TWI880431B - Biological agent for removing pollutant and method of making the same - Google Patents

Abstract

Disclosed is a biological agent for removing pollutant. The biological agent comprises water, a carrier, an active substance, and a calcium chloride cross-linking agent. The carrier includes sodium alginate, sodium acetate, sodium lactate, sucrose, and edible gum which is one or more selected from the group consisting of xylan gum, acacia gum, and pectin. The active substance is one or more selected from the group consisting of probiotic and sugar. A method of preparing the biological agent for removing pollutant is further disclosed.

Description

Biological agent for removing pollutants and preparation method thereof

The present invention relates to a biological agent, and more particularly to a biological agent for removing pollutants and a preparation method thereof.

With the development of human beings, economic growth and progress, chlorinated volatile organic compounds (VOCs) are used as raw materials for the production of daily necessities. The chlorinated pollutants produced in the production process directly affect the environment. Chlorinated pollutants can exist in a variety of environments such as gas phase, liquid phase, soil, river, groundwater, etc., and have been proven to be related to human carcinogenicity. Therefore, restoration of related contaminated land is imperative.

The chlorine-containing pollutant removal technologies currently used in soil and water remediation include adsorption, catalytic combustion, chemical oxidation, electrochemical catalysis, zero-valent metals, and bioremediation. Among them, the bioremediation technology of bioremediation mainly uses biological preparations containing microorganisms to carry out biological dechlorination catalytic reactions to achieve the effect of removing chlorine-containing pollutants. Because this technology has a relatively small impact on the environment and can be maintained for a long time, and in response to the trend of international green energy technology, it has gradually become an important remediation method.

However, in current soil and water remediation practices, due to geological characteristics, such as the excessive permeability of the underground environment, it is difficult for biological agents used for remediation to stay in place and exert their effects, resulting in difficulties in remediation. In addition, improving the preservation and maintenance of biological agents is also a problem to be solved in practice. Therefore, it can be seen that the known biological agents used to remove chlorine-containing pollutants need to be improved.

The main purpose of the present invention is to provide a biological agent for removing pollutants, which can improve the application effect and shelf life.

To achieve the above-mentioned purpose, in one embodiment of the present invention, a biopreparation for removing pollutants is provided, comprising: water; a carrier, based on 100 wt% of the total weight of the biopreparation, comprising 2 to 5 wt% of sodium alginate, 0.01 to 0.05 wt% of sodium acetate, 0.05 to 0.1 wt% of sodium lactate, 0.2 to 5 wt% of sucrose, and an edible gum, based on 100 wt% of the total weight of the biopreparation, wherein the edible gum is selected from 0.1 to 1 wt% of xylan gum, 0.05 to 0.2 wt% of gum arabic, and 0.5 to 2 wt% of pectin; an active substance selected from one or more of the group consisting of probiotics and sugars; and a calcium chloride crosslinking agent. Further, when the active substance is a probiotic, the amount of the probiotic can be 10 ⁸ to 10 ¹⁰ CFU/L; when the active substance is sugar, the content of the sugar can be 0.1 to 4 wt%, for example: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 wt%.

In one embodiment of the present invention, the probiotic is one or more selected from the group consisting of Desulfuromonas, Dehalococcoides, Dehalobacter, Clostridum, Acetobacterium, Rhodobacter, Pseudomonas and Starkeya.

In one embodiment of the present invention, the calcium chloride crosslinking agent does not contain boric acid.

In one embodiment of the present invention, the sugar includes glucose and fructose.

In one embodiment of the present invention, the concentration of the calcium chloride crosslinking agent is 1 to 5 wt % based on 100 wt % of the total weight of the biological preparation, and the calcium chloride crosslinking agent is solidified with the active substance to form a plurality of crystal spheres.

To achieve the above-mentioned purpose, in one embodiment of the present invention, a method for preparing the above-mentioned biopreparation for removing pollutants is provided, comprising: mixing water, 2 to 5 wt% of sodium alginate, 0.01 to 0.05 wt% of sodium acetate, 0.05 to 0.1 wt% of sodium lactate, 0.2 to 5 wt% of sucrose and edible gum based on 100 wt% of the total weight of the biopreparation to form a colloid, wherein the edible gum is selected from 0.1 to 1 wt% of xylan gum, 0.05 to 0.2 wt% of gum arabic and 0.5 to 2 wt% of sucrose based on 100 wt% of the total weight of the biopreparation. wt% of pectin; after adding the colloid to the active substance, stirring evenly to form an active substance colloid; and adding calcium chloride crosslinking agent to the active substance colloid and standing for 1 to 5 hours to solidify and form.

In one embodiment of the present invention, the probiotic is one or more selected from the group consisting of Desulfuromonas, Dehalococcoides, Dehalobacter, Clostridum, Acetobacterium, Rhodobacter, Pseudomonas and Starkeya.

In one embodiment of the present invention, the sugar includes glucose and fructose.

In one embodiment of the present invention, the calcium chloride crosslinking agent does not contain boric acid.

In one embodiment of the present invention, the active substance colloid is added dropwise to the calcium chloride crosslinking agent and solidified to form a plurality of crystal spheres.

The beneficial effects of the invention are: the diameter of the crystal spheres produced is about 5 mm to 30 mm, and the shape can be changed according to the needs. The form and structure are suitable for deployment. After being deployed on the spot, it can effectively remove chlorine-containing pollutants, and the effect lasts for about 2 months, and then dissolves and disappears. It will not cause environmental acidification during the whole period. It does not need to be recycled after use, maintaining the environment naturally without residues, and saving manpower. In particular, it has also shown good remediation effects for sites with specific soil and too fast groundwater flow.

The following will be combined with the attached drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. In addition, in order to better illustrate the present invention, many specific details are given in the specific embodiments below. Those skilled in the art should understand that the present invention can also be implemented without certain specific details.

According to one embodiment of the present invention, a biological preparation for removing pollutants comprises water, a carrier, an active substance and a calcium chloride crosslinking agent. Based on 100 wt% of the total weight of the biological preparation, the water content is 85 to 90 wt% (e.g., 85, 86, 87, 88, 89, 90 wt%), the carrier includes 2 to 5 wt% (e.g., 2, 2.5, 3, 3.5, 4, 4.5, 5 wt%) of sodium alginate, 0.01 to 0.05 wt% (e.g., 0.01, 0.02, 0.03, 0.04, 0.05 wt%) of sodium acetate, 0.05 to 0.1 wt% (e.g., 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 wt%) of sodium lactate, 0.2 to 5 wt% (e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 wt%) of sucrose and edible gum; the edible gum is selected from 0.1 to 1 wt% (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 wt%) of xylan gum, 0.05 to 0.2 wt% (e.g., 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2 wt%) of gum arabic and 0.5 to 2 wt% (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 wt%) of The active substance is selected from one or more of the group consisting of probiotics and sugars. When the active substance is probiotics, the amount of probiotics can be 10 ⁸ to 10 ¹⁰ CFU/L; when the active substance is sugar, the content of sugar can be 0.1 to 4 wt%, for example: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 wt%.

In one embodiment, the probiotics are selected from one or more of the group consisting of Desulfuromonas , Dehalococcoides , Dehalobacter , Clostridum , Acetobacterium , Rhodobacter , Pseudomonas , and Starkeya . Furthermore, in one embodiment, the probiotics may be Pseudomonas LY 2-3, which was deposited at the Food Industry Development Research Institute on November 12, 2014, with the deposit number BCRC 910662. It was screened from the vinyl chloride plant of Taiwan Vinyl Chloride Corporation in the Linyuan Industrial Park in Kaohsiung, which is adjacent to the sea, and can be used to degrade chlorinated organic pollutants.

In one embodiment, the sugars include glucose and fructose.

In one embodiment, the calcium chloride crosslinking agent does not contain boric acid.

In one embodiment, the concentration of the calcium chloride crosslinking agent is 1 to 5 wt %, such as 1, 2, 3, 4, 5 wt %.

According to one embodiment of the present invention, a method for preparing the above-mentioned biological preparation for removing pollutants comprises: based on the total weight of the biological preparation of 100 wt%, 85 to 90 wt% of water, 2 to 5 wt% (for example: 2, 2.5, 3, 3.5, 4, 4.5, 5 wt%) of sodium alginate, 0.01 to 0.05 wt% (for example: 0.01, 0.02, 0.03, 0.04, 0.05 wt%) of sodium acetate, 0.05 to 0.1 wt% (for example: 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 wt%) of sodium lactate, 0.2 to 5 wt% (e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 wt%) of sucrose and an edible gum are mixed to form a colloid, wherein the edible gum is selected from 0.1 to 1 wt% (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 wt%) of xylan gum, 0.05 to 0.2 wt% (e.g., 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2 wt%) of gum arabic and 0.5 to 2 wt% (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2 wt%) of pectin; slowly adding the colloid to the active substance, stirring evenly to form an active substance colloid; finally adding the calcium chloride crosslinking agent to the active substance colloid, and standing for 1 to 5 hours (for example: 1, 2, 3, 4, 5 hours) to solidify and form the biological preparation of the present invention.

In one embodiment, the biological agent is in the form of a spherical particle having a particle size of 5 millimeters (mm) to 30 mm, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 mm.

In one embodiment, the active substance colloid can be dripped into a 4°C calcium chloride crosslinker without boric acid through a dropper or a peristaltic pump, and solidified into a plurality of crystal spheres after being left to stand for 1 to 5 hours. Furthermore, the formed crystal spheres can be washed with phosphate buffered saline (PBS) or RO distilled water before application or storage.

Several embodiments are given below to further illustrate the biological agent for removing pollutants and the preparation method thereof of the present invention, but they are not intended to limit the present invention. Anyone skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention.

Preparation of Biospheres (Biological Agents of the Invention):

Embodiment 1:

85 to 90 wt% of water, 2 to 5 wt% of sodium alginate, 0.2 to 5 wt% of sucrose, 0.1 wt% of glucose and 0.1 to 1 wt% of xylose colloid are mixed to obtain a colloid; the colloid is slowly added to the Pseudomonas LY 2-3 to be embedded, and then stirred evenly to form an active substance colloid, wherein the bacterial count of Pseudomonas LY 2-3 is 1.71 x 10 ⁹ CFU/mL; finally, the active substance colloid is added to 2 to 5 wt% of calcium chloride crosslinking agent at 4°C without boric acid through a peristaltic pump, and is left to stand for 2 hours to solidify and form a plurality of biological crystal balls. After testing, the strain growth is good, and the number of live bacteria after embedding can reach 10 ⁸ CFU/mL.

Embodiment 2:

90 to 95 wt% of water, 2 to 5 wt% of sodium alginate, 0.1 to 1 wt% of xylan gum, 0.1 wt% of gum arabic and 1 wt% of pectin are mixed to obtain a colloid; 1 to 5 wt% of molasses, 0.2 to 2 wt% of glucose, 0.2 to 5 wt% of sucrose and 0.1 to 0.2 wt% of fructose to be embedded, and appropriate amounts of other trace elements and minerals are slowly added to the colloid, and the mixture is stirred evenly to form an active substance colloid; finally, 2 to 5 wt% of calcium chloride crosslinking agent without boric acid at 4° C. is added to the active substance colloid through a dropper, and the mixture is left to stand for 3 hours to solidify and form a plurality of biological crystal balls.

Embodiment 3:

85 to 90 wt% of water, 2 to 5 wt% of sodium alginate, 0.01 to 0.05 wt% of sodium acetate, 0.05 to 0.1 wt% of sodium lactate, 0.005 to 0.01 wt% of B12 and 0.1 wt% of gum arabic are mixed to obtain a colloid; the colloid is slowly added to the anaerobic bacteria to be embedded, including Dehalococcoides , Dehalobacter , Clostridum and Acetobacterium , and stirred to form an active substance colloid; finally, the active substance colloid is added dropwise to 2 to 5 liters of liquid at 4°C without boric acid through a dropper. wt% calcium chloride cross-linking agent, and allowed to stand for 1.5 hours to solidify and form a plurality of biological crystal spheres.

Embodiment 4:

85 to 90 wt% of water, 2 to 5 wt% of sodium alginate, 0.1 wt% of glucose, 0.1 wt% of gum arabic, 1 wt% of pectin, 0.1 wt% of NaCl, 0.1 wt% of NH ₄ Cl, 0.01 wt% of KCl, 0.045 wt% of KH ₂ PO ₄ , 0.09 wt% of K ₂ HPO ₄ , 0.03 wt% of MgSO ₄ ⸳7H ₂ O and 0.2 wt% of trace elements are mixed to obtain a colloid; the colloid is slowly added to the aerobic Pseudomonas LY 2-3 to be embedded, and stirred to form an active substance colloid; finally, the active substance colloid is added dropwise to 2 to 5 % boric acid-free 4°C 4% boric acid-free ... wt% calcium chloride cross-linking agent, and allowed to stand for 2 hours to solidify and form a plurality of biological crystal spheres.

Embodiment 5:

85 to 90 wt% of water, 2 to 5 wt% of sodium alginate, 0.2 to 5 wt% of sucrose, 0.1 to 1 wt% of xylan gum, 0.1 wt% of gum arabic and 1 wt% of pectin are mixed to obtain a colloid; aerobic bacteria to be embedded, which may be Rhodobacter , Pseudomonas , Starkeya , etc., are slowly added to the colloid and stirred evenly to form an active substance colloid; finally, the active substance colloid is dripped into 2 to 5 wt% of calcium chloride crosslinking agent without boric acid at 4°C through a dropper, and is left to stand for 3 hours to solidify and form a plurality of biological crystal balls.

Effect of colloid addition order on biospheres:

The experiment was conducted on the addition method of colloid and active substance. The experiment was divided into two groups, A and B. Group A used colloid as solute and active substance as solvent, and the method of adding colloid into active substance was adopted. Group B used colloid as solvent and active substance as solute, and the method of adding active substance into colloid was adopted. Both groups were stirred at 500 rpm for 30 minutes. The results showed that group B, which used colloid as solvent and active substance as solute, was prone to uneven mixing. This is because the thickness of the colloid makes it difficult for the colloid in the upper layer to be evenly mixed with the active substance. On the contrary, group A, which uses the active substance as the solvent and the colloid as the solute, can be mixed evenly and the mixing time can be effectively shortened.

Furthermore, the above two groups A and B were made into biological crystal balls and tested for their degradation effect on pollutants, namely ethylene dichloride (EDC). Referring to Figure 1, the crystal balls of group A completely degraded ethylene dichloride on the 5th day, whereas no degradation was observed in group B on the 5th day. From the above information, it can be seen that the method of adding active substances into colloids will make it impossible for the active substances and colloids to be mixed evenly, thereby affecting the reaction contact surface, reducing the degradation effect of the biological crystal balls, and even making them ineffective.

Effects of crosslinking agent ingredients on biocrystals:

The biocrystal spheres obtained by using calcium chloride crosslinking agents with or without boric acid were tested. Referring to Figures 2A and 2B, the results show that using calcium chloride crosslinking agents containing boric acid will cause adhesion during the titration process, the contents will easily spill, and the shape of the formed spheres will be irregular, resulting in the failure of the crystal sphere formation. On the contrary, using calcium chloride crosslinking agents without boric acid can smoothly form spheres. The effect of the presence or absence of boric acid on the efficacy of the biocrystal spheres was also tested. Referring to Figure 3, the biospheres obtained by using calcium chloride crosslinking agent containing boric acid affected the degradation effect of ethylene dichloride. No degradation reaction occurred on the first day. On the fifth day, the biospheres without calcium chloride crosslinking agent without boric acid completely decomposed ethylene dichloride, but the biospheres with calcium chloride crosslinking agent containing boric acid still had pollutant residues. In summary, the biospheres prepared by using calcium chloride crosslinking agent without boric acid have a complete shape (Figure 4) and have a good degradation effect on ethylene dichloride.

Bio-crystal activity assessment:

The biospheres obtained in Example 3 were applied to sites contaminated by trichloroethylene (TCE), cis-dichloroethylene (cis-DCE) and vinyl chloride (VC), and the effects of the application of a single bacterial agent (i.e., only the probiotics used in Example 3, not in the form of biospheres) were compared. The test results are shown in Figure 5A (TCE), Figure 5B (cis-DCE) and Figure 5C (VC). The biospheres of the present invention and the single bacterial agent were able to completely degrade trichloroethylene (Figure 5A) and reduce the concentration of vinyl chloride by 50% (Figure 5C) after 1 month, and a biological reaction occurred to produce cis-dichloroethylene (Figure 5B). Furthermore, on the 100th day, the biospheres of the present invention completely decomposed all pollutants and had better effects than the single bacterial agent (Figure 5B, Figure 5C). The above results show the feasibility of colloid encapsulation of anaerobic bacteria and enhance the ability of anaerobic bacteria.

Save test:

The storage test of the biological crystal spheres of the present invention was conducted at three time points: 1 month, 3 months, and 6 months. The method is: directly store the prepared biological crystal spheres in a 50 mL centrifuge tube or other suitable container, and place them in a cool place at room temperature. The results show that after being stored at room temperature for 1 month and 3 months, the crystal spheres are still intact and still have very good biological activity. They can completely degrade the pollutant ethylene dichloride, indicating that the dosage form of the crystal spheres not only has the ability to protect biological preparations, but also can provide strains for growth thereon, and can also achieve the effect of storing products at the same time. In addition, the biological crystal spheres of the present invention do not need to be activated separately when used, and can be directly taken out and used, which increases convenience.

Field Trial:

The biocrystal spheres of Example 2 were placed in a chlorine-contaminated site for testing. The pollutants in the chlorine-contaminated site included vinyl chloride, cis-dichloroethylene, trichloroethylene and dichloroethane. The site was characterized by a fast groundwater flow rate. After three months of continuous monitoring, the data obtained showed that during the application of the biocrystal spheres, the expression of degradation genes and the number of local bacterial flora could be maintained, and the expression of related genes could be increased by 10 to 100 times (Figure 6). At the same time, the pH of the local water quality could still be maintained neutral. This effect could be maintained for at least 2 months. Aceto in Figure 6 represents Acetobacterium , Clo represents Clostridum, DHB represents Dehalobacterium , and bvcA and vcrA are pollutant decomposition genes.

The biological crystal balls of Example 4 and Example 5 were placed in a chlorine-contaminated site for testing. Example 4 was used to treat the pollution of ethylene dichloride (Figure 7A), while Example 5 was used to treat the pollution of vinyl chloride (Figure 7B). Referring to Figures 7A and 7B, BM1, BM11, BM12, and BM18 are the codes of the test wells. One month after placement, the concentrations of pollutants showed a downward trend except for BM12, which showed an increase in concentration. Two months later, both ethylene dichloride and vinyl chloride decreased significantly, and in some test wells, the complete disappearance of pollutants was observed. Referring to Figure 7A, ethylene dichloride began to rise in the third month, which is estimated to be because the active crystal balls have completed their reaction and have lost their activity. In summary, even if the flow rate is faster, the active crystal ball of the present invention can still play a role, so that the active substances therein can interact with the environment uninterruptedly and continuously, and only need to be replenished every 2 months to maintain the protective effect and prevent the concentration of pollutants from rising again, which has practical application value.

Although the present invention has been disclosed in a preferred embodiment, it is not intended to limit the present invention. Anyone skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the attached patent application.

without

Figure 1 shows the results of the effect of the order of colloid addition on the obtained biocrystal spheres, wherein CK shown in other figures represents a control check; Figures 2A to 2B show the results of the effect of the crosslinking agent components on the biocrystal spheres; Figure 3 shows the results of the effect of the crosslinking agent components on the biocrystal spheres; Figure 4 shows the biocrystal spheres prepared using a calcium chloride crosslinking agent that does not contain boric acid; Figures 5A to 5C show the degradation effects of the biocrystal spheres of the present invention on trichloroethylene, cis-dichloroethylene and vinyl chloride; Figure 6 shows the effects of the biocrystal spheres of the present invention in a field test; and Figures 7A and 7B show the effects of the biocrystal spheres of the present invention in a field test.

Pseudomonas sp. LY 2-3 was deposited to Food Industry Development Research Institute on November 12, 2014, with the deposit number BCRC 910662.

Claims (3)

A biopreparation for removing pollutants, comprising: based on 100wt% of the total weight of the biopreparation, 85 to 90wt% of water; a carrier, based on 100wt% of the total weight of the biopreparation, comprising 2 to 5wt% of sodium alginate, 0.01 to 0.05wt% of sodium acetate, 0.05 to 0.1wt% of sodium lactate, and 0.2 to 5wt% of sucrose; and food The edible gum is selected from one or more of the group consisting of 0.1 to 1 wt% xylan gum, 0.05 to 0.2 wt% gum arabic and 0.5 to 2 wt% pectin, based on 100 wt% of the total weight of the biological preparation; the active substance is selected from one or more of the group consisting of probiotics and sugars, wherein the probiotics are selected from Desulfuromonas spp. The invention relates to a probiotic comprising one or more of the following: probiotics selected from the group consisting of Desulfuromonas , Dehalococcoides , Dehalobacter , Clostridum , Acetobacterium , Rhodobacter , Pseudomonas and Starkeya , wherein the amount of the probiotics is 10 ⁸ to 10 ¹⁰ CFU/L, the sugar comprises glucose and fructose, and the content of the sugar is 0.1 to 4 wt% based on 100 wt% of the total weight of the biological preparation; and 1 to 5 wt% of a calcium chloride crosslinking agent based on 100 wt% of the total weight of the biological preparation, wherein the calcium chloride crosslinking agent does not contain boric acid. The biological preparation as described in claim 1, wherein the calcium chloride crosslinking agent and the active substance are solidified into a plurality of crystal spheres. A method for preparing a biopreparation for removing pollutants as described in claim 1, comprising the following steps: Based on the total weight of the biopreparation of 100wt%, 85 to 90wt% of water, 2 to 5wt% of sodium alginate, 0.01 to 0.05wt% of sodium acetate, 0.05 to 0.1wt% of sodium lactate, 0.2 to 5wt% of sucrose and edible gum are mixed to form a colloid, wherein based on the total weight of the biopreparation of 100wt%, the edible gum is selected from 0 .1 to 1 wt % of xylan gum, 0.05 to 0.2 wt % of gum arabic and 0.5 to 2 wt % of pectin; after adding the colloid to the active substance, stirring evenly to form an active substance colloid, wherein the active substance is selected from one or more of the group consisting of probiotics and sugars, wherein the probiotics are selected from Desulfuromonas spp. ( One or more of the group consisting of Desulfuromonas , Dehalococcoides , Dehalobacter , Clostridum , Acetobacterium , Rhodobacter , Pseudomonas and Starkeya , and the amount of the probiotic is 10 ⁸ to 10 ¹⁰ CFU/L, the sugar includes glucose and fructose, and the content of the sugar is 0.1 to 4wt% based on 100wt% of the total weight of the biological preparation; and the active substance colloid is added dropwise to a calcium chloride crosslinking agent at 4°C and allowed to stand for 1 to 5 hours to solidify into a plurality of crystal spheres, wherein the content of the calcium chloride crosslinking agent is 1 to 5wt% based on 100wt% of the total weight of the biological preparation, and the calcium chloride crosslinking agent does not contain boric acid.