TW202346568A - Novel micro bacillus YL-01 and its use for reducing hexavalent chromium having a high reducing ability of 60[mu]M/hour to hexavalent chromium - Google Patents

Abstract

The present invention discloses a novel micro bacillus YL-01 and its use for reducing hexavalent chromium. The novel micro bacillus YL-01 is deposited at the Hsinchu Food Industry Development Research Institute on November 29, 2021. The deposit number is BCRC911083. The novel micro bacillus is highly resistant to hexavalent chromium, can grow normally in a culture media containing 1500ppm hexavalent chromium, and has a high reducing ability of 60[mu]M/hour. Therefore, this novel micro bacillus YL-01 can be used to treat highly concentrated hexavalent chromium pollutions or pollutants containing hexavalent chromium.

Description

Novel Exiguobacterium YL-01 and its use in reducing hexavalent chromium

The present invention relates to novel microorganisms and their uses, in particular to a novel Exiguobacterium YL-01 and its use for reducing hexavalent chromium.

Press, metal chromium has different applications according to its different chemical value. Specifically, chromium with a chemical value of zero is used to make steel; trivalent chromium is an indispensable nutrient for the human body and will be used for Used as a pigment or dye; hexavalent chromium is also used as a compound for material processing or preservation, such as pigments in pigments or dyes; among them, hexavalent chromium is toxic, meaning that when organisms directly or indirectly When people are exposed to high concentrations of chromium in the environment, it will have adverse effects on individual health. For example, inhaling high concentrations of hexavalent chromium can cause breathing problems, and in severe cases, even lung cancer; if the diet contains hexavalent chromium, it can cause Cause gastrointestinal pain, ulcers, or anemia.

When products containing chromium are manufactured or discarded into the environment, chromium will be released into the air, soil, water and other environments. In Taiwan, the wastewater discharged from electroplating factories contains a large amount of hexavalent chromium. Therefore, in order to remove or reduce the concentration of hexavalent chromium in the wastewater, most factories currently choose to use chemical precipitation, ion exchange, and ultra-membrane filtration. , electrolysis and other methods for wastewater treatment. However, the cost of treating wastewater containing hexavalent chromium in the above methods is high, and other hazardous wastes will still be generated during the treatment process.

It can be seen from this that there is still a lack of an efficient and economically valuable method for removing hexavalent chromium pollution in the environment.

The main purpose of the present invention is to provide a novel Exiguobacterium YL-01 and its use for reducing hexavalent chromium. Specifically, the novel Exiguobacterium YL-01 disclosed in the present invention can survive in an environment containing hexavalent chromium. And reduces hexavalent chromium to trivalent chromium. Therefore, the novel Exiguobacterium YL-01 system can be used to treat hexavalent chromium in pollutants, so that the hexavalent chromium content in the final product obtained is in line with or lower than the environment. regulations to effectively treat chromium-containing pollutants without causing secondary pollution to the environment.

The reason is that, in order to achieve the above purpose, the present invention provides a novel Exiguobacterium YL-01, which is deposited at the Hsinchu Food Industry Development Research Institute, the deposit date is November 29, 2021, and the deposit number is BCRC911083, and, the The novel Exiguobacterium YL-01 is able to bind reductase through the cell membrane and reduce hexavalent chromium in a pollutant to trivalent chromium.

One embodiment of the present invention provides a composition, which contains an effective amount of novel Exiguobacterium YL-01 for adding to a pollutant to treat hexavalent chromium in the pollutant.

The composition further includes a fermentation medium, such as LB medium, for providing elements or substances required for the growth and reproduction of the novel Exiguobacterium YL-01.

Another embodiment of the present invention provides a method for treating hexavalent chromium-containing pollutants, which mainly involves adding an effective amount of novel Exiguobacterium YL-01 or a composition containing the effective amount of novel Exiguobacterium YL-01 Incubate with a pollutant for a predetermined time, so that the chromate reductase produced by the novel Exiguobacterium YL-01 can reduce the hexavalent chromium in the pollutant to trivalent chromium.

Specifically, in another embodiment of the present invention, the method for treating hexavalent chromium-containing pollutants includes the following steps:

Step a: Collect the pollutant, where the pollutant must be liquid or soil, and the concentration of hexavalent chromium in the pollutant is preferably 0 ppm and less than or equal to 1500 ppm.

Step b: Take the novel Exiguobacterium YL-01 and a fermentation medium, add them to the pollutant, and perform fermentation and culture for a predetermined time;

Step c: Produce a final product in which the hexavalent chromium content complies with regulations or is almost zero.

Among them, the fermentation culture time in step c can be changed based on the hexavalent chromium content in the pollutant, the amount of bacteria provided, the volume of the pollutant and other factors. For example, the hexavalent chromium content in the pollutant is At the high standard of 1500 ppm, the fermentation culture time is 96 hours.

In order to increase the convenience of processing the final product, the method for processing hexavalent chromium contaminants disclosed in the next embodiment of the present invention further includes a step d, which is located after the step c, and the step d is performed by The acid-base neutralization reaction adjusts the pH value of the final product and forms a solid precipitate of trivalent chromium in it.

The present invention discloses a novel Exiguobacterium YL-01 (Exiguobacterium sp. YL-01) and its use for reducing hexavalent chromium. The novel Exiguobacterium YL-01 is isolated from the soil of the Changhua River. After the genome was desequenced and compared, it was confirmed to be a novel strain that had not been discovered before. As shown in Figure 1, the 16S rRNA gene sequence is SEQ ID No.: 1 and is deposited at the Hsinchu Food Industry Development Research Institute with a deposit date of 2021. On November 29, the deposit number was BCRC911083.

The Exiguobacterium YL-01 is highly tolerant to hexavalent chromium, can grow normally in a medium containing 1500 ppm hexavalent chromium, and has efficient reducing ability, reaching 60 µM/hour. Therefore, the Exiguobacterium YL-01 can Used to treat high-concentration hexavalent chromium pollution or pollutants containing hexavalent chromium.

Furthermore, the isolation procedure of the novel Exiguobacterium YL-01 disclosed in the present invention is as follows:

A. Take 1g of soil sample from Changhua, add 100ml of deionized water, stir evenly, apply it to a solid chromium-resistant LB medium with a 10-fold serial dilution, and culture it at 30°C for 72 hours to obtain cultivable colonies. Among them, Each 100 ml of gold solid chromium-resistant LB medium contains 1 gram of trypsin, 0.5 grams of yeast extract, 1 gram of sodium chloride, 1.5 grams of agar, and contains hexavalent chromium Cr(VI) at a concentration of 1500 ppm.

B. Cultivate the solid chromium-resistant LB medium at 30°C for 72 hours through a drawing plate, screen and isolate the bacterial strain with chromium tolerance. After identification and comparison, it is confirmed that it is the novel Exiguobacterium YL disclosed by the present invention. -01.

Among them, the description of identification and comparison is as follows:

The total DNA of the isolated bacterial strain with chromium tolerance was extracted, and then the 16S rRNA gene was amplified from the total DNA, using 27F of 16S rRNA (SEQ ID No.: 2, 5'-AGAGTTTGATCCTGGCTCAG-3' ) and 1492R (SEQ ID No.: 3, 5'-GGTTACCTTGTTACGACTT-3') primer pair for polymerase chain reaction, and the amplified 16S rRNA fragment was subjected to nucleic acid sequencing. The identification results are based on the test target primers and are compared with the 16S ribosomal RNA sequences (Bacteria and Archaea) database in NCBI. The 16S RNA gene sequence data is analyzed to obtain the 16S rRNA gene sequence of the chromium-tolerant bacterial strain, such as SEQ. ID No.:1. MEGA version 11 software was used to perform multiple sequence alignment analysis (multiple sequence alignment), and the results were used to construct a genetic relationship program. The phylogenetic tree results of the identified strains are shown in Figure 2, and then whole-genome sequencing comparison was performed. , as shown in Figure 1.

The Exiguobacterium YL-01 system can reduce hexavalent chromium [Cr(VI)] with high mobility and toxicity through cell membrane-bound reductase to trivalent chromium [Cr(III) with relatively low toxicity, low solubility and mobility )]. Specifically, Exiguobacterium YL-01 can produce chromate reductase, which participates in the electron transport chain of oxidation and reduction, using chromate as the terminal electron acceptor, and these chromate reductases utilize NAD(P) H acts as an electron donor to reduce hexavalent chromium to trivalent chromium as the final product. Based on this, the Exiguobacterium YL-01 system disclosed in the present invention can be used to improve chromium wastewater through microbial remediation methods, and the final product after treatment almost does not contain hexavalent chromium or already meets the regulatory standards, that is, compared with As the final product of the traditional chemical decontamination method still contains hexavalent chromium, using Exiguobacterium YL-01 disclosed in the present invention to treat hexavalent chromium-containing pollutants can effectively reduce the impact of the final product on the environment. pollution.

In one embodiment of the present invention, the method for treating pollutants with Exiguobacterium YL-01 is to introduce the polluted water to be treated into a fermentation tank, wherein the polluted water to be treated contains high concentration (at least 300 ppm) of VI Hexavalent chromium; then add the Exiguobacterium YL-01 and culture it in LB medium or other media that can support the growth and reproduction of Exiguobacterium YL-01. Within 96 hours of culture, the hexavalent chromium in the pollutants to be treated can be reduced to Trivalent chromium; then use sodium hydroxide to adjust the pH value of the culture solution to a weak alkalinity of about 8.5, so that the trivalent chromium in the pollutants to be treated forms a precipitate: chromium hydroxide (Cr(OH) ₃ ) , to facilitate recycling.

Among them, after adding the Exiguobacterium YL-01 disclosed in the present invention, the OD600mn can be measured every 24 hours to confirm the growth concentration and reduction of hexavalent chromium of the Exiguobacterium YL-01.

In another embodiment of the present invention, a method for treating pollutants with Exiguobacterium YL-01 is disclosed, which is to directly apply the LB culture solution containing the Exiguobacterium YL-01 to the contaminated soil to be treated, so that the Exiguobacterium YL-01 directly reduces hexavalent chromium in contaminated soil; or the contaminated soil to be treated is washed with water, the eluate is collected, and then the contaminated water is treated as disclosed in the above embodiment, and the YL-01 containing Exiguobacterium 01 of LB medium was added to the eluent for culture, allowing Exiguobacterium YL-01 to reduce hexavalent chromium in the eluent.

In the embodiment of the present invention, the LB culture medium containing the Exiguobacterium YL-01 used was prepared in the following manner: 1% log-phase cultured bacterial liquid (about 10 ⁶ CFU/ml) Inoculate into the above LB medium.

The term "pollutants containing hexavalent chromium" or "pollutants" referred to in the present invention refers to water, liquid or soil contaminated by hexavalent chromium and is generally recognized by those with ordinary knowledge in the technical field to which the present invention belongs. The concentration of pollution may be based on regulations or standards set by the competent authority. For example, the soil pollution control standard for rice fields is that the total chromium concentration should not exceed 250 mg/kg; the U.S. FDA stipulates that the concentration of chromium in bottled water should not exceed 0.1 mg/L. wait.

The term "fermentation medium" as used in the present invention refers to a medium suitable for the growth and reproduction of Exiguobacterium YL-01, which contains nutrients and/or raw materials necessary for growth, such as the LB medium used in the embodiments of the present invention.

In the following, in order to illustrate the technical features and effects of the present invention, several experimental examples will be given and explained in detail with drawings.

Example 1: Analysis of chromium tolerance of Exiguobacterium YL-01

The microbacterium YL-01 disclosed in the present invention containing the microbacterium YL-01 cultured in the logarithmic phase was inoculated into the above-mentioned LB liquid culture medium without agar containing different concentrations of hexavalent chromium (500, 1000, 1500, 2000, 3000, 4000ppm). medium (pH 7.0), and culture it at 30°C and 150 rpm for 120 hours. During the culture period, measure OD600nm every 24 hours to observe its growth. And the survival rate was tested, and the results are shown in Figure 3.

It can be seen from the results in Figure 3 that the Exiguobacterium YL-01 disclosed in the present invention can survive in a culture medium with a hexavalent chromium concentration of up to 1500 ppm, which means that the tolerance of Exiguobacterium YL-01 disclosed in the present invention to hexavalent chromium is 1500ppm.

Example 2: Testing the chromium reducing ability of Exiguobacterium YL-01

1% of the microbacterium YL-01 disclosed in the logarithmic phase culture of the present invention was inoculated into LB culture medium containing different concentrations of hexavalent chromium (300, 500, 1000, 1500 ppm) (pH 7.0), and Cultivate for 168 hours at 30°C and 150 rpm. During the cultivation period, detect OD600nm every 24 hours. Observe the growth concentration of Exiguobacterium YL-01 disclosed in the invention and calculate the reduction of hexavalent chromium in the culture medium to trivalent chromium. The reduction rate of chromium, the results are shown in Figure 4.

It can be seen from the results in Figure 4 that the Exiguobacterium YL-01 disclosed in the present invention does have the ability to reduce hexavalent chromium in wastewater or pollutants, and has the reducing ability when the concentration of hexavalent chromium in the pollutants is below 1500 ppm, where , when the concentration of hexavalent chromium in the pollutant is lower, the efficiency of reducing hexavalent chromium by Exiguobacterium YL-01 disclosed in the present invention is better, which means that a higher reduction rate can be achieved in a shorter time.

Example 3: Test on reduction of hexavalent chromium in electroplating wastewater

Obtain the electroplating wastewater from the electroplating manufacturer and dilute it into a solution to be treated at 500 PPM. Add LB culture medium and inoculate 1% of the Exiguobacterium YL-01 bacterial liquid disclosed in the logarithmic phase of the present invention into the solution to be treated. Cultivate at 37°C and 150rpm. 96 hours. Measure OD540nm every 24 hours to observe the reduction of hexavalent chromium by Exiguobacterium YL-01 disclosed in the present invention. The results are shown in Figure 5.

It can be seen from the results in Figure 5 that the Exiguobacterium YL-01 system disclosed by the present invention can reduce the hexavalent chromium contained in the electroplating wastewater. It has a good reduction rate starting from 24 hours of culture and is reduced after 96 hours of culture. Best rate.

without

Figure 1 is a comparison result of the whole genome of Exiguobacterium YL-01 disclosed in the present invention and that of known Exiguobacterium. Figure 2 uses a phylogenetic tree to analyze the genetic relationship between Exiguobacterium YL-01 disclosed in the present invention and other known related bacilli. Figure 3 is a growth curve of Exiguobacterium YL-01 disclosed in the present invention in culture media containing different concentrations of hexavalent chromium. Figure 4 is the result of analyzing the reduction rate of Exiguobacterium YL-01 disclosed in the present invention for different concentrations of hexavalent chromium. Figure 5 shows the analysis of hexavalent chromium in electroplating wastewater treated by Exiguobacterium YL-01 disclosed in the present invention.

Novel Exiguobacterium YL-01, Hsinchu Food Industry Development Research Institute, the deposit date is November 29, 2021, and the deposit number is BCRC911083

Claims (10)

A novel Exiguobacterium sp. YL-01 is deposited at the Hsinchu Food Industry Development Research Institute. The deposit date is November 29, 2021, and the deposit number is BCRC911083. The use of the novel Exiguobacterium YL-01 described in claim 1 is for reducing hexavalent chromium in a pollutant. A composition comprising an effective amount of novel Exiguobacterium YL-01 as described in claim 1. The composition according to claim 3, further comprising a fermentation medium. A method for treating hexavalent chromium-containing pollutants, which is to treat a pollutant with an effective amount of novel Exiguobacterium YL-01 as described in the claim for a predetermined time, so that the novel Exiguobacterium YL-01 can remove the pollutant The hexavalent chromium inside is reduced to trivalent chromium. The method for treating pollutants containing hexavalent chromium as described in claim 5, wherein the concentration of the pollutant is hexavalent chromium is greater than 0 ppm and less than or equal to 1500 ppm. The method for treating hexavalent chromium-containing pollutants described in claim 5 includes the following steps: Step a: Collect the pollutant; Step b: Take the novel Exiguobacterium YL-01 and a fermentation medium, add the contaminant, and perform fermentation culture for a predetermined time; Step c: Produce a final product, wherein the hexavalent chromium content of the final product is almost zero. The method for treating hexavalent chromium-containing pollutants described in claim 7 further includes a step d, located after the step c: Step d: chemically react trivalent chromium in the final product to form a solid precipitate. The method for treating hexavalent chromium-containing pollutants as described in claim 7, wherein the predetermined time in step c is about 96 hours. The method for treating pollutants containing hexavalent chromium as described in claim 5, wherein the concentration of hexavalent chromium in the pollutant is greater than 0 ppm and less than or equal to 1500 ppm.

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