TWI623499B - Bioremediation preparation for mercuric pollution and using method thereof - Google Patents

Abstract

A mercury-contaminated biological remediation preparation comprising: at least one strain having the ability to reduce ionic mercury to elemental mercury and capable of maintaining growth at 3.5 wt% seawater salinity, the strain is selected from the group consisting of: from Enterobacter cloacae (Enterobacter cloacae B37), Pseudomonas (Pseudomonas sp.A45), Pseudomonas (Pseudomonas sp.A46) or any combination thereof, the plurality of strain can not only efficiently reducing the ionic mercury Elemental mercury, which is less toxic and volatile, is diluted by the atmosphere and is also suitable for in situ bioremediation of mercury-contaminated sites on adjacent coasts. Since these strains are all native strains and have not been genetically modified, they will not destroy or interfere with the microbial flora ecology in native water bodies or water-containing soils.

Description

Mercury-contaminated bioremediation preparation and use method thereof

The present invention relates to a mercury-contaminated bioremediation preparation and a method of using the same, and more particularly to a mercury-contaminated bioremediation preparation capable of removing mercury ions and having high salt tolerance and a method of using the same.

Mercury (Hg) is a naturally occurring element found in air, water and soil. Mercury in nature has the following forms: elemental (metal) mercury, inorganic mercury compounds and organic mercury compounds.

Common mercury products in Taiwan include batteries, thermometers, sphygmomanometers, illumination sources (such as fluorescent lamps), amalgams used to fill teeth, paints, specular reflection paints, etc. If mercury-containing products are not properly recycled, they will cause environmental pollution. The main source of pollution in the industry is the alkali chlorine plant. The production method used in the early alkaline chlorine plant in Taiwan to produce caustic soda (sodium hydroxide), chlorine gas, hydrogen gas, hydrochloric acid and bleaching powder is mercury electrolysis. When operating, mercury is used as the cathode and graphite is used as the anode to dehydrate the brine. In the brine, chloride ions are oxidized to chlorine at the anode, and sodium ions are reduced to sodium atoms at the mercury cathode and dissolved in the mercury to form an amalgam. The amalgam is then taken out and reacted with water in another water tank to produce hydrogen and sodium hydroxide. . The sodium hydroxide produced by this method has a high concentration and good purity and was used in large quantities in the early stage. 80% of the mercury imported in the Republic of China in 66 years was used in the operation of the alkali chlorine plant. However, the mercury used as the cathode in this method is often discharged together with the waste brine without proper treatment, and the soil contaminated by these wastewaters is commonly known as "mercury sludge". Due to the rise of environmental awareness, the use of mercury electrolysis to produce alkali chloride was prohibited after 77 years of the Republic of China.

For example, in addition to the pollution of pentachlorophenol and dioxins, the old site of Tainan Sinopec Anshun Plant also has mercury pollution, and the main reason for mercury pollution at this site is that it was previously used as an alkali chlorine plant. As a result, the land near the site was contaminated with mercury. Pollution of these mercury If left untreated, it will not only cause great harm to the ecological environment, but also endanger the health of neighboring residents. For example, mercury ions in inorganic mercury compounds (such as mercury chloride) not only bind to sulfhydryl groups (R-SH), but also to phospholy groups, carboxyl groups (R-COOH). ), amide groups, amine groups. When these groups in the protein react with mercury, the protein is inactivated and affects human health. Exposure to mercury-containing ions (mercury chloride) can cause the following symptoms: (1) irritating the mucous membrane and causing cough and headache; (2) causing skin burning sensation, dermatitis or skin irritation; (3) causing (7), causing a burning sensation in the oropharynx, and abdominal pain, vomiting or diarrhea; and (5), weak pulse beat, pale face respiratory failure or even cause kidney exhaustion and death. Therefore, it is necessary to remediate the soil contaminated with mercury.

The common mercury pollution treatment methods are listed as follows:

(1), fixation method: by adding sulfide (such as sodium sulfide, sodium sulfide, Na ₂ S) to reduce mercury pollutants in the soil to form mercury sulfide precipitation, is a method to stabilize mercury contaminated soil and matter. Mercury sulphide has a relatively low solubility and is less volatile than other forms of mercury, which can reduce the risk of mercury. In addition, the addition of sodium sulfide to an aqueous solution contaminated with mercury can also effectively immobilize mercury ions and remove mercury contaminants from the water by filtration.

(2) Heat treatment method: The heat treatment method uses heating to volatilize the mercury pollutants and separate them from the solid matrix (soil, sludge), and then the separated pollutants are controlled by the exhaust gas treatment system to discharge the exhaust gas. For example, a heat treatment method is used to treat mercury-contaminated soil in an alkaline chlorine plant in Sweden, and the mercury-contaminated soil is heat-treated at 460 ° C for 20 minutes, and the mercury removal efficiency can reach 99%. There is also a heat treatment method to treat mercury-contaminated soil at 600 ° C. The results show that 90% of the mercury contamination in the soil can be successfully removed, and the organic matter in the mercury and exhaust gases can be collected.

(3) Microbial treatment: The genes related to mercury tolerance in bacteria are collectively referred to as the mer gene group. These genes together constitute a mercury operons, which regulate the metabolism of mercury compounds by bacteria, and therefore contain mercury operations. Bacteria have the potential to treat mercury contaminants. The genes on the mercury operator are merA , merB , merP , merT , merE , merG , merR and merD, etc., and the proteins they represent are MerA, MerB, MerP, MerT, MerE, MerG, MerR and MerD, respectively. Has a specific function of metabolizing mercury. For example, by using Mercuric reductase (MerA), which is regulated by the merA gene in bacteria, ionic mercury (Hg ²⁺ ) can be reduced to elemental mercury (Hg), which is less toxic, to the atmosphere or can be recycled. Volatile mercury. In addition, organic mercury lyase (MerB) can catalyze the decomposition of carbon-mercury bonds, and decompose organic mercury into less toxic mercury (Hg ²⁺ ). The mechanism of action of MerA and MerB is as follows:

A related study and application of the known microbial treatment method is described as follows: U.S. Patent No. 3,923,597 discloses a treatment method for significantly reducing the mercury content of mercury-contaminated aqueous liquid by genetically engineering a MER plastid into a strain. ( Pseudomonas putida ) provides bioremediation strains that can be used to treat mercury contamination.

Wang Xiangfei, National Sun Yat-Sen University, in the paper "Study on the Physiology of Mercury-Resistant Bacteria and Anti-Hybrid Genes" in 1997, screened for mercury-resistant strains using mer operon of pHG106 as a probe for the Errenxi and Sanyegong River basins. Six strains were tested for anti-mercury gene similarity, and the merP gene of pHG106 was also cloned into plastid pKK223-3 and returned to E. coli JM109 as a genetically modified mercury-contaminated bioremediation strain.

Xie Junliang of National Chung Hsing University in 2002, "The application of mercury ion binding protein MerP to the bioremediation of heavy metals", the use of mercury-resistant Gram-positive bacteria Bacillus cereus RC607, and the negative bacteria Pseudomonas aerugenosa K-62 mercury The Mercury-binding protein MerP in the resistance genome was expressed in Escherichia coli BL21(DE3)pLysS, respectively, as a genetically modified mercury-contaminated bioremediation strain.

In the paper “Research on the Biological Detoxification Process of Mercury Wastewater by Mercury-Resistant Microorganisms” by Feng Wenxin of Fengjia University in 1997, Pseudomonas aerugoinsa PU21 with anti-mercury plastids (Rip64) was selected from medical wastewater. Anti-mercury bacteria used for detoxification of mercury wastewater.

However, the above research and application still have the following problems: The source of the strain of U.S. Patent No. 3,923,597 is not a native strain of Taiwan, and involves a complicated base reform project. Therefore, if it is to be used in Taiwan, it is necessary to evaluate whether it affects the local. Soil microbial phase ecology. Wang Xiangfei and Xie Ruliang use Escherichia coli as a genetically modified mercury-contaminated bioremediation strain. It involves complex base-change projects and requires time-consuming assessment to destroy or interfere with the soil microbial flora ecology of the native soil. Luo Wenxin's anti-mercury strains selected from medical wastewater are more suitable for wastewater detoxification treatment, and the comprehensive effect on soil environment treatment remains to be evaluated. It is worth noting that none of the above mentioned strains have been evaluated for tolerance to seawater salinity, and it is well known that even cell lines of the same strain have considerable differences in physiological characteristics and functions. . Therefore, for mercury-contaminated sites with high salinity near the coast, these strains or screening strains may have higher organic mercury degradation capacity or ionic mercury reduction capacity, but when used in mercury pollution near the coast After the site, it may not be able to adapt to the water-rich environment and the tolerance to seawater salinity, and it is unable to sustain survival and reproduction, thus reducing the practical effect of applying mercury pollution bioremediation in offshore soil.

Therefore, it is necessary to provide a mercury-contaminated bioremediation preparation and a method for using the same to solve the problems of the conventional technology.

The main object of the present invention is to provide a mercury-contaminated bioremediation preparation and a method for using the same, which are to screen a specific strain from a mercury-contaminated site near the coast of Taiwan, which has a high mercury reductase (MerA) expression amount and At the same time, it has high seawater salinity tolerance, so it can not only reduce the ionic mercury (Hg ²⁺ ) to a less toxic elemental mercury (Hg), which is diluted by the atmosphere, but also can be applied to the adjacent On-site bioremediation of mercury-contaminated sites on the coast. At the same time, the strain is screened and cultured rapidly, the preparation cost is low, the time required to remove mercury ions is short, and the removal effect is remarkable.

A secondary object of the present invention is to provide a mercury-contaminated bioremediation preparation and a method for using the same, which are selected from a local mercury-contaminated site on the local coastal area of Taiwan, which are native strains and have not been genetically modified. Therefore, there is no doubt about the destruction or interference of the microbial flora in the local water body or the watery soil in the field, and the strain can continue to reproduce at the remediation site, and the mercury ion contaminants are continuously removed.

Another object of the present invention is to provide a mercury-contaminated bioremediation preparation and a method for using the same, which are to screen three specific strains locally from a mercury-contaminated site near the coast of Taiwan, and simultaneously use it in a mercury-polluted site for remediation. The combined use of multiple strains is beneficial to ensure that a certain strain can survive and adapt to the local environment, and can continue to reproduce at the remediation site, and continuously remove mercury ion contaminants to relatively improve the remediation effect and reduce the number of remediation.

To achieve the above object, the present invention provides a mercury-contaminated bioremediation preparation comprising: at least one strain having the ability to reduce ionic mercury to elemental mercury and capable of maintaining growth at 3.5 wt% seawater salinity, the strain being selected from the group consisting of Enterobacter cloacae (Enterobacter cloacae B37), Pseudomonas (Pseudomonas sp.A45), Pseudomonas (Pseudomonas sp.A46) or any combination thereof, the plurality of sequentially to strain Accession No. BCRC 910603, BCRC 910604 And BCRC 910605 is hosted by the Center for Bioresource Conservation and Research (BCRC of FIRDI) at the Food Industry Development Institute.

In one embodiment of the invention, the strain is screened from the soil of the old site of the Anshun Plant of Tainan Sinopec.

In one embodiment of the present invention, the mercury bioremediation formulations containing Enterobacter cloacae (Enterobacter cloacae B37) at the same time, the genus Pseudomonas (Pseudomonas sp.A45) and Pseudomonas (Pseudomonas sp.A46) Three strains.

In one embodiment of the present invention, the mixing ratio of the Enterobacter cloacae B37, Pseudomonas A45, and Pseudomonas A46 is from 1 to 10:1 to 10:1 to 10.

In an embodiment of the invention, the mixing ratio is 1:1:1.

In one embodiment of the invention, the strain is embedded in a biocompatible carrier; or coated on a biocompatible carrier particle.

In addition, the present invention provides a method for using a mercury-contaminated bioremediation preparation comprising the steps of: providing a mercury-contaminated biological remediation preparation as described above; and applying the mercury-contaminated bioremediation preparation to a mercury contamination in Taiwan The site is for local bioremediation.

In one embodiment of the present invention, the mercury-contaminated bioremediation preparation is used for bioremediation of groundwater, wastewater, or aqueous soil, sludge or sediment in the mercury contaminated site.

In one embodiment of the invention, the local mercury contaminated site in Taiwan is a mercury contaminated site adjacent to the coast.

In an embodiment of the invention, the mercury contaminated site adjacent to the coast is the old site of the Anshun Plant of Tainan Sinopec.

Figures 1A and 1B: Growth curve of salt tolerance of test strain B37 of the present invention.

Figures 2A and 2B: Growth curves of the salt tolerance of the test strain A45 of the present invention.

Figures 3A and 3B: Growth curves of the salt tolerance of the test strain A46 of the present invention.

Figures 4A and 4B: Growth curves of the ability of the test strain B37 to withstand mercury ion tolerance of the present invention.

Figures 5A and 5B: Growth curves of the ability of the test strain A45 to withstand mercury ion tolerance of the present invention.

Figures 6A and 6B: Growth curve of the ability of the test strain A46 to withstand mercury ion in the present invention.

Figures 7A, 7B, and 7C: The present invention tests a graph of mercury ion removal ability of the six strain groups in three mediums of NB, PMM, and LB containing mercury ions of 60 ppm.

The above and other objects, features and advantages of the present invention will become more <RTIgt; Furthermore, the directional terms mentioned in the present invention, such as upper, lower, top, bottom, front, rear, left, right, inner, outer, side, surrounding, central, horizontal, horizontal, vertical, longitudinal, axial, Radial, uppermost or lowermost, etc., only refer to the direction of the additional schema. Therefore, the directional terminology used is for the purpose of illustration and understanding of the invention.

The invention is used for providing a mercury-contaminated biological remediation preparation and a method for using the same, which is mainly selected from a site contaminated by mercury ions in Taiwan, and can extract a large amount of ionic mercury (Hg ^{2+ )} in a short period of time. Reducing to native Taiwanese strains with low toxicity and volatile elemental mercury (Hg) and high seawater salinity tolerance for bioremediation and remediation in various water bodies or water-containing soils in Taiwan Therefore, the above strains can be applied to the local environment of Taiwan without affecting the indigenous microbial ecosystem, and the rapid and effective microbial remediation treatment effect can be obtained. The present invention screens three native strains, which all contain the merA gene, can exhibit mercury reductase, reduce mercury ions (Hg ²⁺ ) to elemental mercury (Hg), and volatilize to the atmosphere to dilute, Therefore, water bodies and water-containing soils contaminated by mercury ions can be effectively treated; and these native strains can also maintain growth under 3.5% by sea seawater salinity. The present invention will be described in detail below using the experimental examples and the illustrations to explain in detail the detailed experimental procedure and analytical method for screening the strains and confirming the strains of the present invention.

Experimental materials and methods:

(1) Screening method of strains

The soil samples were obtained from the soil of the mercury-contaminated site in southern Taiwan (the old site of Tainan Sinopec Anshun Plant). The screening method of the strain was to take 3 g of the soil into a 15 ml centrifuge tube, and add 8 ml of NB culture solution (nutrient broth), and incubate in a shaking incubator at 30 ° C and a rotation speed of 100 rpm for 16 hr. The cultured bacterial solution was subcultured in 8 ml of a culture solution containing 20 ppm of mercuric chloride (HgCl ₂ ) for 16 hr, and then subcultured in the same manner and gradually increased the concentration of mercury chloride (HgCl ₂ ) to 40 ppm and 60 ppm. . 100 μl of the bacterial solution containing 60 ppm of mercuric chloride was added to 900 μl of the culture solution for serial dilution of 10 ^-1 to 10 ^-8 . 100 μl of each dilution ratio of the bacterial solution was applied to a nutrient agar containing 100 ppm of mercury chloride, and cultured in an incubator at 30 ° C for 16 hr. After 16 hr, a petri dish capable of distinguishing a single colony (colony) was selected, and 95 single colonies were randomly selected and numbered for subsequent detection of the merA gene.

After culturing with 60ppm mercury chloride (HgCl ₂ ) medium and selecting a single bacteria, the total number of strains screened was 95, the initial numbers were A1~48 and B1~47, which can be used to determine the selected ones. The strain is resistant to at least 60 ppm of mercuric chloride (HgCl ₂ ).

(2) Extraction of strain DNA

The strain was cultured in a culture solution containing 60 ppm of mercuric chloride until the absorbance (OD 600 nm) was >0.3. 1.5 ml of the bacterial solution was centrifuged to remove the medium, and the cells were resuspended in 100 μl of TE buffer (1 mM EDTA, 10 mM Tris, pH 8.0), and lysozyme (100 mg/ml) was added thereto. After shaking, the cells were placed in a 37 ° C water bath. The medium was incubated for 30 min and oscillated every 15 min. After 30 min of water bath, 350 μl of TE buffer, 30 μl of 10% SDS, and 5 μl of proteinase K (20 mg/ml) were added, shaken evenly, and placed in a 56 ° C water bath for 1 hr. After 1 hr, add 100 μl of 5 M NaCl solution to mix well, then add an equal volume of phenol 650 μl, and after centrifugation, take the supernatant into a new centrifuge tube and add an equal volume (about 600 μl) of ice chloroform / IAA (24:1), mix well, centrifuge, and take the supernatant into a new centrifuge tube, add two volumes (about 1000 μl) of ice isopropanol and let stand for 12 hours. Thereafter, the supernatant was again centrifuged and removed, and the precipitate was washed with 70% ethanol, and the supernatant was removed by centrifugation and the precipitate was dried in a freeze dryer and then dissolved in deionized water (ddH ₂ O). After re-dissolving, agarose gel electrophoresis can be performed, and the colloid concentration is 0.8%. After the electrophoresis, the colloid is stained with EtBr (ethidium bromide), and the product is confirmed by UV lamp excitation.

(3) Detecting the presence or absence of the merA gene

In primer (primers) PCR detection of gene merA used forward (forward) A1s-nF reverse (reverse) A5-nR, in which series the sequence in Table 1 (Chadhain et al., 2006 , Environ.Microbiol. 8:1746-1752). After the above procedures known PCR primer, PCR product was purified using 1.5% agar gel, electrophoresis (agarose gel electrophoresis), stained with EtBr after electrophoresis ending with excitation light using a UV camera, the size of the PCR Product analysis to detect gene merA There is no.

The 95 strains screened by mercury-contaminated sites, with the merA gene, have the ability to reduce ionic mercury (Hg ²⁺ ) to elemental mercury (Hg) and have potential for remediation of mercury-contaminated sites. Therefore, PCR was first performed with the primers A1s-nF and A5-nR to detect whether the strain carries the merA gene. MerA strain which contained the gene sequence with the merA Gene amplification via PCR, the amplified fragment size is about 285bps, as apparent from the results obtained in the 1.5% agar gel electrophoresis colloid, a strain having a preliminary determination may 285bps It is a strain containing merA gene. A total of 40 strains with merA gene may be screened, numbered A39, A41, A45, A46, B1~7, B9~24, B26, B27, B35, B37~46.

(4) Denaturing gradient gel electrophoresis (DGGE) screening to remove repetitive strains

Since initially randomly selected species, the strains will be selected as part of the same repeating species, thus merA After detecting genes, use a denaturing gradient gel electrophoresis (denaturing gradient gel electrophoresis, DGGE) analysis to The 16S rDNA fragments were obtained for further analysis to determine whether each of them was the same strain, and among the plurality of strains of the same strain, only one of the strains was selected as a representative for subsequent characterization. The procedure for the DGGE assay has been found in the literature (Nübel et al., 1996, J. Bacteriol. 178: 5636-5643) and will not be described in detail herein, and the 16S rDNA fragment PCR primers used are as follows:

After the preliminary screening of the number of strains carrying the merA gene was 40 in the above step (3), the present invention immediately used the DGGE analysis method to obtain the position of the 16S rDNA electrophoresis bright band of each strain, thereby deleting the same repeated strains, and the results showed Nos. B1~2, B4~7, B9~20, B22~25, B27, B35, B37~46 may be the same species, and their 16S rDNA V6~V8 variant region (high variable region) fragment in DGGE The bands formed in the film are all at the same horizontal position, so only one of the strains is selected to represent the sequence of the next merA gene sequence and the 16S rDNA sequence. After deletion by DGGE, the number of different strains was determined to be five strains of B37, A39, A41, A45 and A46. Although the 16S rDNA bright bands of the strains A39, A41, A45 and A46 are in similar positions, there are still some slight differences, so it is initially determined to be different strains.

(5) Sequencing and alignment of strain merA gene

For the B37, A39, A41, A45, A46, the five merA gene sequences amplified by PCR in step (3) are further inserted into the vector (such as pGEM ^® -T Easy Vector) and the implanted strain (such as The method in ECOS ^TM 101 competent cells) was used to culture a clone library. Thereafter, the group entrust Long Meeks (Genomics) sequencing sequencing company merA gene fragment inserted in the vector, merA gene sequences was performed to obtain the following (SEQ ID NO: 5 ~ 9 ).

Next, the merA gene sequences of the above five strains (SEQ ID NOS: 5-9) were further sequenced with the published merA gene sequence from the nucleotides BLAST's Others (nr etc.):Nucleotide collection (nr/nt) database on the NCBI webpage. for comparison, the result of that ratio, wherein merA merA gene and the gene of the Enterobacter aerogenes EA1509E number plastid pEA1509_A B37 is most similar, 97% similarity, and A39, A41, A45, A46 and the Pseudomonas The similarity of putida SP1 is relatively high, and the similarities are 99%, 99%, 99%, and 91%, respectively. Their sequences were further subjected to multiple alignment by the clustalw tool software. It was found that although A39, A41, A45 and A46 were most similar to Pseudomonas putida SP1, there were still some differences between their gene sequences. A39, A41, and A45 are relatively similar to each other, but the merA gene sequences of B37 and A46 are less similar.

The sequence of the B37- merA gene (SEQ ID NO: 5) is as follows:

The sequence of the A39- merA gene (SEQ ID NO: 6) is as follows:

The sequence of the A41- merA gene (SEQ ID NO: 7) is as follows: CGGA-3'

The sequence of the A45- merA gene (SEQ ID NO: 8) is as follows:

The sequence of the A46- merA gene (SEQ ID NO: 9) is as follows:

(6) Identification of the genus name and species name of the strain

For the B37, A39, A41, A45, and A46, these five strains were further preceded by bacterial universal primers 9f and 1525r (Bano and Hollibaugh, 2002, Appl. Environ. Microbiol. 68:505-518.) Perform PCR to amplify a specific 16S rDNA fragment, and then make another clone library in the same manner as described above by inserting the vector (eg pGEM ^® -T Easy Vector) and implanting the strain (eg ECOS ^TM 101 competent cells) (clone library), followed by another two primers T7, SP6 (Tennant et al., 2005, Infect. Immun. 73: 6860-6867) for PCR amplification of a longer 16S rDNA fragment with sufficient supply Identify the gene sequence of the strain species.

Next, the Genomics sequencing company was commissioned to sequence the longer 16S rDNA fragment, and the 16S rDNA gene sequence obtained was as follows (SEQ ID NOs: 14-18); subsequently, it was compared by the NCBI website. The results showed that the 16S rDNA sequences of strains B37, A39, A41, A45 and A46 were respectively associated with Enterobacter cloacae subsp.cloacae ATCC 13047, Pseudomonas sp. BF-2, Pseudomonas sp. HR 26, Pseudomonas sp. BF-2, The 16S rDNA sequence similarity of Pseudomonas sp.HR 26 was 99%, 99%, 100%, 99%, and 99%, respectively. It is pointed out from the past literature that the strain similarity is more than 97%, and the strain is the same strain (Gevers et al., 2005). Therefore, the strain B37 is Enterobacter cloacae , while A39, A45, A41 and A46. All are Pseudomonas sp. (Pseudomonas). The 16S rDNA sequence of the five strains was also sequenced by the clustalw tool software. The A39 and A46 strains were similar; the B37 strain was similar to the other four strains. The relationship is far away. Therefore, the strain characteristics test will be carried out by following the three strains B37, A45 and A46.

The sequence of B37-16S rDNA (SEQ ID NO: 14) is as follows:

The sequence of A39-16S rDNA (SEQ ID NO: 15) is as follows:

The sequence of A41-16S rDNA (SEQ ID NO: 16) is as follows:

The sequence of A45-16S rDNA (SEQ ID NO: 17) is as follows:

The sequence of A46-16S rDNA (SEQ ID NO: 18) is as follows:

(7) Growth characteristics test of mercury-resistant bacteria

(1) Salt tolerance test

The strains B37, A45 and A46 which were screened were cultured in NB medium for one day, and the absorbance (OD 600nm) of the bacteria solution was adjusted to 0.8, and then 50 μl was inoculated with a weight ratio of different salinity (0.5%, 1%, 2%). , 3.5%, 4%, 5%, 6%) in 50ml NB medium, and every 4 hours, 36~72 hours in the interval of 0~36 hours every 4 hours, 72~108 hours At 8 hours, the absorbance (OD 600 nm) was measured separately to prepare a growth curve to observe the growth of the bacteria.

Please refer to the figures 1A and 1B, which reveal the results of the salt tolerance test of B37 strain, wherein the salt concentration of 0.5% is the salt concentration of the NB culture solution itself, and the other salt concentration is achieved by adding NaCl, and In the figure, the growth curve of B37 strain in different salt concentrations can be seen. The B37 strain had no significant effect on its growth when it contained 1% and 2% salt. The growth curve of the strain was almost the same as 0.5% without NaCl, and the lag phase of growth was not prolonged. The maximum absorbance of OD 600nm can reach about 1.4. The delay period begins to increase after the salinity of 3.5%, which means that the strain takes longer to adapt to the environment and then enters the log phase for large-scale replication growth. The growth curve of the strains was similar at salinity of 3.5%, 4%, and 5%, and the logarithmic growth phase was entered between about 4 and 6 hours, and the maximum absorbance of OD 600nm was about 1.3. When the salinity reached 6%, the growth of B37 strain was obviously inhibited, the delay period of the strain was greatly prolonged, and the logarithmic growth phase was entered in about 10 hours, and the highest concentration of strain growth was significantly decreased. The maximum absorbance of OD 600nm could only be reached. About 1.2. From the above results, it can be shown that the salt tolerance of the B37 strain is good, and growth at 0.5% to 5% salt concentration is not significantly inhibited until the salt concentration reaches 6%.

Please refer to the 2A and 2B graphs, which reveal the salt tolerance test results of the A45 strain. The growth curve of the strain in the figure shows that when the salinity is 1%, the growth of the strain is not restricted, and the growth curve and salinity are 0.5%. It is similar, the delay period is not extended, and the maximum absorbance of OD 600nm can reach about 1.2, which is higher than the maximum absorbance of 1.1 at a salinity of 0.5%. In addition, it can be seen from the results graph that although the salt enters the logarithmic growth phase at a salt concentration of 0.5% and 1%, the strain enters the death phase more quickly at 0.5%. At a salt concentration of 2%, the growth of the strain was slightly inhibited, and it was about 2 hours later than the salt concentration of 0.5%. The delayed growth period entered the logarithmic growth phase, but the highest absorbance of the strain could still reach about 1.2 or so. At a salinity of 0.5% of 1.1. At the salt concentration of 3.5%, 4%, 5%, the growth of the strain was significantly inhibited, and the lag phase entered the logarithmic growth phase at about 14, 22, and 42 hours, respectively, and the strain was at the salt concentration of 3.5% and 4%. The highest absorbance value can still reach 1.2, which is higher than the salt concentration of 0.5% of 1.1, until the highest absorbance of the salt concentration of 5% strain is less than 1.1. The salt concentration of 6% strongly inhibited the growth of the strain, and the strain did not begin to grow until 100 hours. From the above results, it was shown that the A45 strain can tolerate a certain salt concentration. After the end of the lag phase of the adaptation period, the absorbance at 4% of the salt concentration can still reach 1.1 at 1.2% above the salt concentration of 0.5%, which also shows that the A45 strain needs A slightly higher salt concentration (1%) will grow better.

Please refer to the figures 3A and 3B, which reveal the growth curve results of the salt tolerance test of A46 strain. It can be seen from the growth curve results in the figure that the A46 strain is produced at a salt concentration of 1%. The long state is the best. At this time, the delay period of the growth curve is consistent with the 0.5% delay period, and the time to enter the death period is also shorter than 0.5%. The maximum absorption value of OD 600nm can reach 1.2 or so. . At the salt concentration of 2%, the growth of the strain began to be slightly affected, and the delay period lasted about 8 hours before it began to enter the logarithmic growth phase, while the maximum absorbance of OD 600nm was still about 1.1. When the salt concentration was 3.5%, 4%, 5%, the growth of the strain began to be significantly inhibited, and the delay period entered the logarithmic growth phase at about 24, 34, and 64 hours, respectively, and the maximum OD 600nm reached approximately 1.2, 1.2, 1.1 or so. When the salt concentration was 6%, the growth of the strain was significantly inhibited until the end of the test (108 hours), and the A46 strain did not grow. From the above results, it can be understood that the A46 strain has a certain tolerance to salt concentration, and although it is longer during the delay period, it can grow under 3.5% seawater salinity.

The results of salt tolerance test showed that the three strains B37, A45 and A46 had certain tolerance to salinity. The salt tolerance ability was strongest to weakest, respectively, B37 was the strongest, A45 was the second, and the weakest was For the A46 strain, but all three strains can grow at a seawater salinity of 3.5%, they should be able to adapt to the salt concentration of the local site.

(2) Mercury resistance test

The strains B37, A45 and A46 which were screened were cultured in NB medium containing 10 ppm Hg ²⁺ for 1 day, and the absorbance (OD 600 nm) of the bacteria solution was adjusted to 0.8, and then 50 μl was inoculated to contain different concentrations of mercury (0 , 20, 40, 60, 80, 100, 150, 200 ppm) in 50 ml NB medium, and every 8 hours, every 4 hours, 48 to 48 hours in the interval of 0 to 24 hours, every 8 hours, every 8 hours The absorbance (OD 600 nm) was used to prepare a growth curve to observe the growth of the bacteria.

Please refer to Figures 4A and 4B for the results of the mercury tolerance test of B37 strain. It can be seen from the results of the growth curve in the figure that the growth of the strain is slightly inhibited at 20 ppm mercury concentration, and the growth curve is delayed. The period of entry into the logarithmic growth phase was delayed by 2 hours from 0 ppm, but the strain was able to grow, and the maximum absorbance of OD 600 nm was about 1.3, which was similar to the maximum absorbance of 0. When the concentration of mercury reached 40ppm and 60ppm, the growth of the strain was obviously inhibited. The strain entered the logarithmic growth phase from 14° to 18 hours, and the highest absorbance of OD 600nm could still reach about 1.5. At 80 ppm and 100 ppm mercury concentration, the growth of the strain was similarly inhibited, and the logarithm was also entered in about 26 hours. During the growth period, the growth situation is similar. When the mercury concentration came to 150 ppm and 200 ppm, the growth of the strain was completely inhibited. From the above results, it is shown that the B37 strain has a certain tolerance to mercury, and the mercury concentration of up to 100 ppm should be able to withstand the mercury concentration of 50 ppm of the in situ pollution site.

Please refer to the 5A and 5B graphs, which reveal the mercury tolerance test results of strain A45. It can be seen from the growth curve in the figure that A45 is well tolerated at mercury concentrations of 20 ppm, 40 ppm and 60 ppm, which are about 8 hours. From lag pahse to the logarithmic growth phase, the maximum absorbance of OD 600nm can reach 1.0 or more. When the concentration of mercury reached 80ppm and 100ppm, the growth of the strain began to be significantly inhibited. It took only about 10 and 12 hours for the strain to enter the logarithmic growth phase from the delayed phase, but the maximum absorbance of the strain at OD 600nm could still reach 1.2. about. When the mercury concentration is 150 ppm and 200 ppm, the growth of the strain is completely inhibited. From the above results, it can be understood that the A45 strain has good mercury tolerance, can grow at a mercury concentration of 100 ppm, and should be able to withstand the mercury concentration of 50 ppm at the local site.

Please refer to Figures 6A and 6B, which reveal the results of the mercury tolerance test of the A46 strain. The results of the growth curve in the figure show that the A46 strain grows only at mercury concentrations of 20 ppm, 40 ppm, 60 ppm and 80 ppm. A slight inhibition, from the delay period to the logarithmic growth phase in about 8 hours, and the maximum absorbance of OD 600nm can reach about 1.2. At the mercury concentration of 100 ppm, the growth of the strain began to be significantly inhibited, and it took about 14 hours to enter the logarithmic growth phase from the delayed phase, but the absorbance at the OD 600 nm could still reach 1.2 or more. When the concentration of mercury is 150 pmm and 200 ppm, the growth of the strain is completely inhibited. From the above results, it is shown that the A46 strain is tolerant to mercury and can withstand a maximum concentration of 100 ppm.

For the three strains B37, A45 and A46 mercury tolerance test can show that the three strains can withstand 100ppm mercury concentration, can grow at this concentration, and the growth of B37 strain is affected by mercury is large, delayed The period of time was significantly prolonged. A45 and A46 were less affected by mercury than B37 strain. The growth curves of mercury concentration of A45 and A46 strains were similar, and the time from the delayed phase to the logarithmic growth phase was approximately the same.

(8) Testing of the ability of strains to reduce mercury ions

After 18 hours of the three strains B37, A45 and A46, respectively, containing 20ml 10ppm Hg ²⁺ medium for pre-culture, the broth medium was added a sterilized OD 600nm absorbance value was adjusted to 0.8, then inoculated into 5% of mercury, The mediums were NB, PMM and LB, respectively, and the Hg ²⁺ concentration after inoculation was 60 ppm. A total of six groups were divided into: (1) B37 single bacteria group, (2) A45 single bacteria group, (3) A46 single bacteria group, and (4) three bacteria mixed group (B37: A45: A46=1). : 1:1), (5) from the original mixed bacteria group S of the soil mixed bacteria of the original mercury contaminated site, and (6) the aseptic control group without any bacteria, after the cultivation, respectively at 0, 1, 2 , 3,6,9,12 days sampling for digestion and extraction (according to the Environmental Protection Agency Environmental Protection Agency, the mercury detection method in water - cold vapor atomic absorption spectrometry, NIEA W330.52A), using mercury analyzer (HIRANUMA) Mercury analyzer HG-310) Analyze the concentration of mercury. The composition of each medium was as follows: NB culture liquid (nutrient broth): animal tissue digest 5 g / L, NaCl 5 g / L, beef extract 1.5 g / L, yeast extract 1.5 g / L.

PMM medium ( Pseudomonas mininal medium): 7 g of K ₂ HPO ₄ , 3 g of KH ₂ PO ₄ , 0.5 g of sodium citrate, 1 g of MgSO ₄ , 7H ₂ O, 1 g of (NH ₄ ) ₂ SO ₄ , 4 g Glucose, 1 liter of water, 0.41 g/L (0.41 mg/ml) of leucine, isoleucine and valine.

LB medium (Luria-Bertani broth): casein hydrolysate 10 g/L, yeast extract 5 g/L, NaCl 10 g/L.

Please refer to Figure 7A for the results of the Hg ²⁺ removal test in a NB medium containing 60 ppm of mercury ions. After being reduced by the strain, Hg ²⁺ was naturally diluted to the environment by elemental mercury atoms volatilized into the atmosphere. The results showed that the best volatile mercury in the six groups was A45 single bacteria group, which could vulcanize mercury after 12 days of treatment. %, while the mercury volatilization rates of the other groups B37 single bacteria group, A46 single bacteria group, three bacteria mixed group and original soil mixed bacteria group were 34.0%, 43.4%, 42.0% and 35.8%, respectively. From the above results, it was found that the strain remained stable after removing a part of the mercury ions and could not process the remaining mercury ions. This may be due to the combination of mercury ions with some components in the NB culture solution, which reduces the ionic state of the mercury in the medium, and the combined product may be formed by the interaction of mercury ions with the thiol group (R-SH). This product may not directly act as a substrate for mercury reductase and cannot be reduced to elemental mercury volatilization and diluted by air. On the other hand, it can be found that the concentration of mercury in the control group without added bacteria also decreases, which may be caused by other substances in the medium component which reduce the ionic mercury to elemental mercury volatilized into the air, or the mercury ions adsorb or diffuse to the container wall. Caused by the above.

Please refer to Figure 7B, which reveals the results of the Hg ²⁺ removal test in PMM medium containing 60 ppm of mercury ions. The results show that the best efficiency of volatile mercury is the three bacteria mixed group, which can be treated after 12 days. The mercury volatilization rate was 89.0%, while the volatilization effect of the A46 single bacteria group could volatilize 87.2% of mercury ions, while the other group B37 single bacteria group, A45 single bacteria group and original soil mixed bacteria group had the mercury volatilization rate of 43.1 respectively. %, 28.1% and 35.5%. The inefficiency of these three groups of volatile mercury may be due to the poor growth of the three groups of bacteria in PMM medium containing 60 ppm of mercury ions. The bacteria liquid was collected after 12 days of sampling. It is in a clarified state, so there is not enough bacteria to volatilize mercury ions. In addition, it can be found that the concentration of mercury in the control group without added bacteria also decreased, and the decrease was 62.1%. This may be due to the presence of phosphates (K ₂ HPO ₄ and KH ₂ PO ₄ ) in the PMM medium, which may react with phosphate to form Hg ₃ (PO ₄ ) ₂ and HgHPO ₄ , both of which are The solubility is not high, 1.4x10 ^-8 mol/L and 2.8x10 ^-7 mol/L, respectively, and it will produce white precipitate in water, so the precipitated part cannot be detected when the mercury concentration is detected.

Please refer to Figure 7C, which reveals the results of the Hg ²⁺ removal test in LB medium containing 60 ppm of mercury ions. The results show that the best volatile mercury is B37 single bacteria group, which can be treated after 12 days. The mercury volatilization rate was 88.6%, while the mercury volatilization rates of other groups A45 single bacteria group, A46 single bacteria group, three bacteria mixed group and original soil mixed bacteria group were 80.3%, 86.2%, 82.7% and 69.4%, respectively. It can be found that these bacteria groups remain stable after removing a part of the mercury ions and can no longer handle the remaining mercury ions. This case is similar to the test results using the NB culture solution group containing 60 ppm of mercury ions, but the amount of mercury ions that cannot be treated is less than that of the NB culture solution group. This result may be that mercury ions are combined with some components in the LB culture solution to reduce the ionic state of the mercury in the medium, and the combined product may be formed by the interaction of mercury ions with the hydrogenthio group (R-SH). This product may not act directly as a substrate for mercury reductase and therefore cannot be reduced to elemental mercury. On the other hand, it can be found that the mercury concentration in the control group without added bacteria also decreased, similar to the test results of the NB culture solution group containing 60 ppm of mercury ions, but the mercury concentration in the LB culture solution was lower than that in the NB culture solution group. The reason for this is that the other substances in the medium component are reduced by the reduction of ionic mercury into elemental mercury volatilized into the air, or by the adsorption or diffusion of mercury ions onto the walls of the container.

The results of the ability to remove mercury ions from the three medium test strains showed that the best removal effect was the use of the three bacteria mixed group in the PMM medium, which removed 89.0% of the mercury ions, but because the medium had the medium composition The problem of reacting with mercury ions to form a precipitate Therefore, it is more objective and feasible to select LB medium to culture strains to remove mercury ion contamination. The B37 single bacteria group with the highest efficiency of removing mercury ions in LB medium can remove 88.6% of mercury ions. From the above comparison, it can be understood that the selected strain has a certain ability to remove mercury ions, and if it is to be strengthened, the bioreactor culture test can be used instead.

According to the above experimental results, in one embodiment of the present invention, the present invention provides a mercury-contaminated bioremediation preparation comprising: at least one strain, which is screened from the soil of the old site of the Anshun Plant of Tainan Sinopec, and has ionic mercury capacity reduced to elemental mercury, as well as to maintain growth at salinity 3.5wt%, the strain is selected from Enterobacter cloacae (Enterobacter cloacae B37), Pseudomonas (Pseudomonas sp.A45) or Pseudomonas (Pseudomonas sp.A46), which filter out some of the experiments via the strain sequence number to register BCRC 910603, BCRC 910604 and BCRC 910605 Storage biological resources conservation and research center Institute for food industry development (BCRC of FIRDI); in addition In another embodiment, the mercury-contaminated bioremediation preparation may also comprise several strains, and the three strains described above are selected to be used at the same time. At this time, Enterobacter cloacae (Enterobacter cloacae B37), Pseudomonas (Pseudomonas sp.A45) and Pseudomonas (Pseudomonas sp.A46) the mixing ratio may be 1 to 10: 1 to 10: 1 Up to 10, preferably 1 to 5:1 to 5:1 to 5, particularly 1 to 2:1 to 2:1 to 2, for example 1:1:1.

In addition, the present invention also provides a method for using a mercury-contaminated bioremediation preparation, comprising: providing a mercury-contaminated biological remediation preparation as described above; and applying the mercury-contaminated bioremediation preparation to a mercury contamination in Taiwan The site is for local bioremediation.

According to an embodiment of the present invention, the contaminated medium usable by the mercury-contaminated bioremediation preparation may be a liquid or aqueous solid environment medium, including: groundwater, waste water, or groundwater containing Or soil, sludge or sediment of wastewater, and in particular mercury contaminated site environmental media adjacent to the coast or estuary. For example, the contaminated medium is selected from the group consisting of drinking water sources (eg, well water), fish culture ponds, factory wastewater, domestic sewage, water-rich plant land soil, agricultural land (eg, Soils in paddy fields, orchards, grazing grasslands, etc., and sludge from sewage treatment plants, especially those selected from adjacent coasts or estuaries, such as seawater, coastal wetlands or adjacent areas of the old site (or nearby) of Tainan Sinopec Anshun Plant (or nearby) Seawater-rich soil.

According to an embodiment of the present invention, the mercury-contaminated bioremediation preparation may optionally contain nutrients beneficial to the growth of microorganisms, such as glycerol, riboflavin, casein, polypeptone ( Polypeptone), meat extract, soybean cake, yeast extract, cellulose, glucose, corn extract, whey powder, starch, vitamins [such as thiamine, biotin, nicotinic acid amide or calcium panthotenate], or containing enzymes such as amylase, protease and/ Or lipase, but not limited to this.

According to an embodiment of the present invention, the mercury-contaminated bioremediation preparation can be manufactured into a form suitable for use by a known technique, including: a culture solution, a suspension, granules, a dry powder, Tablets, pills, capsules, slurries, and/or the like, but are not limited thereto. Further, if necessary, the mercury-contaminated bioremediation preparation may be used by being immobilized on an insoluble support, for example, in a sewage treatment plant for treating mercury-containing sewage.

According to an embodiment of the present invention, the mercury-contaminated bioremediation preparation may further comprise a biocompatible carrier. For example, in one embodiment, one to three specific strains of the mercury-contaminated bioremediation formulation can be entrapped in the biocompatible carrier. At this time, the biocompatible carrier comprises: silica gel, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid. ), polyacrylamide, carrageenan, agarose, gelatin, cellulose, cellulose acetate, dextran, and collagen One or more of them, but is not limited thereto. In another embodiment, one or three specific strains of the mercury-contaminated bioremediation formulation may also be coated on the biocompatible carrier particles. At this time, the biocompatible carrier particles comprise: glass, ceramic, metal oxide, activated carbon, kaolinite, bentonite, zeolite (zeolite) ), aluminum, anthracite, glutaraldehyde, polyacrylic acid, polyurethane, polyvinyl chloride, ion exchange resin ), epoxy resin, photoplastic resin (photosetting) One or more of resin, polyester, and polystyrene, but is not limited thereto.

It should be noted that those skilled in the art should understand that even if different strains of the same strain have considerable differences in physiological characteristics and functions, the scope of protection of the present invention is limited to the above experimental methods. The use of Taiwan native strains B37, A45, and A46, which are screened at specific site locations, alone or in combination, that is, the scope of protection of the present invention is not different from that of the same strains screened by different experimental methods and/or by different site locations. The strains, especially the different strains of the same strains that were not screened at different sites in foreign countries, are described first.

The present invention has been disclosed in its preferred embodiments, and is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.

【Biomaterial Storage】

Domestic registration information [please note according to the registration authority, date, number order]

Food Industry Development Institute 2013-12-31 BCRC 910603

Food Industry Development Institute 2013-12-31 BCRC 910604

Food Industry Development Institute 2013-12-31 BCRC 910603

Organization Applicant

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Claims (7)

A mercury-contaminated bioremediation preparation comprising Enterobacter cloacae B37 having the ability to reduce ionic mercury to elemental mercury and maintain growth at 3.5 wt% seawater salinity, wherein the Enterobacter cloacae B37 line is selected from The soil of the old site of Tainan Sinopec Anshun Plant is deposited with the Bioresource Conservation and Research Center (BCRC of FIRDI) of the Food Industry Development Research Institute under the registration number BCRC 910603. The application of the biological mercury patentable scope of a remediation formulation, wherein the formulation mercury bioremediation further comprising Pseudomonas (Pseudomonas sp.A45), or Pseudomonas (Pseudomonas sp.A46) The Pseudomonas A45 and the Pseudomonas A46 are deposited with the Bioresource Conservation and Research Center (BCRC of FIRDI) of the Food Industry Development Research Institute under the registration numbers BCRC 910604 and BCRC 910605, respectively. The mercury-contaminated bioremediation preparation according to the second aspect of the invention, wherein the mercury-contaminated bioremediation preparation comprises the Enterobacter cloacae B37, Pseudomonas A45 and Pseudomonas A46, and the gutter The mixing ratio of Enterobacter B37, Pseudomonas A45 and Pseudomonas A46 is from 1 to 10:1 to 10:1 to 10. For example, the mercury-contaminated bioremediation preparation described in claim 3, wherein the mixing ratio is 1:1:1. The mercury-contaminated bioremediation preparation according to claim 1, wherein the strain is embedded in a biocompatible carrier; or coated on a biocompatible carrier particle. A method for using a mercury-contaminated bioremediation preparation, comprising the steps of: providing a mercury-contaminated bioremediation preparation according to claim 1; The mercury-contaminated bioremediation preparation was applied to a mercury-contaminated site in Taiwan for on-site bioremediation. The mercury-contaminated site was an old site of Tainan Sinopec Anshun Plant adjacent to the coast. The method for using the mercury-contaminated bioremediation preparation according to claim 6, wherein the mercury-contaminated biological remediation preparation is for the groundwater, waste water, or the soil containing water or sludge of the mercury contaminated site. The sediment is bioremediated.