. The maximum value is reached at 4h of cultivation. After 4h of culture, the biomass of the thallus shows a tendency of reducing along with the prolonging of the culture time (the reason is that the harm toxicity to the bacteria is more and more serious along with the gradual accumulation of the organic acid of the nitrile compound product, and the death rate of the bacteria is gradually larger than the growth rate).
5. The degradation rate of acrylonitrile and the yield of acrylic acid were measured
And (4) centrifuging 1mL of the sampling sample in the step (3) at 8000rpm for 2min, taking supernatant, adding 0.1mL of 2M hydrochloric acid aqueous solution into the supernatant to terminate the reaction, then taking 200 mu L of the supernatant, adding 800 mu L of ethyl acetate, uniformly mixing, standing for 30min, and collecting an ethyl acetate phase.
The acrylonitrile and acrylic acid content in the ethyl acetate phase was determined by gas chromatography. Relevant parameters of gas chromatography: the chromatographic column is a polar HP-FFAP capillary column (30m multiplied by 320 mu m multiplied by 0.25 mu m), the injection port temperature is 260 ℃, the column box temperature is 190 ℃, the FID detector temperature is 260 ℃, the hydrogen flow rate is 40mL/min, the air flow rate is 450mL/min, the nitrogen tail gas flow rate is 40mL/min, the sample injection amount is 0.2 mu L, and the split ratio is 50: 1.
And adopting ethyl acetate as a solvent to prepare acrylic acid standard solutions with various concentrations. The acrylic acid standard solution was checked by gas chromatography (parameters as above). A standard curve of peak area versus acrylic acid concentration was prepared. 2.9507X +6.9485, coefficient of correlation R 2 99.17%; y represents the peak area and X represents the acrylic acid concentration (in mmol/L).
Detecting ethyl acetate standard, acrylonitrile standard and acrylic acid standard by gas chromatography according to the above parameters, and obtaining chromatogram as shown in figure 4. The peak time of ethyl acetate is 1.387min, the peak time of acrylonitrile is 2.187min, and the peak time of acrylic acid is 4.522 min.
The yield of acrylic acid was calculated from the standard curve and the peak area, and the degradation rate of acrylonitrile at each time point of the shaking culture was further calculated. The culture time is 0-32 hours, and the degradation rate of acrylonitrile is gradually improved. The culture time is 32-104 hours, the degradation rate of acrylonitrile is basically unchanged and is maintained at about 75%. The production efficiency of acrylic acid in the first 2 hours was calculated as: about 159mg per hour per gram of stem cells; the production efficiency of acrylic acid in the first 6 hours was: about 381mg per hour per gram of stem cells.
6. Detecting the degradation rate of mandelonitrile and the yield of mandelic acid
Single colonies were picked from the surface of TSA solid medium and inoculated into seed medium, shake-cultured in a constant temperature shaker (30 ℃, 150rmp) for two days, then inoculated into the activated medium of example 2 at an inoculum size of 3%, shake-cultured using shaker for 48h, separately added with mandelonitrile at concentrations of 10mM, 30mM, and 50mM, i.e., 1.33g/L, 3.99g/L, and 6.65g/L, sampled once every 12h, centrifuged using a high speed centrifuge (30 ℃, 12000rmp) for 20min, and the supernatant was taken for HPLC assay, and each experiment was repeated three times.
Detecting mandelonitrile and mandelic acid content in the reaction solution by HPLC, wherein the chromatographic column is reverse C18 column, and the mobile phase is methanol: 0.1% H 3 PO 4 The flow rate is 0.5mL/min at 40: 60, the ultraviolet detection wavelength is 220nm, the column temperature is 40 ℃, the sample injection amount is 20 mu L, and the detection time is 20 min. HPLC analysis of substrate mandelonitrile and product mandelic acid is shown in FIG. 5, where the retention time of mandelonitrile is 13.901min and the retention time of mandelic acid is 10.318 min.
The degradation rate of mandelonitrile gradually decreases with time, which may be caused by the fact that the produced mandelic acid is itself a biologically toxic organic substance, which, as mandelic acid accumulates, hinders bacterial growth. When 10mM mandelonitrile was added, full conversion doubled within 72 h; upon addition of 30mM mandelonitrile, conversion was complete within 84 h; upon addition of 50mM mandelonitrile, all was converted to mandelic acid in 108h, i.e.: the production efficiency of mandelic acid is: about 453mg per hour per gram of stem cells (fig. 6).
Example 3 degradation of other nitriles by Leuconostoc DLY11
Nitrile degradation medium: the acrylonitrile in example 2 was replaced by: mandelonitrile, 3-cyanopyridine, adiponitrile, and n-valeronitrile (20 mM each). The culture conditions were: culturing at 30 deg.C for 2-8h in a shaker at 150 rpm.
The degradation rate of different nitriles is determined by adopting a phenol-sodium hypochlorite method: sampling 1mL of the solution to perform a color reaction; taking a 10mL centrifuge tube, sequentially adding 1mL of sampling solution, 2mL of sodium phenolate solution, 3mL of sodium nitroferricyanide solution and 3mL of sodium hypochlorite solution, then supplementing to 10mL with water, reacting at 37 ℃ for 15min, and then determining OD 630nm The value is obtained. Samples were taken every 2 h.
The degradation rate of 3-cyanopyridine is about 60 percent, and the degradation rates of adiponitrile and n-valeronitrile are 53 percent and 21 percent respectively.
Sequence listing
<110> China university of Petroleum (east China)
<120> heavy metal tolerant nitrile degradation bacterium and application thereof in production of organic acid
<130> 16S rDNA sequence of Strain
<141> 2020-12-24
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1393
<212> DNA
<213> Lightbacterium (Leuconobacter sp.)
<400> 1
tgcagtcgaa cgctgaagtc agagcttgct ctggtggatg agtggcgaac gggtgagtaa 60
cacgtgagta acctgcccct gactctggga taagcgctgg aaacggtgtc taatactgga 120
tatgagcaac ggtcgcatgg cctgttgttg gaaagattta tcggttgggg atggactcgc 180
ggcctatcag cttgttggtg aggtaatggc tcaccaaggc gacgacgggt agccggcctg 240
agagggtgac cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag 300
tggggaatat tgcacaatgg gcgcaagcct gatgcagcaa cgccgcgtga gggatgacgg 360
ccttcgggtt gtaaacctct tttagtaggg aagaagcgta agtgacggta cctgcagaaa 420
aagcaccggc taactacgtg ccagcagccg cggtaatacg tagggtgcaa gcgttgtccg 480
gaattattgg gcgtaaagag ctcgtaggcg gcttgtcgcg tctgctgtga aatcccggag 540
ctcaactccg ggcctgcagt gggtacgggc aggctagagt gcggtagggg agattggaat 600
tcctggtgta gcggtggaat gcgcagatat caggaggaac accgatggcg aaggcagatc 660
tctgggccgt aactgacgct gaggagcgaa agcatgggga gcgaacagga ttagataccc 720
tggtagtcca tgccgtaaac gttgggaact agatgtaggg cctgttccac gggttctgtg 780
tcgtagctaa cgcattaagt tccccgcctg gggagtacgg ccgcaaggct aaaactcaaa 840
ggaattgacg ggggcccgca caagcggcgg agcatgcgga ttaattcgat gcaacgcgaa 900
gaaccttacc aaggcttgac atattcgaga acgctctaga aatagagaac tctttggaca 960
ctcgtttaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt cggttaagtc 1020
cggcaacgag cgcaaccctc gtcctatgtt gccagcacgt tatggtggga actcatggga 1080
tactgccgtg gtcaacacgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg 1140
tcttgggctt cacgcatgct acaatggccg gtacaatggg ctgcgatacc gcgaggtgga 1200
gcgaatccca aaaagccggt ctcagttcgg attggggtct gcaactcgac cccatgaagt 1260
cggagtcgct agtaatcgca gatcagcaac gctgcggtga atacgttccc gggccttgta 1320
cacaccgccc gtcaagtcat gaaagtcggt aacacccgaa gccgatggcc taacccttgt 1380
ggagggagtc gtc 1393
Claims (4)
1. A strain of heavy metal tolerant nitrile degradation and mandelic acid, acrylic acid producing bacterium Leuconostoc (Leuconobacter sp.) DLY11 is characterized in that: the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (the address: China, Beijing City, Tokyo ward, West Lu No. 1 institute of microbiology No. 3, China academy of sciences; zip code: 100101) 6.17.2020, and the preservation number is CGMCC No: 1.16127.
2. the leucine bacterium (Leucobacter sp.) of claim 1 having a CGMCC No: 1.16127 in the degradation of nitrile compounds, characterized in that:
the preparation method of the culture medium for degrading the nitrile compounds, the degradation substrate and the culture conditions are respectively as follows:
(1) the formula of the activation medium is as follows: tryptone soy broth 30g in ddH 2 Dissolving O, metering to 1L, and adjusting the pH value to 7.0;
(2) the preparation method of the basic culture medium for degrading the nitrile compounds comprises the following steps: dissolving 2.0g of disodium hydrogen phosphate, 1.0g of potassium dihydrogen phosphate, 0.5g of yeast powder, 0.2g of magnesium sulfate and 0.03g of ferrous sulfate in 800mL of Na 2 HPO 4 /NaH 2 PO 4 Buffer, then add glycerol 3.25mL and mix well, then add Na 2 HPO 4 /NaH 2 PO 4 The buffer solution is added to the volume of 1L;
(3) the degradation substrate is: any one of mandelonitrile, acrylonitrile, 3-cyanopyridine, adiponitrile and n-valeronitrile, wherein the addition amount of the substrate is 20 mM;
(4) the culture conditions are as follows: the culture was carried out at 30 ℃ in a shaker at a rotation speed of 150 rpm.
3. The leucine bacterium (Leucobacter sp.) of claim 1 having a CGMCC No: 1.16127A process for the production of acrylic acid, characterized in that:
(1) preparing a bacterial suspension: inoculating seed solution of Leuconostoc DLY11 to basic culture medium containing 10mM acrylonitrile for degrading nitrile compound of claim 2, culturing for 48 hr under shaking, collecting thallus precipitate and preparing bacterial suspension;
(2) acrylic acid production from acrylonitrile: the basic culture medium for nitrile degradation according to claim 2 containing 20mM acrylonitrile was added to the suspension and cultured at 30 ℃ under shaking at 150rpm for 104 hours. Sampling 5mL each time for detection; 20mM acrylonitrile was added to the culture system after each sampling.
4. The leucine bacterium (Leucobacter sp.) of claim 1 having a CGMCC No: 1.16127A process for the production of mandelic acid, characterized in that:
(1) preparing a bacterial suspension: selecting a single colony, inoculating the single colony into an activation culture medium, carrying out shaking culture in a constant temperature shaking table at 30 ℃ and 150rmp for 48h, repeating the activation step with the inoculum size of 3%, collecting thalli and preparing bacterial suspension;
(2) the mandelonitrile is used for producing mandelic acid: the basic culture medium for nitrile degradation according to claim 2, wherein the bacterial suspension and mandelonitrile are added to the culture medium at a concentration of 10mM, 30mM, or 50mM, respectively, i.e., 1.33g/L, 3.99g/L, or 6.65g/L, and the mixture is subjected to shaking culture at 30 ℃ and 150 rpm.
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