TWI701324B - Immobilized microorganism gel, preparation method thereof, and composition for degrading chlorinated-organic substance - Google Patents

Abstract

A method of manufacturing an immobilized microorganism gel is provided, including providing alkoxysilane (AS); providing tetraalkyl orthosilicate (TAOS); mixing the AS, the TAOS and acid solution to obtain an alkoxide solution; providing a bacterial suspension including a hydrogen-producing bacteria; mixing the alkoxide solution, bacterial suspension and silica nanoparticles to obtain the immobilized microorganism gel after gel formation. The immobilized microorganism gel manufactured by the above mentioned method is also provided. A composition for degrading chlorinated-organic substance is also provided, comprising the immobilized microorganism gel and a dechlorination bacteria.

Description

Embedding microbial colloid, its preparation method and reducing Composition for decomposing chlorinated organic matter

The invention relates to a colloid and a preparation method thereof, and particularly relates to a colloid embedded with microorganisms and a preparation method thereof.

Trichloroethylene (TCE) is a volatile, transparent and colorless liquid, and it is one of the substances that commonly cause groundwater pollution. The traditional method of dechlorination is to provide bacterial nutrients on site to carry out reductive dechlorination reactions. However, under hypoxic conditions, although there are many environmental bacteria that can dechlorinate tetrachloroethylene and trichloroethylene to cis-dichloroethylene (cis-DCE), it still requires dechlorination bacteria to completely dechlorinate Dichloroethylene and vinyl chloride are reductively dechlorinated into environmentally friendly ethylene.

In recent years, studies have found that hydrogen often plays a role of directly supplying electrons in the reductive dechlorination reaction, and is considered to be a key factor in promoting the reductive dechlorination reaction. In sites contaminated with chlorinated organic matter, hydrogen can help strengthen the effectiveness of dechlorinating bacteria in reducing dichloroethylene and vinyl chloride to ethylene.

Therefore, how to provide hydrogen on-site to assist dechlorination bacteria to carry out reductive dechlorination reactions requires improvement in the existing technology.

The purpose of the present disclosure is to provide an embedded microbial colloid to achieve the effect of making hydrogen-producing bacteria stable in an environment containing chlorinated organic matter and providing a large amount of hydrogen.

The present disclosure provides a method for preparing microbial-embedded colloids, including: providing alkoxysilane (AS); providing tetraalkyl orthosilicate (TAOS); mixing alkoxysilane, tetraalkylene Base orthosilicate and acid solution to hydrolyze alkoxysilane and tetraalkylorthosilicate to obtain alkoxy solution; provide bacteria solution, including hydrogen-producing bacteria; and mix alkoxy silane, bacteria solution and silicon Nanoparticles are condensed with alkoxy solution, bacterial liquid and silicon nanoparticle to obtain embedded microbial colloid.

In some embodiments, the hydrogen-producing strains comprise Clostridium ( Clostridium ) strains, Enterobacter ( Enterobacter ) strains, or a combination thereof.

In some embodiments, the Clostridium species includes Clostridium butyricum .

In some embodiments, the alkoxysilane (AS) includes methyltrimethoxysilane (MTMS), methyl triethoxysilane (MTES), or a mixture thereof.

In some embodiments, the tetraalkyl orthosilicate (TAOS) includes tetraethyl orthosilicate (TEOS), tetramethoxysilane (TMOS), or a combination thereof.

In some embodiments, in the step of mixing the alkoxysilane, the tetraalkylorthosilicate and the acid solution, the volume ratio of the alkoxysilane to the tetraalkylorthosilicate is 1:0.01-100.

In some embodiments, the volume ratio of the alkoxysilane and the tetraalkylorthosilicate to the acid solution is 1:0.1-10.

In some embodiments, in the step of mixing the alkoxy solution, the bacterial solution and the silicon nanoparticle, the silicon nanoparticle is formed by covalently bonding a plurality of silicon dioxide.

The present disclosure also provides an embedded microbial colloid, which is prepared by the aforementioned preparation method.

The present disclosure also provides an embedded microbial colloid, which includes a plurality of silicon nano particles and a plurality of hydrogen-producing bacteria. These silicon nano-particles are covalently bonded in an oxygen-silica-oxygen arrangement to surround the embedded hydrogen-producing bacteria. Among them, the embedded microbial colloid has multiple holes scattered on the inside and surface of the embedded microbial colloid.

In some embodiments, the methyltrimethoxysilane and the tetramethoxysilane are covalently bonded in a silicon-oxygen-silicon (siloxane bond) arrangement.

The present disclosure also provides a composition for degrading chlorine-containing organic matter, including the aforementioned embedded microbial colloid and dechlorinating bacteria.

In some embodiments, the bacteria comprising dechlorination Dehalococcoides, Dehalobacter, Dehalospirillum, the genus off sulfite (Desulfitobacterium), Candida desulfurization (Desulfomonile) or combinations thereof.

In some embodiments, the composition further includes a medium that provides a carbon source.

In order to make the above and other objects, features, advantages and embodiments of the present invention more comprehensible, the description of the accompanying drawings is as follows: Figure 1 shows the seven different groups of the present disclosure that generate hydrogen over time Concentration; Figure 2 shows the concentration of degraded trichloroethylene in seven different groups of the present disclosure over time; Figure 3 shows the increase of cis-dichloroethylene in seven different groups of the present disclosure over time Concentration; Figure 4 shows the seven different groups of this disclosure increasing the concentration of 1,1-dichloroethylene over time; Figure 5 shows the seven different groups of this disclosure increasing vinyl chloride over time The concentration of ethylene; and Figure 6 shows the seven different groups of the present disclosure that increase the concentration of ethylene over time.

In order to make the description of this disclosure more detailed and complete, the following An illustrative description is provided for the implementation aspects and specific embodiments of the present invention, but this is not the only way to implement or use the specific embodiments of the present invention. The embodiments disclosed below can be combined or substituted with each other under beneficial circumstances, and other embodiments can also be added to an embodiment without further description or description. In the following description, many specific details will be described in detail so that the reader can fully understand the following embodiments. However, the embodiments of the present invention can also be practiced without these specific details.

In this article, unless the article is specifically limited in the context, "一" and "the" can generally refer to one or more. It will be further understood that the terms "include", "include", "have" and similar words used in this article indicate the recorded features, regions, integers, steps, operations, elements and/or components, but do not exclude Other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

The hydrogen-producing strains used in the present disclosure include, but are not limited to, currently known hydrogen-producing strains, such as Clostridium species or Enterobacter species. In some embodiments, the hydrogen-producing strains are selected from in-situ groundwater of the contaminated site, and the hydrogen-producing strains are embedded in the colloid using immobilization technology to increase the stability of hydrogen-producing strains , Hydrogen production activity and tolerance to poisons, thereby enhancing the effectiveness of remediation of chlorinated organic compounds in groundwater in the wild.

In some embodiments, Clostridium butyricum with the ability to produce hydrogen is screened out from groundwater of the contaminated site. In the process of reducing and degassing trichloroethylene as a pollutant, Clostridium butyricum provides hydrogen as a supplier of electrons to assist environmental dechlorination bacteria to accelerate the degradation of chlorine-containing organic matter. Hydrogen plays an important role in the process of anaerobic reduction and removal rate. Among the microorganisms in groundwater, hydrogen-producing strains Due to competition, insufficient carbon sources, etc., it is often impossible to stably provide hydrogen as a source of electrons for dechlorination bacteria to perform reductive dechlorination reactions, resulting in low efficiency of reductive dechlorination reactions.

In some embodiments, strain embedding technology can enhance the tolerance and stability of strains as a means of bioaugmentation. In the present disclosure, silica gel is used as the embedding carrier, which can permeate organic matter, so that the embedded strain can effectively use the environmental matrix and stably produce hydrogen. The embedded microbial colloid prepared by the present disclosure can be used to regenerate organisms in the groundwater environment, and it can produce hydrogen stably for at least 30 days (720 hours). Therefore, adding the embedded microbial colloid prepared by the present disclosure can increase the rate of environmental degradation of chlorine-containing organic matter.

In this article, the term "biological augmentation" refers to the process of increasing the rate of biodegradation by adding microorganisms. Microbes originating from contaminated areas may already be able to break down waste, but they may be inefficient and slow. Therefore, it is usually necessary to study the characteristics of local microbial species to determine whether biological stimulation is possible.

In some embodiments, the present disclosure provides a method for preparing microbial-embedded colloids, including providing alkoxysilane; providing tetraalkylorthosilicate; mixing alkoxysilane, tetraalkylorthosilicate and acid Solution, hydrolyze alkoxysilane and tetraalkylorthosilicate to obtain alkoxy solution; provide bacteria solution, including hydrogen-producing bacteria; mix alkoxy solution, bacteria solution and silicon nano particles to make alkoxy solution , Bacteria liquid and silicon nano particles are condensed to obtain embedded microbial colloid.

In this article, the term "condensation" (condensation) is A type of scientific reaction in which two molecules combine to form a new molecule through changes in functional groups, and small molecules are lost in the process. The small molecules lost are usually water, hydrogen chloride, methanol or acetic acid. In the present disclosure, the small molecule lost is methanol.

In some embodiments, a method for preparing microbial-embedded colloids of the present disclosure, wherein the alkoxysilane is methyltrimethoxysilane, the tetraalkylorthosilicate is tetramethoxysilane, silicon nanoparticle It is formed by covalent bonding of multiple silicon dioxides, and multiple hydroxyl groups are exposed on the periphery of silicon nano particles. First, methyltrimethoxysilane, tetramethoxysilane, and hydrochloric acid are mixed to hydrolyze methyltrimethoxysilane and tetramethoxysilane to obtain an alkoxy solution. Then, the alkoxy solution, the bacteria solution and the silicon nano particles are mixed, and the alkoxy solution, the bacteria solution and the silicon nano particles are condensed to obtain an embedded microbial colloid. In detail, in the condensation reaction, the hydroxyl group on the silicon nanoparticle undergoes a nucleophilic substitution reaction with the silicon atom of methyltrimethoxysilane and the silicon atom of tetramethoxysilane, so that the silicon nanoparticle can react with methyltrimethoxysilane. Silane and tetramethoxysilane are covalently bonded and produce methanol (CH ₃ OH) as a by-product. Silicon nano-particles are covalently bonded in an oxygen-silicon-oxygen arrangement by condensing with methyltrimethoxysilane and/or tetramethoxysilane, and methyltrimethoxysilane and tetramethoxysilane are covalently bonded. Silanes are covalently bonded in a silicon-oxygen-silicon (siloxane bond) arrangement. When the condensation continues, it surrounds the embedded strain and forms multiple holes in the inside and surface of the embedded microbial colloid. After the gel is formed, it can be washed with PBS to remove methanol.

In one embodiment, the diameter of each hole is about 5 nanometers to about 15 nanometers.

In some embodiments, after mixing methyltrimethoxysilane (MTMS) and tetramethoxysilane (TMOS) in a volume ratio of 1:0.01 to 100, then (MTMS+TMOS) and acidic solution are mixed at 1: Mix in a volume ratio of 0.1-10 to obtain an alkoxy solution. In some embodiments, the acidic solution is hydrochloric acid. In some embodiments, the ratio of MTMS to TMOS is 1:0.01, 1:0.05, 1:0.1, 1:0.2, 1:0.5, 1:0.7, 1:0.9, 1:1, 1:2, 1: 3. 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100. In some embodiments, the ratio of (MTMS+TMOS) to acidic solution is 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1. : 0.9, 1: 1, 1: 1.1, 1: 1.2, 1: 1.3, 1: 1.4, 1: 1.5, 1: 1.6, 1: 1.7, 1: 1.8, 1: 1.9, 1: 2, 1: 3 , 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

Example

Although a series of operations or steps are used in the following to illustrate the method disclosed herein, the sequence of these operations or steps should not be construed as a limitation of the present invention. For example, certain operations or steps may be performed in a different order and/or simultaneously with other steps. In addition, it is not necessary to perform all the illustrated operations, steps and/or features to realize the embodiments of the present invention. In addition, each operation or step described herein may include several sub-steps or actions.

Preparation of embedded microbial colloid

TMOS is a 98% pure liquid, MTMS is a 98% pure liquid (purchased from Sigma-Aldrich), silica nanoparticles (Ludox TM-40 colloidal silica nanoparticles, 22mm diameter silica), and the bacterial solution contains Clostridium butyricum 1×10 ⁹ or 5×10 ⁹ and suspended in phosphate buffered saline (PBS). After mixing TMOS and MTMS in a volume ratio of 2:3 (60% MTMS), then mix (TMOS+MTMS) and 5mM hydrochloric acid in a volume ratio of 1:1 to hydrolyze MTMS and TMOS to obtain an alkoxy solution. And stir at room temperature for about 2 hours. Mix the alkoxide solution, silicon nanoparticle and bacteria liquid in a volume ratio of 2.5:2.5:1, so that the alkoxide solution, bacteria liquid and silicon nanoparticle will begin to condense, and pour it into a mold to form an embedding Microbial colloid. After shaping, the embedded microbial colloid can be washed with PBS to remove the by-product methanol. In one embodiment, the microbial-embedded colloid is spherical.

Example 1 Hydrogen production capacity

Collect aquifer sediments (such as soil) from the site containing trichloroethylene pollution and add it to synthetic groundwater to make the trichloroethylene content approximately 2.5 mg/L. Synthetic groundwater components include potassium dihydrogen phosphate (KH ₂ PO ₄ ) 326.4 mg, sodium monohydrogen phosphate (Na ₂ HPO ₄ ) 1263.8 mg, magnesium sulfate heptahydrate (Mg ₂ SO ₄ 7.7H ₂ O) 98.6 mg, and CaCl ₂ . 2H ₂ O ₂ 44.1 mg, ammonium chloride (NH ₄ Cl) 10.7 mg, trace elements (trace elements) 3.35 mg (initial pH=7.5), please refer to Kao et al. in J. Harzard. Mater. 254-255, 107- 115, papers published in 2013.

The culture medium for culturing strains is mainly to provide carbon source. In one embodiment, the culture medium is a slow polycolloid matrix (slow polycolloid -releasing substrate, SPRS). SPRS includes surfactants, vegetable oils, cane molasses, water and multivitamin complexes. Please refer to Liang et al. in Water Res. 37(1), 27-38, 2013 presented paper.

The experiment was divided into seven groups as shown in Table 1, respectively: (1) LC group: natural degradation control group; (2) OC group: SPRS control group; (3) SC group: SPRS and colloid control group; 4) HA group: low-dose Clostridium butyricum without colloid group (non-embedded strain); (5) HB group: high-dose Clostridium butyricum without colloid group (non-embedded strain); (6) SHA group : Low-dose Clostridium butyricum colloid-containing group (embedded strain); and (7) SHB group: High-dose Clostridium butyricum colloid-containing group (embedded strain).

Cultivate the above seven groups for 30 days, and measure from 0 to 30 days The concentration of hydrogen dissolved in water.

Please refer to Figure 1, the SHA group and SHB group reached the highest level of hydrogen production on the 4th to 6th day. As for the HA group and HB group, the maximum amount of hydrogen production must be reached on the 15th day, where HB The hydrogen concentration of the group was only half of that of the SHB group. In other words, compared with the case of non-embedded strains, the embedded microbial colloid of the present disclosure not only reduces the hydrogen-producing time of hydrogen-producing strains by more than half, but the maximum hydrogen production can surpass that of non-embedded strains. About 1 times the degree. This shows that hydrogen-producing bacteria can stably survive in the colloid composed of TMOS, MTMS and silicon nanoparticle, and enable the hydrogen-producing bacteria to maintain the function of producing hydrogen.

Example 2 Ability to degrade chlorine-containing organic matter

The experimental group was the same as in Example 1. The above seven groups were cultured for 30 days, and the levels of trichloroethylene, cis-dichloroethylene, 1,2-dichloroethylene, vinyl chloride and ethylene in water from 0 to 30 days were measured. concentration.

Please refer to Figure 2, except for the LC group (in the case of natural degradation), the trichloroethylene in the other groups has a certain degree of reduction, and the SHB group has the highest decrease. Please refer to Figures 3 and 4, the content of intermediate products cis-dichloroethylene and 1,2-dichloroethylene after the degradation of trichloroethylene increases as the degradation time of trichloroethylene increases. It is worth observing that only the SHA group and SHB group of vinyl chloride (Figure 5) and ethylene (Figure 6) can increase with time, and other groups cannot effectively decompose the intermediate products of trichloroethylene into Ethylene which is harmless to the environment. It can be seen that the groups of non-embedded strains (HA and HB groups) cannot provide sufficient activity in the environment of chlorinated organic matter and provide sufficient hydrogen, so that they cannot provide on-site collection of The dechlorination bacteria in the sediments of the water layer decompose the chlorinated organic matter into ethylene which is harmless to the environment.

This shows that the embedded microbial colloid of the present disclosure can effectively improve the efficiency of the reductive dechlorination reaction and accelerate the dechlorination of trichloroethylene by providing a large amount of hydrogen. If the hydrogen-producing bacteria are also added but treated in a non-embedded manner, the efficiency of the reductive dechlorination reaction will be limited due to insufficient hydrogen production, and the trichloroethylene cannot be degraded to environmentally harmless ethylene.

Therefore, the embedded microbial colloid of the present disclosure can make hydrogen-producing bacteria stable in the environment of chlorinated organic matter, so that hydrogen can be produced quickly, continuously and in large quantities, and act as a supplier of electrons to assist in the removal of pollution. Chlorine bacteria accelerate the degradation of chlorine-containing organic matter.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention. Anyone familiar with the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection of the present invention The scope shall be subject to those defined in the attached patent scope.

Claims (13)

A preparation method of embedded microbial colloid, comprising: providing alkoxysilane (AS); providing tetraalkyl orthosilicate (TAOS); mixing the alkoxysilane and the tetraalkyl orthosilicate Hydrolyze the alkoxysilane and the tetraalkylorthosilicate to obtain an alkoxy solution; provide a bacteria solution including a hydrogen-producing bacteria species; and mix the alkoxysilane, The bacterial liquid and a silicon nanoparticle are condensed to obtain the embedded microbial colloid. The method according to claim 1, wherein the hydrogen-producing strain comprises Clostridium ( Clostridium ) strain, Enterobacter ( Enterobacter ) strain or a combination thereof. The method according to claim 2, wherein the Clostridium species comprises Clostridium butyricum . The method according to claim 1, wherein the alkoxysilane (AS) comprises methyltrimethoxysilane (MTMS), methyl triethoxysilane (MTES) or a mixture thereof. The method of claim 1, wherein the tetraalkyl orthosilicate (TAOS) comprises tetraethyl orthosilicate (TEOS), tetramethoxysilane (TMOS), or a combination thereof. The method according to claim 1, wherein in the step of mixing the alkoxysilane, the tetraalkylorthosilicate and the acid solution, the volume ratio of the alkoxysilane to the tetraalkylorthosilicate is 1: 0.01~100. The method according to claim 1, wherein the volume ratio of the alkoxysilane and the tetraalkylorthosilicate to the acid solution is 1:0.1-10. The method according to claim 1, wherein in the step of mixing the alkoxy solution, the bacterial solution and the silicon nanoparticle, the silicon nanoparticle is formed by covalently bonding a plurality of silicon dioxide. An embedded microbial colloid prepared by the preparation method described in claim 1. An embedded microbial colloid, comprising: a plurality of silicon nano particles; and a plurality of hydrogen-producing bacteria, wherein the silicon nano particles are covalently bonded in an oxygen-silicon-oxygen arrangement, surrounding the embedded particles Hydrogen bacteria, Wherein, the embedded microbial colloid has a plurality of holes scattered on the inside and surface of the embedded microbial colloid. A composition for degrading chlorinated organic matter, comprising: the embedded microbial colloid as described in claim 10; and a dechlorinating bacteria. The composition of item 11, wherein the bacteria comprises a dechlorination Dehalococcoides, Dehalobacter, Dehalospirillum, the genus off sulfite (Desulfitobacterium), Candida desulfurization (Desulfomonile) or combinations thereof request. The composition according to claim 11, further comprising a medium for providing a carbon source.

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