Preparation method for bioremediation enhancer, bioremediation enhancer, and biotreatment method

Abstract

This invention relates to a method for preparing a biological remediation aid, the biological remediation aid itself, and a biological treatment method. The method for preparing the biological remediation aid includes: implanting an algal seed source into a culture medium, placing it in a photosynthetic acclimation tank, continuously introducing gas, and acclimating it for at least 7 days to obtain photosynthetically acclimated decomposition-aiding algae; and further processing the aforementioned decomposition-aiding algae to obtain a biological remediation aid suitable as a carbon source and electron supply source for organic matter degrading bacteria. Therefore, the method for preparing the biological remediation aid and the biological treatment method of this invention can remove algae from water bodies, help control eutrophication of water bodies, convert algal waste into valuable biological remediation aids, and ultimately reduce greenhouse gas emissions.

Description

Technical Field

[0001] This invention relates to a method for preparing a biological remediation adjuvant, the biological remediation adjuvant, and a biological treatment method, particularly to a biological remediation adjuvant for removing algae that cause eutrophication from water bodies as a halogenated organic solvent pollutant in groundwater, its preparation method, and its biological treatment method. Prior Technology

[0002] Halogenated organic pollutants (HOPs) are organic compounds containing halogen atoms (such as chlorine, bromine, fluorine, and iodine), characterized by high stability, high biotoxicity, and reluctance to degrade. These pollutants are widespread in the global environment, posing a serious threat to ecosystems and human health. HOPs decompose extremely slowly in the natural environment, easily accumulating gradually in the food chain, thus posing a threat to top predators and humans.

[0003] Halogenated organic compounds (HOCs) are a major component of groundwater pollution. Examples include trichloroethylene. Halogenated hydrocarbons are frequently found in industrial wastewater and municipal sewage, posing a threat to aquatic ecosystems. Due to their persistence, toxicity, and global impact, HOCs have become a major challenge to global environmental pollution and ecological health. While progress has been made in remediation technologies and international regulation, challenges remain in terms of technology and resources, necessitating strengthened research and development of innovative technologies and international cooperation to address this issue.

[0004] Current treatment technologies include activated carbon adsorption, high-efficiency membrane separation, chemical oxidation (such as ozone oxidation and Fenton's reagent), supercritical water oxidation, and the use of microorganisms to degrade or transform halogenated organic pollutants (such as anaerobic dehalogenation). However, halogenated organic pollutants are diverse in type and properties, making unified treatment difficult. Some pollutants enter remote areas and polar regions, or are discharged into soil and groundwater aquifers, making treatment challenging. Furthermore, the detection and removal of low-concentration pollutants are costly.

[0005] Furthermore, the largest carbon emission source in the anaerobic reduction technology currently used for the biological remediation of halogenated organic pollutants is methane, which has a greenhouse effect equivalent of 34 times that of carbon dioxide, resulting in higher carbon emissions from biological remediation compared to conventional physicochemical methods. Secondly, providing a large amount of carbon source beyond demand leads to the proliferation of other anaerobic bacteria that do not contribute to pollutant degradation, resulting in high carbon dioxide emissions.

[0006] Some organic compounds, such as nitrogen- or phosphorus-containing compounds, can be directly or indirectly utilized by algae as nutrients for growth, promoting the rapid proliferation of cyanobacteria and green algae. Although this process absorbs carbon dioxide from the atmosphere, it also leads to eutrophication of water bodies. Eutrophication results in the accumulation of large amounts of biomass, and the decomposition of this biomass by aerobic microorganisms leads to dissolved oxygen consumption, creating an anaerobic environment that favors the growth of anaerobic microorganisms. The decomposition of organisms by anaerobic microorganisms releases hydrogen sulfide, ammonia, and thiols, further causing the death of aquatic organisms and, in severe cases, forming dead zones. Therefore, removing nitrogen and phosphorus from water has become the most important issue in addressing eutrophication. Currently, algae removal is the most direct and effective method, but the lack of economic incentives is the biggest challenge, and algae waste disposal requires additional costs; moreover, current waste disposal costs have risen to at least NT$10,000 per metric ton (and if it is hazardous, the entry fee can reach over NT$40,000 per metric ton). Therefore, how to effectively utilize carbon sinks such as algae as remediation aids and reduce methane generation is an important research topic in bioremediation.

[0007] Previous technologies offer various methods for eutrophication control, but lack follow-up utilization for the most effective algae removal. Furthermore, the raw materials used in trichloroethylene bioremediation adjuvants must be obtained, manufactured, and distributed in ways that increase carbon emissions, and excessive methane emissions are not considered during on-site use. A solution is needed that integrates carbon reduction, eutrophication control, circular economy, and minimizing the carbon footprint of adjuvants during manufacturing and use.

[0008] In summary, in order to overcome the aforementioned shortcomings, the inventors of this case have devoted considerable research and development energy and spirit to continuous breakthroughs and innovations in this field, hoping to solve the deficiencies of conventional methods with novel technical means, thereby bringing better products to society and promoting industrial development. Summary of the Invention

[0009] The main objective of this invention is to provide a method for preparing a biological remediation adjuvant, the biological remediation adjuvant itself, and a biological treatment method. The biological remediation adjuvant of this invention is mixed algae found in eutrophic water bodies in Taiwan, and the cultivation environment is a naturally eutrophic water body. The reasons for using algae as a biological remediation adjuvant in this invention are as follows: First, algae are rich in carbohydrates, which can act as fermenting bacteria to produce acetic acid. Acetic acid is the carbon source for Dehalococcoides, the most effective bacteria for degrading chlorinated organic matter. Second, algae are rich in carbohydrates, which can act as fermenting bacteria to produce hydrogen. Hydrogen is the electron donor for Dehalococcoides, the most effective bacteria for degrading chlorinated organic matter. Third, Dehalococcoides require vitamin B12 when degrading halogenated organic solvents, and cyanobacteria in algae are one of the main sources of vitamin B12 extracted industrially. Fourth, microbial growth requires trace elements, which can be provided by the algal bodies after the algae die.

[0010] To achieve the above objectives, the present invention provides a method for preparing a biological remediation aid, comprising: preparing an algal culture medium, wherein 1500.0 mg of sodium nitrate, 40.0 mg of dipotassium hydrogen phosphate, 75.0 mg of magnesium sulfate, 36.0 mg of calcium chloride, 6.0 mg of ferric ammonium citrate, 6.0 mg of citric acid, 1.0 mg of disodium EDTA, and 20.0 mg of sodium carbonate, and other trace elements are added to every 1 liter of deionized water; the medium is sterilized under high temperature and high pressure (121°C, 1.5 atm) for 15 minutes; nitrogen gas is introduced to cool the medium; and the pH is adjusted to 7.0 using a pH adjuster. Next, an algal germplasm is cultivated, which is implanted into the culture medium and placed in a photosynthetic cultivation tank, with continuous gas introduction for cultivation for at least 7 days, thereby obtaining photosynthetically cultivated decomposition aid algae. Furthermore, a biological remediation adjuvant is prepared, and the aforementioned decomposition-aiding algae are further processed to obtain a biological remediation adjuvant suitable as a carbon source and electron supply source for organic matter degrading bacteria; wherein the algae source is a natural algae growing in eutrophic water bodies; the yield of the biological remediation adjuvant is at least 500g dry weight per cubic meter of algae; the acid-base buffer includes at least one selected from NaHCO3, KH2PO4, K2HPO4, and combinations thereof; the acid-base adjuster includes at least one selected from HCl and NaOH.

[0011] In the preparation method of the biological remediation adjuvant of the present invention, the algae seed source is implanted into the algae culture medium after undergoing a pretreatment; the pretreatment includes at least one selected from the following: crushing treatment, crushing and sterilization treatment, fermentation treatment, and combinations thereof.

[0012] The preparation method of the bioremediation adjuvant of the present invention further includes: a step of concentrating the bioremediation adjuvant to obtain a high concentration of bioremediation adjuvant of 30.0 g / L or more; the concentration includes at least one selected from gravity sedimentation, filtration concentration, centrifugal concentration, vacuum concentration, freeze drying, and combinations thereof.

[0013] In the preparation method of the biological remediation adjuvant of the present invention, the algae species belongs to the genus *Leptolyngbya boryana* of *Cyanobacteria*.

[0014] In the preparation method of the biological remediation adjuvant of the present invention, the introduced gas is carbon dioxide.

[0015] To achieve the other objective mentioned above, the present invention provides a bioremediation adjuvant, which is prepared by the preparation method described above, and the bioremediation adjuvant is suitable for treating halogenated organic pollutants (HOPs).

[0016] To achieve another objective mentioned above, the present invention provides a biological treatment method for halogenated organic pollutants (HOPs) in water, comprising: inoculating a biological treatment adjuvant and an HOP-degrading bacterium into a water body containing halogenated organic pollutants, and acclimating them for at least 3 days; wherein the halogenated organic pollutants are 1,1,2,2-tetrachloroethane (1,1,2,2-PCA), 1,1,1-trichloroethane (1,1,1-TCA), 1,1,2-trichloroethane (1,1,2-TCA), 1,2-dichloroethane (1,2-DCA), 1,1-dichloroethane (1,1-DCA), chloroethane (CA), carbon tetrachloride (C2C), and chloroethane (CA). The bioremediation adjuvant is selected from at least one of the following: CT), chloroform (CF), methylene chloride (MC), chloromethane (CM), tetrachloroethylene (PCE), trichloroethene (TCE), cis-1,2-dichloroethene (cis-1,2-DCE), trans-1,2-dichloroethene (trans-1,2-DCE), 1,1-dichloroethene (1,1-DCE), vinyl chloride (VC), chlorobenzene (MCB), dichlorobenzene (DCB), and combinations thereof. Furthermore, the bioremediation adjuvant is prepared by the method described above, or is a bioremediation adjuvant as described above. Furthermore, the HOP-degrading bacterial strain was acclimated in an anaerobic medium with an HOP concentration of at least 1.0 mg / L for at least one month. The strain was obtained by selecting the bacterial group with the lowest methane production during the acclimation process, using thermal screening temperature, pH value, salinity and water content as control factors and methane production as the screening factor.

[0017] In the preparation method of the biological treatment adjuvant of the present invention, the anaerobic culture medium is selected from at least one of Resazurin, (NH4)2HPO4, CaCl2·2H2O, MgCl2·6H2O, KCl, MnCl2·4H2O, CoCl2·6H2O, H3BO3, CuCl2·2H2O, Na2MoO4·2H2O, ZnCl2, FeCl2·4H2O, Na2S·7H2O, Biotin, Folic Acid, Pyridoxine HCl, Riboflavin, Thiamin, Pantothenic Acid, Nicotinic Acid, p-Aminobenzoic Acid, Thioctic Acid, Vitamin, yeast extract, NaHCO3, KH2PO4, and K2HPO4.

[0018] In the preparation method of the biological treatment adjuvant of the present invention, when the biological treatment adjuvant is obtained by acclimating algae seed sources that have been crushed into the algae culture medium and then processing them, the degradation rate of HOP in the biological treatment method is 90.0% or higher.

[0019] In the preparation method of the biological remediation adjuvant of the present invention, when the biological remediation adjuvant is obtained by acclimating algae seed sources that have undergone crushing and high-temperature and high-pressure sterilization into the algae culture medium and then processing them, the degradation rate of the halogenated organic pollutants is 89.5% or higher. Simple Explanation of the Diagram

[0020] Figure 1 is a flowchart of the preparation method of the biological remediation adjuvant of the present invention. Figure 2 is a side view of the culture facility for the biological treatment aid of the present invention. Figure 3 shows the trichloroethylene degradation curves of experimental examples 1-4 of the biological treatment method of the present invention. Figure 4 shows the trichloroethylene degradation curves of experimental examples 5-8 of the biological treatment method of the present invention. Figure 5 shows the trichloroethylene degradation curves of experimental examples 9-12 of the biological treatment method of the present invention. Figure 6 is a graph showing the degradation curve of trichloroethylene compared with three commercially available agents using the bio-treatment adjuvants used in this invention. Figure 7 shows the degradation curves of cis-1,2-dichloroethylene compared with three commercially available agents using the bio-treatment adjuvants used in this invention. Figure 8 is a graph showing the degradation curves of vinyl chloride compared with three commercially available agents using the bio-treatment adjuvants used in this invention. Figure 9 is a graph showing the ethylene degradation curves of the bioremediation adjuvant used in this invention compared with three commercially available agents. Figure 10 is a comparison curve of dissolved organic carbon concentration in Experimental Examples 13 to 18 of the present invention. Figure 11 is a comparison chart of methane concentrations in Experimental Examples 13 to 18 of this invention. Figure 12 is a comparison chart of phosphate concentrations in Experimental Examples 13 to 18 of this invention. Implementation

[0021] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the invention are now described in detail with reference to the accompanying drawings. The detailed description and technical content of the present invention are illustrated below in conjunction with the accompanying drawings; however, the accompanying drawings are provided for reference and illustration only and are not intended to limit the scope of the invention.

[0022] Please refer to Figure 1, which is a flowchart of the preparation method of the biological remediation adjuvant of the present invention.

[0023] First, the algal strains used in this invention can be cultured using a specific culture medium or commercially available culture media. Commercially available culture media include, but are not limited to, BG-11 medium, BG11 medium for cyanobacteria, BG11 medium for cyanobacteria, and cyanobacterial medium. Alternatively, for example, commercially available culture media can be used for ease of acquisition; however, from the viewpoint of obtaining more efficient algal cultivation, it is preferable to use a specific culture medium.

[0024] In one embodiment of the present invention, a specific algal culture medium containing multiple components is used to cultivate algal seed sources to obtain a biological treatment adjuvant. For example, the algal culture medium suitable for use in the present invention may have the components shown in Table 1, which is obtained by adding 1500.0 mg of sodium nitrate, 40.0 mg of dipotassium hydrogen phosphate, 75.0 mg of magnesium sulfate, 36.0 mg of calcium chloride, 6.0 mg of ferric ammonium citrate, 6.0 mg of citric acid, 1.0 mg of disodium EDTA, and 20.0 mg of sodium carbonate and other trace elements to every 1 liter of deionized water, sterilizing at high temperature and high pressure (121°C, 1.5 atm) for 15 minutes, cooling it by purging with nitrogen gas, and adjusting the pH to 7.0 using an acid-base adjuster.

[0025] Table 1 Chinese drug name Chemical formula Dosage per liter (mg / L) Sodium nitrate NaNO3 1500.0 dipotassium hydrogen phosphate K 2HPO 4‧3H 2O 40.0 Magnesium sulfate MgSO₄‧7H₂O 75.0 Calcium chloride CaCl₂‧2H₂O 36.0 Ferric ammonium citrate (NH₄)₅[Fe(C₆H₈O₇)₂] 6.0 Citric acid C 6H 8O 7 6.0 Disodium-EDTA Na 2EDTA 1.0 Sodium carbonate Na₂CO₃ 20 Trace elements H₃BO₃ (boric acid) 0.5286 MnCl₂‧4H₂O (manganese chloride tetrahydrate) 0.3345 ZnSO₄·7H₂O (Zinc sulfate heptahydrate, ferrous sulfate) 0.0410 Na₂MoO₄‧2H₂O (Sodium molybdate dihydrate) 0.0721 CuSO₄·5H₂O (copper sulfate pentahydrate) 0.0146 Co(NO3)2‧6H2O (cobalt nitrate hexahydrate[II]) 0.0091

[0026] Furthermore, according to literature reports, this species belongs to the genus *Cyanobacteria* and is widely distributed in terrestrial environments and freshwater. Due to environmental factors such as oxygen content, available water content, and differences in light intensity, soil surface, and water bodies, terrestrial *Leptolyngbya* likely exhibits genomic differences from freshwater species to adapt to terrestrial habitats. *Leptolyngbya* can synthesize tetrapyrrole under microaerobic conditions. Tetrapyrrole is the molecular center structure of vitamin B12 and phycobilins, and also serves as a major structural molecule for porphyrins (such as heme) and chlorophyll. It possesses genes for trehalose biosynthesis, and some strains have the ability to produce spore-like amino acids and spore-like glycine.

[0027] The following describes the identification of algal strains suitable for use in this invention. First, for algae, the 16S rRNA gene sequence was sequenced using sequencing technology, and then compared with a database of biological strains with known 16S rRNA gene sequences. The results showed that the strain similarity was over 97%, with the closest strain being *Leptolyngbya boryana*. Therefore, it can be inferred that the algal strain used in this invention is a cyanobacterium, *Leptolyngbya boryana*.

[0028] Furthermore, in this invention, cyanobacteria, *Leptolyngbya boryana*, are used as the algal seed source. After large-scale cultivation, the relevant experiments in the following embodiments are performed. In one embodiment of this invention, for example, natural algae growing in eutrophic water bodies may be used as the algal seed source, including but not limited to.

[0029] For example, in the step of domesticating an algal germplasm in this invention, the algal germplasm, belonging to the genus *Leptolyngbya boryana* of *Cyanobacteria*, is implanted into the culture medium and placed in a photosynthetic domestication tank. Carbon dioxide is continuously introduced into the tank, and domestication is carried out for at least 7 days to obtain photosynthetically domesticated decomposition-aiding algae. The algal germplasm is implanted into the algal culture medium after undergoing a pretreatment; the pretreatment includes at least one selected from: crushing treatment, crushing and sterilization treatment, fermentation treatment, and combinations thereof.

[0030] Furthermore, in the step of preparing a bioremediation aid in this invention, the aforementioned decomposition-aiding algae are further processed to obtain a bioremediation aid suitable as a carbon source and electron supply source for organic matter degrading bacteria.

[0031] Furthermore, the subsequent processing methods applicable to this invention are not particularly limited, for example, including but not limited to: concentration, dehydration, heating and drying, natural air drying, sun drying, film drying, drum drying, freeze drying, vacuum drying, freeze-vacuum drying, expansion drying, film drying, hot air drying, etc., at least one or more of various industrial methods.

[0032] The yield of the biological remediation agent is at least 500g dry weight per cubic meter of algae; the acid-base buffer includes at least one selected from NaHCO3, KH2PO4, K2HPO4, and combinations thereof; the acid-base adjuster includes at least one selected from HCl and NaOH.

[0033] Furthermore, the biological treatment agent is concentrated to obtain a high concentration of algae, at least 500 g or 600 g dry weight per m³. For example, in one embodiment, after culturing the algae seed in the aforementioned algal culture medium for one week, 10 L is concentrated to 200 mL. The concentration before concentration is 0.624 g / L, and the concentration after concentration is 31.2 g / L. Approximately 60 g dry weight algae can be produced from 0.09588 m³ per week. In other words, in large-scale industrial production, 624 g dry weight algae can be obtained per m³ per week.

[0034] In one embodiment, the concentration means suitable for the present invention include at least one selected from gravity sedimentation, filtration concentration, centrifugal concentration, vacuum concentration, freeze drying, and combinations thereof.

[0035] To achieve the other objective mentioned above, the present invention provides a bioremediation adjuvant, which is prepared by the preparation method described above, and the bioremediation adjuvant is suitable for treating halogenated organic pollutants (HOPs).

[0036] Please refer to Figure 2, which is a side view of the culture facility for the biological treatment adjuvant of the present invention.

[0037] As shown in Figure 2, in a preferred embodiment of the present invention, a plurality of photosynthetic acclimatization tanks 1 are provided. Each photosynthetic acclimatization tank has a diffuser plate with a plurality of diffuser holes at its bottom. Each photosynthetic acclimatization tank is connected to another photosynthetic acclimatization tank and is provided with a water level gradient, so that liquid can overflow from one photosynthetic acclimatization tank to another photosynthetic acclimatization tank.

[0038] To achieve the aforementioned other objective, the present invention provides a biological treatment method for halogenated organic pollutants (HOPs) in water, comprising: inoculating a biological treatment agent and an HOP-degrading bacterium into a water body containing halogenated organic pollutants, and acclimating them for at least 3 days; preferably between 3 and 9 days, more preferably between 3 and 7 days, and particularly preferably between 3 and 5 days.

[0039] The halogenated organic pollutant applicable to the method of the present invention is 1,1,2,2-tetrachloroethane (1,1,2,2-PCA), 1,1,1-trichloroethane (1,1,1-TCA), 1,1,2-trichloroethane (1,1,2-TCA), 1,2-dichloroethane (1,2-DCA), 1,1-dichloroethane (1,1-DCA), chloroethane (CA), carbon tetrachloride (CT), chloroform (CF), methylene chloromethane (MC), chloromethane (CM), tetrachloroethylene (PCE), and trichloroethylene (PCE). TCE), cis-1,2-dichloroethene (cis-1,2-DCE), trans-1,2-dichloroethene (trans-1,2-DCE), 1,1-dichloroethene (1,1-DCE), vinyl chloride (VC), chlorobenzene (MCB), dichlorobenzene (DCB), and at least one combination thereof.

[0040] In one embodiment, the bioremediation adjuvant is prepared by the preparation method described above, or is a bioremediation adjuvant as described above.

[0041] Furthermore, the HOP-degrading bacterial strain was acclimated in an anaerobic medium with an HOP concentration of at least 1.0 mg / L for at least one month. The strain was obtained by selecting the bacterial group with the lowest methane production during the acclimation process, using thermal screening temperature, pH value, salinity and water content as control factors and methane production as the screening factor.

[0042] The following describes the domestication and screening of TCE biodegrading bacteria used in this invention. For example, trichloroethylene (TCE) degrading bacteria from three different sites (NT, KS, and GS) preserved in the inventor's laboratory were used to screen for TCE biodegrading bacteria with low methane (CH4) production.

[0043] Furthermore, the TCE biodegrading microbial community at the aforementioned NT site can completely degrade 3.5 mg / L of trichloroethylene into cis-1,2-dichloroethylene (cis-1,2-DCE) within one day, producing only less than 0.004 m / L of vinyl chloride (VC). The TCE degradation half-life is 1.5 hours.

[0044] Secondly, the TCE biodegrading microbial community at the aforementioned KS site can completely degrade TCE into ethylene within 3.0 days.

[0045] Furthermore, the TCE biodegrading bacteria at the aforementioned GS site are the same bacteria found at contaminated sites in central Taiwan, and can completely degrade TCE into ethylene within 28 days.

[0046] In one embodiment of the present invention, for example, the anaerobic culture medium may be composed of at least one of the following components: Resazurin, (NH4)2HPO4, CaCl2·2H2O, MgCl2·6H2O, KCl, MnCl2·4H2O, CoCl2·6H2O, H3BO3, CuCl2·2H2O, Na2MoO4·2H2O, ZnCl2, FeCl2·4H2O, Na2S·7H2O, Biotin, Folic Acid, Pyridoxine HCl, Riboflavin, Thiamin, Pantothenic Acid, Nicotinic Acid, p-Aminobenzoic Acid, Thioctic Acid, Vitamin, yeast extract, NaHCO3, KH2PO4, and K2HPO4.

[0047] For example, the anaerobic culture medium may have the components shown in Table 2.

[0048] Table 2 Element Added per liter Resazurin 1.0 mg (NH4)2HPO4 72.1 mg CaCl₂·2H₂O 225.5 mg MgCl₂·6H₂O 1.6 g KCl 1.2 g MnCl₂·4H₂O 18.0 mg CoCl₂·6H₂O 27.0 mg H 3BO 3 5.1 mg CuCl 2·2H 2O 2.4 mg Na₂MoO₄·2H₂O 2.3 mg ZnCl 2 1.9 mg FeCl 2·4H 2O 37.0 g Na 2S·7H 2O 50.0 g Biotin 20.0 g Folic Acid 20.0 g Pyridoxine HCl 100.0 g Riboflavin 50.0 g Thiamin 50.0 g Pantothenic Acid 50.0 g Nicotinic Acid 50.0 50.0 g​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Please refer to Figures 3 to 5. Figure 3 is a trichloroethylene degradation curve for experimental examples 1 to 4 of the biological treatment method of the present invention; Figure 4 is a trichloroethylene degradation curve for experimental examples 5 to 8 of the biological treatment method of the present invention; and Figure 5 is a trichloroethylene degradation curve for experimental examples 9 to 12 of the biological treatment method of the present invention.

[0050] Example 1

[0051] Example 1 of this invention involves placing the algal seed source into an algal culture and domestication facility, and then inoculating the HOP-degrading bacteria into a water body containing halogenated organic pollutants to perform a biological treatment method for the halogenated organic pollutants in the water body. The halogenated organic pollutant is trichloroethene (TCE). Please refer to Table 3. C represents the TCE concentration at a specific time point, C0 represents the initial TCE concentration, and C / C0 represents the relative concentration, which is the ratio of the TCE concentration to the initial concentration during the degradation process, used to measure the degradation effect of TCE over time. The experimental examples of Example 1 are Experimental Example 1, Experimental Example 5, and Experimental Example 9 in Table 3.

[0052] Example 2

[0053] The steps of Example 2 of this invention are largely the same as those of Example 1, except that the algal seed source in Example 2 is domesticated after undergoing a pretreatment process involving crushing and sterilization. The experimental examples for Example 2 are Example 2, Example 6, and Example 10 in Table 3.

[0054] Example 3

[0055] The steps of Example 3 of this invention are largely the same as those of Example 2, except that the algal seed source in Example 3 is domesticated after undergoing a pre-treatment of fermentation. The experimental examples of Example 3 are Experimental Example 3, Experimental Example 7, and Experimental Example 11 in Table 3.

[0056] Example 4

[0057] The steps in Example 4 of this invention are largely the same as those in Example 2, except that the algal seed source in Example 4 is domesticated after undergoing a pretreatment involving crushing. The experimental examples for Example 4 are Example 4, Example 8, and Example 12 in Table 3.

[0058] Table 3 Experimental Example name Time (days) 0 1 3 4 5 6 Experimental Example 1 Culture medium domestication only 100% 92.3% 88.8% - 119.7% 116.4% Experiment Example 2 Broken and sterilized algae domestication 100% 150.8% 154.2% - 1.3% 0.0% Experimental Example 3 Fermentation product domestication 100% 148.9% 165.7% - 167.1% 171.7% Experiment Example 4 Broken algae domestication 100% 205.7% 182.7% - 0.0% 0.0% Experimental Example 5 Culture medium domestication only 100% 91.2% 97.3% 98.2% 77.3% 41.3% Experimental Example 6 Broken and sterilized algae domestication 100% 109.9% 105.8% 12.9% 0.0% 0.0% Experimental Example 7 Fermentation product domestication 100% 129.3% 110.7% 104.6% 77.8% 29.2% Experimental Example 8 Broken algae domestication 100% 115.3% 83.5% 9.7% 0.0% 0.0% Experimental Example 9 Culture medium domestication only 100% 88.3% 84.2% 71.5% 62.3% 0.0% Experimental Example 10 Broken and sterilized algae domestication 100% 74.0% 48.2% 0.0% 0.0% 0.0% Experimental Example 11 Fermentation product domestication 100% 86.7% 82.9% 81.3% 70.7% 6.4% Experimental Example 12 Broken algae domestication 100% 90.1% 14.8% 0.0% 0.0% 0.0%

[0059] As shown in Table 3, the trichloroethylene degradation experiments in Examples 1-4 showed that using algal culture medium and algal fermentation products was not very effective, with almost no degradation throughout the process. Examples 5-8, however, began degradation after the fourth day, and Examples 9-12 also began degradation after the fourth day, completing degradation by the sixth day. This effect is likely due to the gradual adaptation of the bacterial community caused by long-term repeated exposure. However, using algae digested in a crushed and sterilized autoclave, or using only crushed algae for acclimatization, resulted in complete degradation of trichloroethylene in all three experiments, with significantly better results than using culture medium and algal fermentation products. A careful comparison of the results of Examples 2 and 4 shows that Example 4 may have performed slightly better than Example 2. Therefore, it is evident that only the algae removed from the eutrophic water body needs to be ground for use, eliminating the need for electricity and facilities for sterilization, thus significantly reducing carbon emissions from the process.

[0060] In summary, when the biological treatment adjuvant used in this invention is obtained by acclimating crushed algae seed into the algae culture medium and then processing it, the degradation rate of HOP in this biological treatment method is above 90.0%, preferably between 90.0% and 99.9%, more preferably between 93.0% and 99.9%, and most preferably between 96.0% and 99.9%.

[0061] Secondly, when the biological remediation adjuvant used in this invention is obtained by acclimating algae seed sources that have undergone crushing and high-temperature and high-pressure sterilization into the algae culture medium and then processing them, the degradation rate of the halogenated organic pollutants is 89.5% or higher, preferably between 90.0% and 99.9%, more preferably between 93.0% and 99.9%, and most preferably between 96.0% and 99.9%.

[0062] Example 5

[0063] To maintain commercially viable mixed algae, the isolated algal strains were subsequently cultured in an open environment. A comparative experiment was then conducted using locally sourced TCE-contaminated groundwater with added TCE (not a laboratory-prepared, fixed-concentration TCE solution) and commercially available bioremediation adjuvants. The results of Experiments 13 to 18 are shown in Table 4 below. The buffer solutions were both 0.075 g / L NaHCO3 and 0.075 g / L Na2CO3, and the initial TCE concentration was 5.0 mg / L.

[0064] Table 3 Experimental Example Group medicine Increased dosage microorganism Experimental Example 13 Blank group none none Sterile Experimental Example 14 control group none none Local microorganisms Experimental Example 15 Test A Commercially available drug A 1000 mg / L Local microorganisms Experimental Example 16 Test B Commercially available drug B 1000 mg / L Local microorganisms Experimental Example 17 Test C Commercially available drug C 1000 mg / L Local microorganisms Experimental Example 18 Test F1 algae 500 mg / L Local microorganisms

[0065] Please refer to Figures 6 to 12. Figure 6 is a graph comparing the degradation curve of trichloroethylene with three commercially available agents using the bioremediation adjuvant of the present invention; Figure 7 is a graph comparing the degradation curve of cis-1,2-dichloroethylene with the bioremediation adjuvant of the present invention with three commercially available agents; Figure 8 is a graph comparing the degradation curve of vinyl chloride with the bioremediation adjuvant of the present invention with three commercially available agents; Figure 9 is a graph comparing the degradation curve of ethylene with the bioremediation adjuvant of the present invention with three commercially available agents; Figure 10 is a graph comparing the concentration of dissolved organic carbon in Experimental Examples 13 to 18 of the present invention; Figure 11 is a graph comparing the concentration of methane in Experimental Examples 13 to 18 of the present invention; and Figure 12 is a graph comparing the concentration of phosphate in Experimental Examples 13 to 18 of the present invention.

[0066] From Experiments 13-18 and Figures 6 to 9, it can be seen that algae, as a bioremediation adjuvant, outperforms commercially available products. Figure 10 also shows that the organic carbon released by algae is relatively low but persistent, which is consistent with the characteristic of dehalococcoides, which are oligotrophs requiring only low levels of TOC. Figures 11 and 8 show that algae adjuvants have the lowest methane production among all adjuvants, and their greenhouse effect is lower than that of all commercially available adjuvants.

[0067] Furthermore, the preparation method of the bioremediation adjuvant of the present invention is a bioremediation technology for halogenated organic pollutants in the environment based on carbon negative technology. As can be seen from Figure 10, because the organic carbon released by this algal source is relatively limited, the actual methane production is also the lowest among all tested agents, and the phosphate release is also quite low.

[0068] In summary, the preparation method of the biological remediation adjuvant of the present invention uses algae that cause eutrophication as a biological remediation adjuvant for halogenated organic solvent pollution in groundwater. This biological remediation adjuvant can achieve the following objectives: control of water eutrophication, with removing algae from the water body being the most effective means of eutrophication remediation; algal growth absorbs carbon dioxide from the atmosphere, achieving net zero carbon emissions by 2050; converting waste algae into valuable resources; and achieving sustainable green remediation through low-carbon biological remediation methods.

[0069] Furthermore, the biological treatment method for halogenated organic pollutants (HOPs) in water bodies of the present invention can completely decompose TCE within 5.0 days, which translates to a half-life of 0.5 days; and, under more intensive sampling conditions, it was found that the degradation half-life is 0.25 days, compared with the previously disclosed TCE degradation rate of 1.0 day, thus the degradation efficiency of the present invention is 4 times that of the prior art.

[0070] The above embodiments are merely illustrative of the technology and effects of the present invention and are not intended to limit the present invention. Any person skilled in the art can modify and change the above embodiments without departing from the technical principles and spirit of the present invention; therefore, the scope of protection of the present invention should be as described in the patent application scope below.

[0071] 1: Photosynthesis Acclimation Tank

Claims

1. A method for preparing a biological remediation adjuvant, comprising: An algal culture medium was prepared by adding 1500.0 mg of sodium nitrate, 40.0 mg of dipotassium hydrogen phosphate, 75.0 mg of magnesium sulfate, 36.0 mg of calcium chloride, 6.0 mg of ferric ammonium citrate, 6.0 mg of citric acid, 1.0 mg of disodium EDTA, and 20.0 mg of sodium carbonate, along with other trace elements, to every 1 liter of deionized water. The medium was then sterilized under high temperature and high pressure (121°C, 1.5 atm) for 15 minutes, cooled by purging with nitrogen gas, and the pH was adjusted to 7.0 using an acid-base adjuster. An algal seed source is domesticated, implanted into a culture medium, placed in a photosynthetic domestication tank, and continuously acclimated with gas for at least 7 days to obtain photosynthetically domesticated decomposition-aiding algae; and a biological remediation adjuvant is prepared, which is then further processed to obtain a biological remediation adjuvant suitable as a carbon source and electron supply source for organic matter degrading bacteria; wherein the algal seed source is a natural algae growing in eutrophic water; the yield of the biological remediation adjuvant is at least 500g dry weight per cubic meter of algae; the pH adjuster includes at least one selected from HCl and NaOH.

2. The method for preparing the biological treatment adjuvant as described in claim 1, wherein the algal seed source is implanted into the algal culture medium after undergoing a pretreatment; the pretreatment includes: At least one of the following: crushing treatment, crushing and sterilization treatment, fermentation treatment, and combinations thereof.

3. The method for preparing the biological treatment adjuvant as described in claim 1, further comprising: The biological remediation adjuvant is concentrated to obtain a high concentration of biological remediation adjuvant of 31.2 g / L or higher; The concentration includes at least one of gravity sedimentation, filtration concentration, centrifugal concentration, vacuum concentration, freeze drying, and combinations thereof.

4. The method for preparing the biological remediation adjuvant as described in claim 1, wherein the algae species belongs to the genus *Leptolyngbya boryana* of *Cyanobacteria*.

5. The method for preparing the biological treatment adjuvant as described in claim 1, wherein the introduced gas is carbon dioxide.

6. A bioremediation adjuvant prepared by any one of the methods described in claims 1 to 5, the bioremediation adjuvant being suitable for treating halogenated organic pollutants (HOPs).

7. A biological treatment method for halogenated organic pollutants (HOPs) in water, comprising: A biological remediation adjuvant and a HOPs-degrading bacterium were inoculated into water bodies containing halogenated organic pollutants and acclimated for at least 3 days. The halogenated organic pollutants included 1,1,2,2-tetrachloroethane (1,1,2,2-PCA), 1,1,1-trichloroethane (1,1,1-TCA), 1,1,2-trichloroethane (1,1,2-TCA), 1,2-dichloroethane (1,2-DCA), 1,1-dichloroethane (1,1-DCA), chloroethane (CA), carbon tetrachloride (CT), chloroform (CF), and methylene chloride (CF). The bioremediation adjuvant is selected from at least one of the following: MC, chloromethane (CM), tetrachloroethylene (PCE), trichloroethene (TCE), cis-1,2-dichloroethene (cis-1,2-DCE), trans-1,2-dichloroethene (trans-1,2-DCE), 1,1-dichloroethene (1,1-DCE), vinyl chloride (VC), chlorobenzene (MCB), dichlorobenzene (DCB), and combinations thereof; the bioremediation adjuvant is prepared by the method described in any one of claims 1 to 5, or the bioremediation adjuvant described in claim 6; the HOPs-degrading bacterial culture is in an anaerobic medium with an HOPs concentration of at least 1.0 mg / L. After acclimatization in an anaerobic environment for at least one month, the bacteria with the lowest methane production during the acclimatization process were selected, with thermal screening temperature, pH value, salinity and moisture content as control factors and methane production as the screening factor.

8. A biological treatment method for halogenated organic pollutants (HOPs) in water as described in claim 7, wherein the anaerobic culture medium is selected from at least one of the following: Resazurin, (NH4)2HPO4, CaCl2·2H2O, MgCl2·6H2O, KCl, MnCl2·4H2O, CoCl2·6H2O, H3BO3, CuCl2·2H2O, Na2MoO4·2H2O, ZnCl2, FeCl2·4H2O, Na2S·7H2O, Biotin, Folic Acid, Pyridoxine HCl, Riboflavin, Thiamin, Pantothenic Acid, Nicotinic Acid, p-Aminobenzoic Acid, Thioctic Acid, Vitamin, yeast extract, NaHCO3, KH2PO4, and K2HPO4.

9. In the biological treatment method for halogenated organic pollutants (HOPs) in water bodies as described in claim 7, where the biological treatment adjuvant is obtained by acclimating crushed algae seed into the algae culture medium and then processing it, the degradation rate of HOPs by the biological treatment method is 90.0% or higher.

10. The biological treatment method for halogenated organic pollutants (HOPs) in water as described in claim 7, wherein the biological treatment adjuvant used is obtained by acclimating algae seed sources that have undergone crushing and high-temperature and high-pressure sterilization into the algae culture medium and then processing them, wherein the degradation rate of the halogenated organic pollutants is 89.5% or higher.

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