CN111996138B

CN111996138B - Reduced strain Ylb10 and application thereof in reduction of Cr (VI)

Info

Publication number: CN111996138B
Application number: CN202010797607.2A
Authority: CN
Inventors: 吕育财; 左群; 郭金玲; 任立伟; 龚大春
Original assignee: China Three Gorges University CTGU
Current assignee: China Three Gorges University CTGU
Priority date: 2020-08-10
Filing date: 2020-08-10
Publication date: 2022-03-08
Anticipated expiration: 2040-08-10
Also published as: CN111996138A

Abstract

The invention provides a strainAlicycliphilusAnd sp.YLB10, which is preserved in China Center for Type Culture Collection (CCTCC) M2020415 at 8 months and 7 days in 2020, and the preservation address is Wuhan university in China. The application of the strain Ylb10 in repairing Cr (VI) heavy metal pollution and reducing Cr (VI) into Cr (III). The strain has better Cr (VI) reducing capability, and 96.45 percent of Cr (VI) is reduced within 18 hours when the concentration of environmental Cr (VI) is 50 mg/L. When the concentration of the environmental Cr (VI) is 200mg/L, the reduction rate of the Cr (VI) in 60 hours reaches 99.06 percent. The pH value of the optimal reduced Cr (VI) of the strain is 8-9, and the culture condition is static culture. The strain can utilize amino acids such as L-alanine, L-aspartic acid, L-glutamic acid, L-serine, etc., and organic acids such as alpha-hydroxy-butyric acid, L-lactic acid, propionic acid, acetic acid, etc. Ylb10 bacteria is an effective hexavalent chromium reducing bacteria, and can be applied to the field of biological remediation of water samples and soil polluted by chromium.

Description

Reduced strain Ylb10 and application thereof in reduction of Cr (VI)

Technical Field

The invention relates to a strain Alicyclophilius sp.YLb10 and application thereof in reduction of Cr (VI), belonging to the technical field of biology.

Background

In recent years, chromium salts, leathers, and textile printing have been accompanied byAnd electroplating, etc., are involved in the development of the chromium industry, the occurrence of chromium (Cr) pollution of soil is increasing, environmental pollution caused by chromium is also becoming more serious, and the health of human beings is also becoming threatened due to bio-amplification. Cr (VI) is easily dissolved in water and strong in oxidation, damages to nervous system, liver, kidney and the like can be caused by long-term exposure of human bodies to heavy metal chromium pollution, the Cr (VI) is listed as carcinogenic substance, skin contact can cause allergy or hereditary genetic defect, and Cr (VI) has lasting harm to the environment. Cr (III) has low solubility and mobility, is a necessary trace element for human bodies and animal bodies, and is mainly involved in the metabolic process of sugar and fat of the human bodies. Therefore, the treatment of Cr (VI) pollution, and the reduction of Cr (VI) to Cr (III) is the main way. The microorganism can continuously grow and reproduce, so that the method has strong environment adaptation capability, and the method for treating heavy metal chromium pollution by using the microorganism has the characteristics of low investment cost, low energy consumption, no secondary pollution and the like. The microbiological method opens up a new field with great potential for the treatment of chromium (VI) polluted soil and industrial chromium (VI) containing wastewater. Many microorganisms capable of repairing Cr (VI) contamination are currently isolated. Including Pseudomonas mandelii, Bacillus pumilus, Cellulosimicrobium celllilans, sulfate-reducing bacteria, Desulfovibrio deguelcans, Mor ganella Mor ganii, etc. The repairing mode is mainly that the microorganism reduces Cr (VI) into Cr (III), and a bacterial enzyme system with NADH as a reducing agent converts the Cr (VI) into soluble NAD⁺-cr (III) complex, chromate reduced to soluble chromium (III) complex. However, cr (vi) has a toxic effect on microorganisms, and many microorganisms cannot effectively reduce chromium or grow in a high-concentration cr (vi) environment, so that the isolation of cr (vi) reducing strains with high performance from the environment or the acquisition of cr (vi) reducing strains with high performance by genetic engineering means is still an important issue in the field of heavy metal bioremediation. The research aims at obtaining effective Cr (VI) reducing strains, separates a Cr (VI) reducing strain from a group of floras, and researches the Cr (VI) reducing capability, the optimal reducing condition and the substrate metabolism characteristics of the strain.

Disclosure of Invention

According to the technical scheme, a Cr (VI) reducing strain Ylb10 is successfully separated from YEM001 flora and is identified as gram-negative denitrifying Alicyclophilius densificans. The strain Alicyclophilius sp.YLb10 is preserved in China Center for Type Culture Collection (CCTCC) at 8 months and 10 days in 2020, the preservation number is CCTCC M2020415, and the classification name is as follows: alicylilus dentifrians YLb10 with the storage address of Wuhan university in China.

YEM001 flora is a group of flora with Cr (VI) reducing capability obtained by taking Soxhlet sediment of the university of Sanxia as an initial bacterial source and carrying out enrichment culture for 5 generations by using a PCS-Cr culture medium in 2018 in 9 months.

And (3) separating the Cr (VI) reducing strain by using a gradient dilution coating plate method by using YEM001 as a bacterial source, and purifying to finally obtain a pure culture strain Ylb10 with Cr (VI) capacity. The culture medium used for separation and culture is a PCS-Cr culture medium, and comprises the following main components: peptone 5g/L, yeast extract 1g/L, sodium chloride 5g/L, pH 8.5-9.0, K₂Cr₂O₇100 mg/L. The strain is cultured biochemically at 28 ℃. Solid medium, 1.5% agar powder was added.

The reduced strain Ylb10 is preserved in China center for type culture Collection (CCCCTCC) at 8 months and 7 days in 2020, and the preservation address is university of Wuhan, China.

The application of the strain Ylb10 in reducing Cr (VI) into Cr (III).

Preferably, the reduced strain Ylb10 is applied to the reduction of Cr (VI) into Cr (III) in water samples or soil.

The strain Ylb10 can be used for growth of amino acids and organic acids in water samples or soil.

The amino acid comprises L-alanine, L-aspartic acid and L-glutamic acid, and the organic acid comprises any one or combination of more of alpha-hydroxy-butyric acid, L-lactic acid, beta-hydroxy-D, L-butyric acid, alpha-keto-butyric acid, propionic acid and acetic acid. But shows no metabolic capability on various sugars such as glucose, galactose, mannose, sucrose, lactose, dextrin and maltose.

The reduced strain Ylb10 is applied to reducing Cr (VI) into Cr (III) in a water sample under a standing condition.

The pH value of the environment of the reduced strain Ylb10 is 8-9 in the application process of reducing Cr (VI) into Cr (III).

Ylb10 strain can reduce 100mg/L Cr (VI) by 98.70% in 36 hours under the condition of static culture. In contrast, the Ylb10 strain biomass rapidly grows under the condition of shaking table (aerobic) but Cr (VI) is difficult to reduce, and 100mg/L of Cr (VI) is reduced by only 18.24% in 72 hours.

Ylb10 has high Cr (VI) reducing power. At concentrations of Cr (VI) of 50, 100, 200mg/L and 300mg/L, the reduction rates of Cr (VI) were 96.45%, 93.83%, 99.06% and 2.15% respectively at 18, 24, 60 hours and 84 hours. Ylb10 the best growth pH of the bacteria is 7, and the best pH for reducing Cr (VI) is 8-9.

Drawings

Fig. 1 is a morphology of Ylb10 under different circumstances, wherein the growth morphology of a. yleb 10 bacteria on PCS-Cr medium is the gram stain of b.yleb 10 bacteria.

FIG. 2 is a phylogenetic tree based on sequencing of Ylb10 bacterial 16S rDNA.

FIG. 3 shows the growth curve of Ylb10 bacteria under chromium-free conditions.

FIG. 4 is a graph comparing the resting and shake culture conditions of Ylb10 bacteria with hexavalent chromium reduction, wherein A is a growth graph of Ylb10 under the resting and shake culture conditions, and B is a graph showing the reduction effect of Ylb10 on Cr (VI) under the resting and shake culture conditions.

FIG. 5 shows the effect of different starting concentrations of hexavalent chromium on the reduction of Cr (VI) by Ylb10 bacteria.

FIG. 6 shows the effect of different pH on Ylb10 growth and ability to reduce Cr (VI), where A is the effect on Ylb10 growth under different pH conditions, B is the effect on Ylb10 broth pH during growth, and C is the effect on Cr (VI) reducing ability under different pH conditions.

Detailed Description

Example 1

Ylb10 separation and purification

YEM001 is a group of floras capable of effectively reducing Cr (VI), and is obtained by taking seeking brook sediment of the university of three gorges as a bacterial source and carrying out enrichment culture on 5 generations by using PCS culture solution (containing Cr (VI)).

Taking YEM001 flora as a separating bacterial source, separating Cr (VI) reducing strain by using a gradient dilution coating plate method, and purifying to finally obtain pure culture strain Ylb10 with Cr (VI) capacity. The culture medium used for separation and culture is a PCS-Cr culture medium, and comprises the following main components: peptone 5g/L, yeast extract 1g/L, sodium chloride 5g/L, pH 8.5-9.0, K₂Cr₂O₇100 mg/L. The strain is cultured biochemically at 28 ℃. On the solid medium, 1.5% agar powder (Solebao, Beijing) was added.

The strain morphology and gram stain results of the obtained strain Ylb10 are shown in FIG. 1 the results show that the strain can grow on solid media containing Cr (VI). The colonies were clear, round and slightly smaller (FIG. 1-A). Gram stain red, belonging to gram-negative bacteria (FIG. 1-B).

Example 2

Ylb10 phylogenetic analysis of Strain

Ylb10 bacteria are cultured for about 48 hours, 3OD bacteria are collected, total DNA of the bacteria is extracted, and the obtained DNA is subjected to 16S rDNA sequencing analysis. The sequencing results were compared to the existing 16S rDNA sequences in the NCBI database and phylogenetic tree analysis was performed using MEGA X, and the constructed phylogenetic tree is shown in FIG. 2. The results showed that the 16S rDNA sequence of this strain had 98.07% similarity to the 16S rDNA sequence of the A.denitrificans Alicyclophilus denitificans in the database. The strain was identified as Alicyclophilius dentifricans.

Example 3

Growth characteristics of Ylb10 bacterium

Ylb10 shows the growth tendency of bacteria in PCS medium without Cr (VI) as shown in FIG. 3. Ylb10 was inoculated into fresh PCS broth with an initial OD of 0.15, and the strain started to revive growth in a very short time and ended the exponential growth phase within 40h, entering the stationary phase of growth. 87h enter the decline phase. The bacteria have relatively fast growth capacity, which is favorable for treating environmental pollutants.

Example 4

Ylb10 analysis of Strain Using substrate

The substrate used by Ylb10 was analyzed using Biolog's GEN III identification plate. Ylb10 cells were activated in TSA medium, and colonies 3mm in diameter were picked up from the agar plate where cells grew using an Inoculatorz swab and added to the IF-A inoculum solution to give a cell concentration of 90% to 98% T. Adding the bacterial suspension into all the holes of the GEN III identification plate according to the amount of 100 mu L per hole; after the lid of the GEN III microplate was closed, the GEN III plate inoculated with Ylb10 cells was incubated at 33 ℃ for 24 hours, and then the color change of the reaction of different substrates on the GEN III plate was measured to characterize the use of Ylb10 for different substrates (see Table 1). Ylb10 the main substrates used are: methyl pyruvate, L-alanine, alpha-hydroxy-butyric acid, L-lactic acid, beta-hydroxy-D, L-butyric acid, L-aspartic acid, alpha-keto-butyric acid, L-glutamic acid, propionic acid, acetic acid, L-serine, and exhibit no metabolic capacity for various sugars such as glucose, galactose, mannose, sucrose, lactose, dextrin, maltose, and trehalose.

TABLE 1 results of carbon Source utilization by Strain Ylb10

Note: "+" positive, "-" negative

Growth characteristics of Ylb10 bacterium

Example 5

Effect of oxygen on Ylb10 reduction of Cr (VI)

Ylb10 was inoculated into fresh PCS-Cr (100mg/L Cr (VI)) culture solution with an initial culture concentration of 02OD, and cultured in a biochemical incubator and a constant temperature shaker (200r/min) at 28 ℃ under standing and shaking conditions, respectively. Comparison Ylb10 Biomass (OD) at different oxygen content₆₀₀) And efficiency of reducing cr (vi) (fig. 4). The results showed Ylb10 that the biomass was significantly higher in the shake condition than in the stationary culture, and that the OD of the strain increased with the increase of the culture time in the shake culture condition and reached 2.0 at 72 hours, whereas the OD increased slowly in the stationary culture condition and reached only 0.58 at 72 hours. It can be seen that Ylb10 can grow under the condition of different oxygen content, and the sufficient oxygen is more favorable for growth of Ylb10 (FIG. 4-A).

In contrast to the biomass trend, Ylb10 showed very poor Cr (VI) reduction capacity under shaking conditions, and 100mg/L Cr (VI) was reduced by 18.24% over 72 hours. While in the quiescent state, Ylb10 was able to reduce 98.70% over 36 hours (FIG. 4-B). It can be seen that Ylb10 has low ability to reduce Cr (VI) under sufficient oxygen conditions, and oxygen has an inhibitory effect on Ylb10 reduction of Cr (VI). And the Cr (VI) is reduced with high efficiency under anaerobic condition.

Example 6

Effect of different Cr (VI) concentrations on Cr (VI) reduction of Ylb10 bacteria

Ylb10 bacteria were inoculated to a medium containing 0, 50, 100, 200mg/L and 300mg/L K, respectively₂Cr₂O₇In the PCS culture solution of (1), the initial culture OD thereof₆₀₀0.2, the Cr (VI) concentration and OD in the sample were measured periodically₆₀₀The value of (c).

Ylb10 ability to reduce Cr (VI), the results are shown in FIG. 5. When the concentrations of Cr (VI) in the culture broth were 50, 100, 200mg/L and 300mg/L, the reduction rates of Cr (VI) at 18, 24, 60 hours and 84 hours were 96.45%, 93.83%, 99.06% and 2.15%, respectively. The time required for the total reduction is extended with increasing cr (vi) concentration. When the concentration of Cr (VI) in the culture solution reaches 300mg/L, the strain can not reduce Cr (VI). It can be seen that Ylb10 bacteria have high Cr (VI) reducing ability.

Example 7

Effect of different initial pH on Ylb10 reduction of Cr (VI)

The pH had a significant effect on Ylb 10's ability to grow and reduce cr (vi). Ylb10 were cultured in PCS-Cr (100mg/L Cr (VI)) at initial pH's of 5, 6, 7, 8, and 9, respectively, and it was found that Ylb10 was able to grow in an environment at pH's of 7, 8, and 9, with an optimum growth pH of 7. Whereas when the pH was less than 6, the growth of the strain was severely inhibited (FIG. 6-A). During the cultivation, the growth of the strain affects the pH of the culture broth. When the initial pH of the culture was 7 and 8, the pH of the culture broth increased with the extension of the culture time, while when the initial pH was 9, the pH of the culture broth decreased, and finally, the pH tended to stabilize between pH 8 and 9 (FIG. 6-B). The culture solution has the capacity of metabolizing acid, and the capacity of metabolizing ammonia and other substances is weak. This corresponds to Ylb10 detection of substrate utilization.

The pH of the environment also affects the Ylb10 reduction efficiency for cr (vi). When the pH of the culture broth was 5 or 6, the reducing ability of Cr (VI) was not exhibited, probably due to the inhibition of the growth of the microorganism. The ability to reduce Cr (VI) was demonstrated at

pH

7, 8, and 9. And Ylb10 reduced Cr (VI) more efficiently at

pH

8 and 9, with 98.47% and 95.54% for 36 hours, and 94.78% at pH 7 for 48 hours (FIG. 6-C). Therefore, the optimum pH range for the Ylb10 strain hexavalent chromium reduction is 8-9. Under the alkalescent condition, Ylb10 bacteria are more favorable for reducing Cr (VI).

Claims

1. The application of a reduced strain Ylb10 in repairing Cr heavy metal pollution and reducing Cr (VI) into Cr (III), wherein the strain is classified and named as: denitrifying Alicyclophilic bacterium (a)Alicycliphilus denitrifians) Ylb10, preserved in China Center for Type Culture Collection (CCTCC) at 8 months and 10 days in 2020, with the preservation number of CCTCC M2020415 and the preservation address of Wuhan university, China; the Cr (VI) concentration is less than 200mg/L, and the environmental pH is 7-9.

2. The use of the strain Ylb10 in repairing Cr heavy metal pollution in water or soil, and reducing Cr (VI) to Cr (III) according to claim 1.

3. The use according to claim 1, wherein strain Ylb10 is grown using an amino acid or an organic acid in the environment; the amino acid comprises L-alanine and L-glutamic acid, and the organic acid comprises any one or combination of more of alpha-hydroxy-butyric acid, L-lactic acid, beta-hydroxy-D, L-butyric acid, alpha-keto-butyric acid, propionic acid and acetic acid.

4. The use according to claim 1, wherein strain Ylb10 reduces Cr (VI) to Cr (III) in a water sample under standing conditions.