CN108102942A - One plant of bacterial strain and its application for being used to purify molasses alcohol waste water - Google Patents

Abstract

The present invention relates to one plant of microbial strains and its applications, and in particular to a kind of lysine bacillus (Lysinibacilus sp.) S6 and its application in molasses alcohol waste water belong to technical field of microbe application.Being the object of the present invention is to provide a kind of bacterial strain can be in microbial strains lysine bacillus (Lysinibacilus sp.) S6 grown using molasses alcohol waste water as sole carbon source, and its corresponding condition of culture and in the case of being disclosed in and not adding any trace element, COD removal effects are ideal, and 48h is up to 64.22%.In addition, the bacterium also has certain denitrification dephosphorization function.

Description

A bacterial strain for purifying molasses alcohol wastewater and its application

technical field

The invention belongs to the field of microbes, in particular to a bacterial strain for purifying molasses alcohol wastewater and its application.

Background technique

Molasses, a by-product of sugar production, is often used for fermentation to produce alcohol. During the production of alcohol, a large amount of high-concentration organic wastewater is released, which is called molasses alcohol wastewater. Direct discharge of a large amount of wastewater without treatment will cause eutrophication of water bodies and damage water bodies and terrestrial ecosystems. Molasses alcohol wastewater is directly used for agricultural irrigation, which will cause soil acidification and compaction. If it is used to irrigate sugarcane fields, it will increase the yield of sugarcane, but will reduce the sugar content in sugarcane. The current methods for treating molasses alcohol wastewater include physical and chemical methods and biological treatment methods. Compared with physical and chemical methods, biological treatment methods are safer and more efficient, have a shorter cycle time, lower cost, and will not cause secondary pollution. Biogas energy can also be generated during the treatment process, and the treated bacteria are good feed protein. These advantages make the biological treatment method become the focus and difficulty of current research. For biological treatment methods, good microbial strains are the key to ensuring the success of wastewater biological treatment, and screening excellent microbial strain resources from the environment is an important way. Therefore, in recent years, researchers have screened out microorganisms (bacteria, fungi, etc.) that have high biodegradability for molasses alcohol wastewater as the starting point for research. Wang Weijia isolated two strains of bacteria PSB-C and PSB-D from the oxidation pond of molasses alcohol wastewater, which belong to the genus Rhodopseudomonas of photosynthetic bacteria. Under the optimal reaction conditions (COD concentration is 20800mg/L, reaction temperature is 30℃, reaction time is 3d), the COD removal rates of the two strains to wastewater are 55% and 54% respectively. Liu Jianfu screened out 8 strains of bacteria from the reactor granular sludge of UASB, namely Pyrobacterium, Corynebacterium, Exiguobacterium, Lactobacillus, Cellulomonas, Propionibacterium, and Rhododendron , when the wastewater COD concentration is 24000mg/L, the COD removal rate is 25.1% when treated at 37°C (Question: see the literature, the COD removal rate of the compound bacterial agent is over 90%, it is best to provide literature information, the information corresponds more be more specific). The bacterial flora enriched by Fan Yanxia from the sludge of the IC reactor belonged to 7 orders of Erysipelotrichales, Clostridiales, Lactobacillales, Xanthomonadales, Burkholderiales, Enterobacteriales, and Bacteroidales. The removal rate of COD is 38.5%. The bacterial flora DMC obtained by Mohana was inoculated into the alcohol wastewater added with glucose and inorganic salt molasses at the same time, and treated at 37°C for 4 days, the COD removal rate was 51%, and the wastewater decolorization rate was 67%. Pseudomonas aeruginosa PAO1, stenotrophomonas maltophila, Proteus mirabilis. chopra treated molasses alcohol wastewater with white rot fungi and C. versicolor, added glucose and peptone as nutrients, and the removal rates of COD and chroma were both 53% after 8 days. Ghosh used Pseudomonas putida and Aeromonas sp. to treat molasses alcohol wastewater in two steps, adding 1% glucose to the wastewater. Finally, after 24 hours of treatment, the COD removal rate of Pseudomonas putida was 44.4%, the colorimetric removal rate was 60%, and the COD removal rate of Aeromonas was 44.4%.

At present, there is no report on the application of Bacillus lysinus to reduce the COD of molasses alcohol wastewater.

Contents of the invention

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a bacterial strain for purifying molasses alcohol wastewater.

Another object of the present invention is to provide the application of the bacterial strain for purifying molasses alcohol wastewater.

The object of the present invention is achieved through the following technical solutions: a bacterial strain for purifying molasses alcohol wastewater, called Lysinibacilus sp. S6, with a preservation number of CCTCC M2017085, preserved on March 6, 2017 at the Chinese Type Culture Collection at Wuhan University, Wuhan, Hubei, China.

The application of the bacterial strain for purifying molasses alcohol wastewater in the treatment of molasses alcohol wastewater preferably comprises the following specific steps:

(1) inoculating the described bacterial strain for purifying molasses alcohol wastewater in the fermentation medium to ferment;

(2) Inoculate the bacterium liquid obtained after the fermentation of step (1) into molasses alcohol wastewater, and perform fermentation treatment.

The composition of the fermentation medium described in the step (1) is preferably as follows: the consumption of molasses alcohol waste water is to calculate with fermentation medium COD being 100000～110000mg/L, the MgSO of 1mol/L 2.00mL, the CaCl of 1mol/LO .10mL, 200mL of 5×M9 salt solution, dilute to 1000mL, pH 7.0. When the COD concentration of the molasses alcohol wastewater used is high, it is necessary to use distilled water for constant volume.

The amount of molasses alcohol wastewater in the fermentation medium is calculated based on the COD of the fermentation medium being 105851.15 mg/L.

The composition of the 5×M9 salt solution is as follows: Na2PO4·7H2O 12.80g, KH2PO4 3.00g, NaCl 0.50g, NH4Cl 1.00g, distilled water to 200mL.

The fermentation conditions are preferably 37° C. and 200 rpm/min rotating speed in an aerobic environment shaker.

Compared with the prior art, the present invention has the following advantages and effects:

The bacterial strain provided by the invention can grow with molasses alcohol wastewater as the only carbon source, and the COD concentration of molasses alcohol wastewater is 105851.15 mg/L, the temperature is about 37 ° C, and no trace elements are added. Ideally, it can reach 64.22% in 48 hours. In addition, the bacteria also has a certain denitrification and dephosphorization effect.

Description of drawings

Fig. 1 is the morphological photo figure of the bacterial strain that is used for purifying molasses alcohol waste water that the present invention provides; Wherein, figure (A) is the photomicrograph figure (100 ×/1.25oil) of Gram stain, and figure (B) is bacterium colony photo graph.

Fig. 2 is the phylogenetic tree of the bacterial strain that is used for purifying molasses alcohol waste water that the present invention constructs with multiple sequence alignment software MEGA5.0, wherein, branch value represents the confidence degree after 1000 calculations, scale bar represents genetic distance, and scale bar represents genetic distance.

Fig. 3 is the growth curve of the bacterial strain used for purifying molasses alcohol wastewater provided by the present invention.

Fig. 4 is a graph showing the detection results of removing COD from molasses alcohol wastewater by the bacterial strain used for purifying molasses alcohol wastewater provided by the present invention.

Fig. 5 is a graph showing the detection results of removing ammonia nitrogen from molasses alcohol wastewater by the bacterial strain used for purifying molasses alcohol wastewater provided by the present invention.

Fig. 6 is a graph showing the detection results of removing total phosphorus from molasses alcohol wastewater by the bacterial strain used for purifying molasses alcohol wastewater provided by the present invention.

Detailed ways

The present invention will be described in further detail below in conjunction with the examples and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

The used culture medium of the present invention is as follows:

The acclimatization medium is: molasses alcohol wastewater and filtered pig manure water are prepared according to the COD ratio of 3:2 to make a mixed culture solution with a total COD concentration of 4000mg/L. The mixed culture medium and the rich medium were mixed at a volume ratio of 1:4 (acclimation medium A), 2:4 (acclimation medium B), 3:4 (acclimation medium C), 4:0 (acclimation medium D ) and mix evenly, adjust the pH to 7.00, and sterilize at 115° C. for 15 minutes with low-pressure damp heat to obtain acclimatization media A, B, C, and D in turn.

The rich medium is: peptone 10.00g, beef extract 3.00g, yeast powder 5.00g, glucose 3.00g, sodium chloride 3.00g, dipotassium hydrogen phosphate 2.50g, soybean broth 3.00g, water to 1000mL, pH7. 00.

LA medium is: tryptone 10.00g, yeast powder 5.00g, sodium chloride 5.00g, agar 11g, water to 1000mL, pH 7.00.

The fermentation liquid medium is: the amount of molasses alcohol wastewater (COD=105851.15mg/L) is calculated based on the COD of the fermentation medium being 105851.15mg/L, 1mol/L MgSO ₄ 2.00mL, 1mol/L CaCl ₂ 0.10mL , 5×M9 saline solution 200 mL, total volume 1000 mL, pH 7.0.

The composition of 5×M9 salt solution is as follows: Na ₂ HPO ₄ ·7H ₂ O 12.80g, KH ₂ PO ₄ 3.00g, NaCl 0.50g, NH ₄ Cl 1.00g, distilled water to 200mL.

The fermented solid medium is: adding agar to the fermented liquid medium, and the concentration of the agar is 1.1% (w/v).

Example 1

(1) Acclimatization culture: extract sludge from the bottom of the IC reactor (effective volume is about 20L) from operation to the stable period (unacclimated sludge is taken from the bottom of the IC reactor in Guangxi Guitang Co., Ltd., rich in Microbial flora), mash the activated sludge with a sterile glass rod, mix it evenly, let it stand for 5 minutes, and take its supernatant. Inoculated into rich medium with 5% (w/v) inoculation amount, placed in an aerobic (37° C., 200 rpm shaking culture) environment and cultured for 2 days. Inoculated into acclimatization medium A with an inoculation amount of 5% (v/v), and cultured for 2 days under the same conditions. Shake well and transfer to acclimatization medium B at an inoculum size of 5% (v/v), and culture for 3 days under the same conditions. Shake well and transfer to acclimatization medium C at an inoculum size of 5% (v/v), and culture for 3 days under the same conditions. Finally, shake well and transfer to the acclimatization medium with only molasses alcohol wastewater and pig manure water mixed solution with 5% (v/v) inoculum size for cultivation (ie acclimatization medium D) for 3 days, and the acclimatization stage ends.

(2) Separation and purification culture: samples are taken from the fermentation broth in the final stage of acclimatization, and the samples are serially diluted by the dilution coating plate method and then coated on LA plates; 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^{- 6} Four gradient samples, set three parallels for each gradient, smear the plate with 1% inoculum, set a blank control, place it at 37°C for shaking culture and observe the daily growth; when the colony abundance on the plate is more, observe the colony Morphology, use the plate streaking method to pick out the strains with different shapes on the plate as much as possible, then transfer them to fresh rich medium, and pick out single colonies continuously for streaking purification, and place them in an aerobic environment at 37°C , 150 ~ 200rpm culture, at least repeat the above marking operation 3 times or more.

(3) Morphological identification: Gram-negative/positive bacteria were identified by the Gram staining method for isolated and purified cultured strains. The strains were cultured on LA medium at 37°C for 1-2 days, and then the colony size, shape, color and surface characteristics were observed and described. Gram staining and colony morphology are shown in Figure 1. The Gram-stained strain was red, rod-shaped, and determined to be a negative rod-shaped bacterium. When growing on LA solid medium, the single colony is round, flat, with uneven edges, wet, smooth surface, easy to pick, and both sides are light brown.

(4) Molecular biological identification: Using genomic DNA as a template, 16S rDNA was amplified by PCR using universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-TACGGTTACCTTGTTACGACT T-3'). The products were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. The sequence obtained by sequencing is shown below. The 16S rDNA fragment obtained by sequencing was compared with the known bacteria in the Genbank database to obtain the information of the sample at the level of genus and classification. A phylogenetic tree was constructed using the multiple sequence alignment software MEGA5.0, as shown in Figure 2.

GCTGGCTCCAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGACTTTATCGGATTAGCTCCCTCTCGCGAGTTGGCAACCGTTTGTATCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTAAATGATGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGTTGCCCCCGAAGGGGAAACTATATCTCTACAGTGGTCAACGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCAAATCTCTACGCATTTCACCGCTACACTTGGAATTCCACTTTCCTCTTCTGCACTCAAGTCCCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAAGGACCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTAATAAGGTACCGTCAAGGTACAGCCAGTTAC TACTGTACTTGTTCTTCCCTTACAACAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGGCTTTCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGCCCATCCTATAGCGACAGCCGAAACCGTCTTTCAGTCTTTCACCATGAAGCAAAAGAGATTATTCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAAACTATAGGGTAGGTTGCCCACGTGT。

After identification, it was determined that the isolated bacteria belonged to Lysinibacilus sp., named it Lysinibacilus sp. S6, and the preservation number was CCTCC M2017085. It was preserved in Wuhan, Hubei, China on March 6, 2017. Chinese Type Culture Collection Center of Wuhan University.

Example 2

Take the Lysinibacilus sp. S6 strain to measure the growth curve: inoculate the bacteria on the LA plate in the rich medium, and cultivate them in an aerobic environment shaker at 37°C with a rotation speed of 200rpm/min. It can be observed that the growth and reproduction of bacteria has a certain regularity, and the OD ₆₀₀ value is used as the vertical axis, and the culture time is used as the horizontal axis to draw a growth curve.

The growth curve in the rich medium is shown in Figure 3. It can be seen that the domesticated microorganisms have no adjustment period or the adjustment period is very short, and enter the logarithmic phase soon after inoculation, and the microorganisms enter the stable phase after 48 hours.

Example 3

The acclimatization medium for Lysinibacilus sp. S6 strain was taken and cultivated at 200 rpm for 3 days. Then inoculate the aerobic bacteria liquid into the fermentation liquid medium with an inoculation amount of 1%, and culture them in an aerobic shaker at 37°C and 200 rpm; measure their COD, ammonia nitrogen, and total phosphorus every day for a total of 6 days. Observe the changes of COD, ammonia nitrogen and total phosphorus.

The COD removal effect in the fermentation medium is shown in Figure 4: the COD removal rate on the 1st day was 45.06%, and the COD removal rate on the 2nd day was 64.22%. With the prolongation of treatment time, the increase of COD removal rate showed a downward trend. After 6 days of fermentation treatment, the COD removal rate only reached 71.00%. In the 3rd to 6th days, as the fermentation time prolongs, the nutrients in it decrease, and the activity of some bacteria may decrease.

The removal effect of ammonia nitrogen in the fermentation medium is shown in Figure 5: the removal rate of ammonia nitrogen gradually increases with the prolongation of the fermentation time within 1d to 4d. A maximum of 21.90% is reached at 4d. Compared with the 4th day, the removal rate of ammonia nitrogen decreased in the 5th to 6th day.

The removal effect of total phosphorus in the fermentation medium is shown in Figure 6: in the first 1d to 2d of fermentation, the microbial activity is high, the absorption effect on total phosphorus is good, and the removal rate is high. In the subsequent treatment time, the removal rate of total phosphorus decreased. Overall, the strain's removal rate of total phosphorus was less than 5%, and the removal effect was not ideal.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Guangxi University

<120> A bacterial strain used to purify molasses alcohol wastewater and its application

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<170> PatentIn version 3.5

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agagtttgat cctggctcag 20

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<223> Primer 1492r

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tacggttacc ttgttacgac tt 22

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gctggctcca aaggttacct caccgacttc gggtgttaca aactctcgtg gtgtgacggg 60

cggtgtgtac aaggcccggg aacgtattca ccgcggcatg ctgatccgcg attacktagcg 120

attccggctt catgtaggcg agttgcagcc tacaatccga actgagaacg actttatcgg 180

attagctccc tctcgcgagt tggcaaccgt ttgtatcgtc cattgtagca cgtgtgtagc 240

ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg tttgtcaccg 300

gcagtcacct tagagtgccc aactaaatga tggcaactaa gatcaagggt tgcgctcgtt 360

gcgggactta acccaacatc tcacgacacg agctgacgac aaccatgcac cacctgtcac 420

cgttgccccc gaaggggaaa ctatatctct acagtggtca acgggatgtc aagacctggt 480

aaggttcttc gcgttgcttc gaattaaacc acatgctcca ccgcttgtgc gggcccccgt 540

caattccttt gagtttcagt cttgcgaccg tactccccag gcggagtgct taatgcgtta 600

gctgcagcac taaggggcgg aaacccccta acacttagca ctcatcgttt acggcgtgga 660

ctaccagggt atctaatcct gtttgctccc cacgctttcg cgcctcagcg tcagttacag 720

accagaaagt cgccttcgcc actggtgttc ctccaaatct ctacgcattt caccgctaca 780

cttggaattc cactttcctc ttctgcactc aagtccccca gtttccaatg accctccacg 840

gttgagccgt gggctttcac atcagactta aaggaccgcc tgcgcgcgct ttacgcccaa 900

taattccgga caacgcttgc cacctacgta ttaccgcggc tgctggcacg tagttagccg 960

tggctttcta ataaggtacc gtcaaggtac agccagttac tactgtactt gttcttccct 1020

tacaacagag ttttacgatc cgaaaacctt cttcactcac gcggcgttgc tccatcaggc 1080

tttcgcccat tgtggaagat tccctactgc tgcctcccgt aggagtctgg gccgtgtctc 1140

agtcccagtg tggccgatca ccctctcagg tcggctacgc atcgtcgcct tggtgagccg 1200

ttacctcacc aactagctaa tgcgccgcgg gcccatccta tagcgacagc cgaaaccgtc 1260

tttcagtctt tcaccatgaa gcaaaagaga ttattcggta ttagccccgg tttcccggag 1320

ttatcccaaa ctataggggta ggttgcccac gtgt 1354

Claims (6)

1. a bacterial strain for purifying molasses alcohol wastewater is characterized in that: the name of the bacterial strain used for purifying molasses alcohol wastewater is lysine bacillus (Lysinibacilus sp.) S6, and the preservation number is CCTCC M2017085. Deposited on March 6, 2017 at the Chinese Type Culture Collection Center located at Wuhan University, Wuhan, Hubei, China. 2. the application of the bacterial strain for purifying molasses alcohol wastewater according to claim 1 in the treatment of molasses alcohol wastewater. 3. the application of the bacterial strain for purifying molasses alcohol wastewater according to claim 2 in the treatment of molasses alcohol wastewater is characterized in that comprising the following specific steps: (1) inoculating the described bacterial strain for purifying molasses alcohol wastewater in the fermentation medium to ferment; (2) Inoculate the bacterium liquid obtained after the fermentation of step (1) into molasses alcohol wastewater, and perform fermentation treatment. 4. the application of the bacterial strain for purifying molasses alcohol wastewater according to claim 3 in the treatment of molasses alcohol wastewater is characterized in that: The composition of the fermentation medium described in the step (1) is as follows: the consumption of molasses alcohol wastewater is calculated based on the fermentation medium COD being 100000～110000mg/L, _the MgSO of 1mol/L 2.00mL, the CaCl of 1mol/L ₂ 0.10mL, 200mL of 5×M9 salt solution, dilute to 1000mL, pH 7.0. 5. the application of the bacterial strain for purifying molasses alcohol wastewater according to claim 4 in the treatment of molasses alcohol wastewater is characterized in that: The amount of molasses alcohol wastewater in the fermentation medium is calculated based on the COD of the fermentation medium being 105851.15 mg/L. 6. the application of the bacterial strain for purifying molasses alcohol wastewater according to claim 3 in the treatment of molasses alcohol wastewater is characterized in that: The fermentation conditions are preferably 37° C. and 200 rpm/min rotating speed in an aerobic environment shaker.