TWI756791B - Colloidal composition and method for in situ remediation of hexavalent chromium - Google Patents

Abstract

The present invention provides a colloidal composition for in situ remediation of hexavalent chromium, in which the colloidal composition includes sulfate, emulsified oil, and a carboxymethyl cellulose solution with a specific range of viscosity. The concentration of the released sulfate is controlled by modulating the viscosity of the carboxymethyl cellulose solution in the present invention, so that the growth of indigenous sulfate-reducing bacteria can be stably enhanced, and in situ remediation of hexavalent chromium can be performed continuously and effectively.

Description

Gel composition and method for on-site bioremediation of hexavalent chromium

The present invention relates to a composition for rectifying hexavalent chromium, in particular to a gel composition for on-site biological rectification of hexavalent chromium and a method for on-site biological rectification of hexavalent chromium.

Chromium is an important metal element in industry. In addition to elemental chromium, trivalent chromium and hexavalent chromium compounds are also widely used, and are often used in electroplating, kneading, pigments, wood anticorrosion and iron and steel industries.

However, chromium compounds are toxic, and hexavalent chromium compounds such as chromate and dichromate are the most toxic. Hexavalent chromium is also highly irritant. After exposure to and/or inhalation of hexavalent chromium, organisms may cause irritation, inflammation, and even ulcers of the skin, eyes and/or respiratory mucosa. Accidental ingestion of hexavalent chromium can easily cause symptoms such as vomiting, stomach pain, and even damage to the kidneys and/or liver, and may also cause cancers such as lung cancer and sinus cancer.

Common hexavalent chromium remediation methods include physical remediation and chemical remediation, among which physical remediation is to avoid hexavalent chromium by physical adsorption. Chromium diffusion. The chemical remediation method reduces hexavalent chromium to trivalent chromium with less solubility and toxicity by means of chemical reduction. However, both the physical remediation method and the chemical remediation method have problems such as secondary pollution, regional limitations and/or inability to perform for a long time.

As for the biological remediation law, the metabolism of organisms is used to reduce hexavalent chromium. Common biological remediation methods include: adding microorganisms, nutrients, electron donors or electron acceptors to directly increase the content of microorganisms that can reduce hexavalent chromium, or indirectly to promote the growth of microorganisms by providing reactants for metabolism and metabolism. The above-mentioned biological remediation methods can also be used in combination, in which the addition of emulsified oil containing nutrient salts can simultaneously provide carbon sources and electron donors or electron acceptors, which are often used in biological remediation methods. However, the oil droplets of emulsified oil are small, so it is easy to diffuse into the pores of different types of soil, and at the same time release nutrients, resulting in rapid loss of nutrients, resulting in less than expected biological remediation effect and short timeliness.

In view of this, it is necessary to provide a composition and/or method that can improve the bioremediation of hexavalent chromium to solve the above problems.

Therefore, one aspect of the present invention is to provide a gel composition for in-situ bioremediation of hexavalent chromium, which adjusts the viscosity of the gel composition by adding carboxymethyl ether of cellulose (CMC) to delay the The release of sulfate in the gel composition promotes the growth and metabolism of sulfate-reducing bacteria, thereby effectively reducing the concentration of hexavalent chromium in polluted areas.

Another aspect of the present invention is to provide a method for in-situ bioremediation of hexavalent chromium, comprising applying the gel composition to a polluted area to stably release a specific concentration of sulfate, thereby stably promoting the growth of sulfate-reducing bacteria and Metabolize, and then form permeable reactive biobarriers (PRBBs) for long-term and effective remediation of hexavalent chromium.

According to the above aspect of the present invention, there is provided a gel composition for on-site bioremediation of hexavalent chromium, wherein based on 1L of the gel composition, the gel composition may contain 30g to 50g of carboxymethyl cellulose (carboxymethyl ether). of cellulose, CMC), more than 0.03L but not more than 0.05L of emulsified oil, and a balanced amount of sulfate aqueous solution. The viscosity of the above-mentioned carboxymethyl cellulose can be, for example, 0.3 Pa.s (Pa.s) to 0.6Pa. s. Based on 100 weight percent (wt %) of the above emulsified oil, the emulsified oil may contain 50 to 55 wt % of vegetable oil, 15 to 20 wt % of surfactant, and a balance of water. The concentration of the above sulfate aqueous solution may be, for example, 3.0 g/L to 4.0 g/L.

According to an embodiment of the present invention, the glucose polymerization degree of carboxymethyl cellulose may be, for example, 100 to 2000.

According to an embodiment of the present invention, the concentration of the aqueous sulfate solution may be, for example, 3.0 g/L.

According to an embodiment of the present invention, the sulfate may include alkali metal salts and/or alkaline earth metal salts.

According to an embodiment of the present invention, the emulsified oil may further comprise 0.001wt% to 0.002wt% of multivitamins.

According to an embodiment of the present invention, the emulsified oil may further comprise 0.4 wt % to 0.5 wt % of lactate.

According to the above aspects of the present invention, there is provided a method for in-situ bioremediation of hexavalent chromium, which may include applying a gel composition to a contaminated area, so that the gel composition continuously releases sulfate for a period of time, thereby promoting sulfate The reducing bacteria grow and decompose the hexavalent chromium pollutants, wherein the gel composition can be, for example, the above-mentioned gel composition, and the contaminated area contains the hexavalent chromium pollutants and the sulfate reducing bacteria.

According to an embodiment of the present invention, the gel composition continuously releases at least 100 mg/L of sulfate, and the period may be, for example, at least 30 days.

According to an embodiment of the present invention, the gel composition continuously releases 200 mg/L to 400 mg/L of sulfate, and the period may be, for example, not less than 20 days.

Applying the gel composition of the present invention for bioremediation of hexavalent chromium and the method thereof, the viscosity is increased by adding carboxymethyl cellulose, so as to control the concentration of released sulfate, thereby stably promoting the growth of native sulfate-reducing bacteria , and then sustainable and effective bioremediation of hexavalent chromium.

101,103,105,107,109,111,201,203,205,207,209,211,501,503,505,601,603,605,701,703,705,801,803,805,901,903,905,1001,1003,1005: Folding line

301, 303, 305, 1101, 1103, 1105, 1201, 1203, 1205: Straight

400: Device

410: Feed chute

420: Peristaltic Pump

430: First String

440: Second String

450: Third column

432,442,452: Sampling points

445: Injection port

460: outlet tank

In order to make the above-mentioned and other objects, features, advantages and embodiments of the present invention more obvious and easy to understand, the detailed description of the accompanying drawings is as follows: [Fig. 1] is related to different preparation examples described in one embodiment of the present invention. Line graph of time versus hexavalent chromium concentration.

[ Fig. 2 ] is a line graph of sulfide concentration versus time in different preparation examples described in one embodiment of the present invention.

[ Fig. 3 ] is a bar graph of time versus sulfite reductase (dsr A) gene content for different preparations described in one embodiment of the present invention.

[FIG. 4] is a schematic diagram of an apparatus for a pipe string test according to an embodiment of the present invention.

[ Fig. 5 ] is a line graph of time versus hexavalent chromium concentration in Comparative Example 1 according to an embodiment of the present invention.

[ Fig. 6 ] is a line graph of time versus sulfate concentration for Comparative Example 1 described in one embodiment of the present invention.

[ Fig. 7 ] is a line graph of time versus hexavalent chromium concentration for Comparative Example 2 described in one embodiment of the present invention.

FIG. 8 is a line graph of time versus sulfate concentration for Comparative Example 2 described in one embodiment of the present invention.

[ Fig. 9 ] is a line graph of time versus hexavalent chromium concentration in Example 1 according to an example of the present invention.

[ Fig. 10 ] is a line graph of time versus sulfate concentration in Example 1 according to an example of the present invention.

[ Fig. 11 ] is a bar graph of the content of drsA gene at different time points in different substrates according to an embodiment of the present invention.

[ Fig. 12 ] is a bar graph of the 16S rRNA gene content at different time points in different substrates according to an example of the present invention.

References herein to the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. range of values (e.g. A) of 10%~11% includes the upper and lower limit values unless otherwise specified (ie 10%≦A≦11%); if the value range does not define the lower limit (such as B lower than 0.2%, or 0.2% The following B) means that the lower limit may be 0 (ie 0%≦B≦0.2%). The above terms are used to describe and understand the present invention, but not to limit the present invention.

The present invention provides a gel composition for on-site biological remediation of hexavalent chromium, wherein the gel composition comprises sulfate and carboxymethyl ether of cellulose (CMC), so as to stably release sulfate to native natives in the soil. Sulfate-reducing bacteria, thereby forming permeable reactive biobarriers (PRBBs), and then long-term and effective remediation of hexavalent chromium.

The term "in situ" is relative to "ex situ", where "in situ" refers to the direct treatment of pollutants on-site at the contaminated site to avoid the spread of pollutants, and "ex situ" refers to the removal of contaminants. excavate or take out contaminated soil or groundwater, and then move it to another place or on the ground for disposal. The present invention belongs to the former, and relative to the latter, the former can avoid problems such as the movement of pollutants, environmental changes, waste and/or waste water discharge and the like. In some embodiments, the texture of the soil is not particularly limited, and can be, for example, but not limited to clay, sandy clay, loamy clay, sandy clay loam, clay loam, loamy clay, sandy loam, loam, loamy soil, loamy soil, loamy sandy soil and sandy soil, etc.

The "hexavalent chromium" is chromium with an oxidation number of +6 and its compounds, such as chromium trioxide, chromic acid and its salts, dichromic acid and its salts.

The "remediation" refers to reducing the content of hexavalent chromium in soil or groundwater, and the remediation method may include adsorption of hexavalent chromium and/or reduction of hexavalent chromium to toxic substances. Less volatile and less water-insoluble trivalent chromium. The "biological remediation" is to use the metabolism of microorganisms to remediate hexavalent chromium, wherein the microorganisms can be selected from, for example, indigenous microorganisms, because microorganisms that can survive in a high-concentration hexavalent chromium environment are usually resistant to hexavalent chromium. Tolerance, eg: chromate reducing bacteria (CRB). Chromate reducing bacteria is a general term for bacteria that can reduce hexavalent chromium to trivalent chromium, and the enzymes used for chromate reduction to reduce hexavalent chromium are called chromate reductases (such as hydrogenases) and cytochrome c3).

Some chromate-reducing bacteria are also sulfate-reducing bacteria (sulfate reducing bacteria, SRB). Sulfate-reducing bacteria use sulfate as the final electron acceptor in the electron transport chain of respiration, where sulfate will form sulfide after accepting electrons. Since sulfide is a strong reducing agent, sulfate-reducing bacteria can indirectly reduce hexavalent chromium through sulfide. On the other hand, some sulfate-reducing bacteria possess hydrogenase and cytochrome c3, which can directly reduce hexavalent chromium.

In addition, sulfate-reducing bacteria can also reduce free hexavalent chromium through biosorption. First, the structure of chromic acid is similar to that of sulfuric acid, so chromic acid can enter sulfate-reducing bacteria through the sulfate transport system of sulfate-reducing bacteria. Second, chromic acid can also be adsorbed by functional groups of the cell walls of sulfate-reducing bacteria. As can be seen from the above, the use of sulfate-reducing bacteria is a good method for biological remediation of hexavalent chromium.

In one embodiment, bioremediation is to apply nutrients such as nutrients and carbon sources in the midstream area of the hexavalent chromium-contaminated site, thereby promoting the growth and metabolism of native bacteria (or microorganisms), thereby forming a water-permeable biological reaction zone. (permeable reactive biobarriers, PRBBs). When groundwater flows through the permeable biological reaction zone, the content of hexavalent chromium can be reduced due to the reduction or adsorption of native microorganisms such as sulfate-reducing bacteria. In a preferred embodiment, the hexavalent chromium concentration can be reduced to below 0.5 g/L.

The "gel composition" can include sulfate, emulsified oil and thickener, wherein sulfate provides electron acceptor, emulsified oil provides carbon source, and thickener increases the viscosity of the gel composition, which is beneficial to control sulfuric acid. The rate at which salts and emulsified oils are released to avoid rapid loss of sulfates and emulsified oils. In one embodiment, based on 1 L of the gel composition, the gel composition comprises 30 g to 50 g of thickening agent, more than 0.03 L but not more than 0.05 L of emulsified oil, and a balanced amount of aqueous sulfate solution, wherein thickening The viscosity of the agent can be, for example, 0.3 Pascal seconds (Pa.s) to 0.6Pa. s.

The "sulfate" refers to the salts formed by sulfate radicals and metal ions, such as alkali metal sulfates and/or alkaline earth metal sulfates. In one embodiment, the sulfate can be selected from, for example, the group consisting of sodium sulfate, potassium sulfate, magnesium sulfate, and any combination thereof. Sulfate administration to hexavalent chromium-contaminated sites can promote the growth and metabolism of native sulfate-reducing bacteria.

However, if too much sulfate is provided, the sulfate-reducing bacteria will over-metabolize and produce too much sulfide, thereby poisoning the sulfate-reducing bacteria, but cannot effectively reduce the hexavalent chromium content. If the supplied sulfate is too low, the effect of promoting the growth and metabolism of sulfate-reducing bacteria cannot be achieved, and the hexavalent chromium cannot be effectively reduced. Experiments have confirmed that when the concentration of released sulfate is less than or equal to 100mg/L, the growth and metabolism of sulfate-reducing bacteria cannot be effectively stimulated, and if the concentration of released sulfate is greater than or equal to 500mg/L, It will lead to the excessive growth and metabolism of sulfate-reducing bacteria, resulting in the formation of a high concentration of sulfide adversity, which in turn leads to the death of in-situ microorganisms (including sulfate-reducing bacteria), and thus cannot effectively remediate hexavalent chromium. Therefore, how to maintain the sulfate concentration released from the gel composition is the key to bioremediation of hexavalent chromium.

In the present invention, the release of high-concentration sulfate is delayed by adjusting the viscosity of the gel composition, thereby stably releasing sulfate concentration greater than 100 mg/L but less than 500 mg/L. In one embodiment, the concentration of the aqueous sulfate solution may be, for example, 3.0 g/L to 4.0 g/L. In a preferred embodiment, the concentration of the aqueous sulfate solution may be, for example, 3.5 g/L. In the above embodiment, it is necessary to add a viscosity of 0.3Pa to each 1L of the gel composition. s to 0.6Pa. 30g to 50g of thickener for s to increase the viscosity of the gel composition, thereby achieving the effect of effectively delaying the release of sulfate.

If the content of the thickener in the gel composition is too low, and/or the viscosity of the thickener is too low, the gel composition cannot effectively delay the release of sulfate. If the content of the thickener is too high, and/or the viscosity of the thickener is too high, the sulfate concentration released from the gel composition will be too low to stimulate the growth and metabolism of a sufficient amount of sulfate-reducing bacteria, It can also lead to increased turbidity in groundwater, which in turn increases remediation costs.

In one embodiment, in order to avoid secondary pollution, the thickener is preferably a substance that is non-toxic and easily biodegradable. In one embodiment, the thickener may be, for example, carboxymethyl ether of cellulose (CMC). In one embodiment, the glucose polymerization degree of carboxymethyl cellulose can be, for example, 100 to 2000 to reach 0.3Pa. s to 0.6Pa. s viscosity.

The "emulsified oil" is a mixture of water, surfactant and long-chain fatty acid oil. The oil droplets of emulsified oil are small and uniform, and can diffuse into the pores of different types of soil to provide nutrients for the growth of native microorganisms. In one embodiment, the gel composition may contain more than 0.03L but not more than 0.05L of emulsified oil. If the content of emulsified oil is too low, the gel composition cannot provide sufficient carbon source, and cannot effectively promote the growth and metabolism of native microorganisms. If the content of emulsified oil is too high, it will lead to the turbidity of the groundwater body and the increase of chromaticity, which will increase the cost of remediation. In one embodiment, based on 100 weight percent (wt %) of the emulsified oil, the emulsified oil may comprise 50 to 55 wt % of long chain fatty acid oils, 15 to 20 wt % of a surfactant and a balance of water.

The above-mentioned long-chain fatty acid oil can be, for example, vegetable oil, wherein the vegetable oil can be selected from, for example, the group consisting of soybean oil, hydrogenated cottonseed oil, solid edible shortening, olive oil, palm oil, coconut oil and any combination of the above, wherein the long-chain fatty acid After fermentation, fats and oils can produce small-molecule fatty acids (such as acetate, lactate, propionic acid, and butyric acid) to provide carbon sources and energy for metabolism by anaerobic organisms such as sulfate-reducing bacteria.

The above-mentioned surfactant is preferably a biodegradable surfactant, which can be selected from, for example, alkyl polyglucoside, polyaspartic acid, phospholipid, polysorbate, monoglyceride, diglyceride, The group consisting of triglycerides, oleic acid glycerides, polaxamers, plant extracts and any combination thereof, the plant extracts can be selected from, for example, citrus, sapindus, japonica, saponin , horse chestnut and any combination of the above-mentioned groups of extracts. In one embodiment, the surfactant is Simple Green ^(TM ) manufactured by California Sunshine Manufacturing Company.

In one embodiment, the emulsified oil may contain, but is not limited to, 0.001 wt % to 0.002 wt % of multivitamins to assist the metabolism of sulfate-reducing bacteria.

In one embodiment, the emulsified oil may contain, but is not limited to, 0.4 wt % to 0.5 wt % of lactate to directly provide small-molecule fatty acids to sulfate-reducing bacteria.

When the gel composition is applied to the contaminated area, the gel composition can continuously release sulfate for a period of time, thereby promoting the growth and metabolism of sulfate-reducing bacteria, thereby forming a water-permeable biological reaction area. The "contaminated area" may include hexavalent chromium contaminated sites and the middle and lower reaches of the groundwater basin of the contaminated sites.

In a column experiment, the gel composition continued to release at least 100 mg/L of sulfate for at least 30 days. Furthermore, the period during which the gel composition continues to release 200 mg/L to 400 mg/L of sulfate can be, for example, at least 20 days. The soil type used in the above-mentioned string experiment can be, for example, sandy loam.

The "effective" means that the application of the gel composition at the contaminated site with a specific residence time can reduce the concentration of hexavalent chromium at the contaminated site to the groundwater control standard in a relatively short period of time. In one embodiment, the shorter time may be, for example, within half a month, and the groundwater control standard may be, for example, less than or equal to 0.5 mg/L, that is, less than the first type of groundwater control standard. The "residence time of application" refers to at least 60 PV after the gel composition is applied to the contaminated site. Even after the gel composition is applied, the contaminated site is still contaminated by hexavalent chromium, and the hexavalent chromium is still in the contaminated site. The permeable biological reaction zone can still effectively reduce hexavalent chromium.

Several embodiments are used below to illustrate the application of the present invention, but they are not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various changes and modifications without departing from the spirit and scope of the present invention. retouch.

Example one, preparation of emulsified oil

In this embodiment and the following examples, based on 100wt% of the emulsified oil, the emulsified oil contains 50wt% of soybean oil and 6.67wt% of a commercially available biodegradable surfactant [trade name: Simple Green ^TM ); Manufacturer: Sunshine Makers.Inc.], 8.33wt% soybean lecithin, 4.17wt% sodium lactate, 7g to 10g multivitamins, and a balanced amount of ultrapure water.

Example 2. Assessing the effect of different emulsified oil concentrations on the bioremediation of hexavalent chromium

Digging a chromium-contaminated site in Taiwan to excavate sandy soil and native bacteria. Sterilized soil was obtained after sterilizing the soil at 121° C. and 15 lb/in ² for 20 minutes. Hereinafter, the unsterilized soil is referred to as the in situ soil, and the sterilized soil is referred to as the sterilized soil.

A hexavalent chromium solution is prepared to simulate the groundwater of the polluted site, wherein the hexavalent chromium solution contains 80mg/L hexavalent chromium, buffer substances (including: 326.4mg/L KH ₂ PO ₄ , 1263.8mg/L Na ₂ HPO _4. 98.6mg/L of Mg ₂ SO ₄ . 7H ₂ O, 44.1mg/L of CaCl ₂ . 2H ₂ O, 10.7mg/L of NH ₄ Cl), trace metals and balance water, and trace metals For the detailed composition and content, please refer to the article published by Mr. Gao Zhiming's team in the journal Water Research in 2003, which is listed as a reference here.

Prepare a pilot test sample, wherein the pilot test sample contains 130mL of hexavalent chromium solution and 4g/L sodium bicarbonate. The pilot test samples of experimental groups 1 to 3 also contained 10 g of sterilized soil, and contained 1 wt %, 3 wt % and 5 wt % of emulsified oil, respectively. The pilot test samples of the control group also contained 10 g of sterilized soil and 500 mg/L of mercuric chloride to maintain the sterile state.

Next, RNA extraction was carried out on the 0th day, the 4th day, the 20th day and the 36th day, respectively, to obtain the bacterial RNA in the pilot test sample, and then use the sequence identification number (SEQ ID NO.): 1 and 2. Primer pairs were subjected to Real-time quantitative polymerase chain reaction (qPCR) for the 16S rRNA quantification step. RNA extraction and qPCR are well known to those skilled in the art to which the present invention pertains, and will not be described in detail here. In addition, the hexavalent chromium quantification step was carried out according to the method of No. NIEA W320.52A published by the Environmental Inspection Institute.

The results showed that the 16S rRNA content of experimental groups 1 to 3 increased with time. On the 20th day, the 16S rRNA content per liter of experimental groups 1 to 3 was 7.72×10 ⁹ gene copies, respectively. ), 1.69×10 ¹¹ gene copy number and 9.80×10 ¹¹ gene copy number. On the 36th day, the 16S rRNA contents of experimental groups 1 to 3 were 2.18×10 ¹¹ gene copies per liter, 8.32×10 ¹¹ gene copies and 1.72×10 ¹³ gene copies per liter, respectively, indicating that emulsification The higher the oil concentration, the higher the number of native bacteria that grow.

The concentration of hexavalent chromium in the control group did not change with time, while the concentration of hexavalent chromium in the experimental groups 1 to 3 decreased with the increase of time, and the higher the concentration of emulsified oil, the higher the reduction ratio of hexavalent chromium. At 36 days, the content of hexavalent chromium in the pilot test samples of experimental groups 1 to 3 were 63.9 mg/L, 60.8 mg/L and 49.7 mg/L, respectively.

The above results show that the higher the concentration of emulsified oil, the more the growth of native bacteria (including chromate reducing bacteria) can be promoted, and the more hexavalent chromium can be reduced. However, too high concentration of emulsified oil will cause water turbidity and pollution. Therefore, in the following examples, the concentration of emulsified oil is 5wt%.

Embodiment three, assess the influence of different sulfate concentrations on reducing hexavalent chromium

Configure batch test samples, wherein the batch test samples include 130mL of hexavalent chromium solution and 4g/L sodium bicarbonate aqueous solution. The batch test samples in the first blank group also contained 10 g of sterilized soil, 5 wt % of emulsified oil and 500 mg/L of mercuric chloride. The batch test samples of the second blank group also contained 10 g of field soil. The batch test samples of Comparative Example 1 also included 10 g of in situ soil and 5 wt % of emulsified oil. The batch test samples from Preparation Example 1 to Preparation Example 3 also contained 10 g of in situ soil, and respectively contained 100 mg/mL, 300 mg/mL or 500 mg/ml of sodium sulfate.

On the 20th day, the 40th day, the 60th day and the 80th day, the hexavalent chromium quantification step was carried out according to the method described in the second implementation, and the US national test was carried out according to the reagent group HACH Method 8131 of HACH Company in the United States. The sulfide content analysis step was carried out by the methylene blue method announced by the Environmental Protection Agency (EPA). Next, RNA extraction and 16S rRNA quantification steps were performed, and qPCR was performed using the primer pair shown in SEQ ID NO.: 3 and 4 to perform the dissimilatory sulfite reductase gene (dsrA) quantification step. The above-mentioned dsrA is a key enzyme in the sulfate reduction reaction. The results are shown in FIGS. 1 , 2 and 3 .

1 is a line graph of time versus hexavalent chromium concentration for different preparation examples described in one embodiment of the present invention, wherein the horizontal axis represents time, the vertical axis represents hexavalent chromium concentration, and broken lines 101 , 103 , and 105 , the broken line 107 , the broken line 109 and the broken line 111 respectively represent the first blank group, the second blank group, the preparation comparative example 1, and the preparation examples 1 to 3.

As shown in FIG. 1 , in the first blank group (broken line 101 ), the native bacteria died after being sterilized, so the concentration of hexavalent chromium hardly changed with the increase of time. The protobacteria in the second blank group (polyline 103) were not inhibited, but had no additional nutrient source, so only a slight reduction of hexavalent chromium was possible. Preparation of Comparative Example 1 (broken line 105) contains emulsified oil, which can promote the growth and metabolism of native bacteria, thereby reducing hexavalent chromium, thereby reducing the concentration of hexavalent chromium. In addition to emulsified oil, the batch test samples of Preparation Example 1 to Preparation Example 3 (polyline 107, polyline 109, and polyline 111) also contain sulfate, which can promote the growth and metabolism of sulfate-reducing bacteria, thereby effectively reducing batch testing. The hexavalent chromium concentration in the samples, where on day 80, the hexavalent chromium concentration of Preparation Example 2 (polyline 109) was almost zero. It is worth noting that, compared with Preparation Example 1 (polyline 107 ), the concentration of hexavalent chromium in Preparation Example 3 (polyline 111 ) is higher on the contrary.

Figure 2 is a different preparation example described in relation to one embodiment of the present invention A line graph of sulfide concentration versus time, in which the horizontal axis represents time, the vertical axis represents sulfide concentration, and broken line 201, broken line 203, broken line 205, broken line 207, broken line 209, and broken line 211 indicate the first blank group, the second Blank group, Preparation Comparative Example 1, and Preparation Examples 1 to 3. As shown in FIG. 2 , there is almost no sulfide in the first blank group (polyline 201 ), the second blank group (polyline 203 ), and Comparative Preparation Example 1 (polyline 205 ), while in Preparation Examples 1 to 3, sulfide The concentration is positively correlated with the sulfate concentration.

Fig. 3 is a bar graph of time versus dsr A gene content for different preparations according to an embodiment of the present invention, wherein the horizontal axis represents the concentration, and from left to right are the control group (ie, the second blank group above) , 0 mg/mL (preparation comparison 1), 100 mg/mL (preparation example 1), 300 mg/mL (preparation example 2) and 500 mg/ml (preparation example 3), the vertical axis represents the dsr A gene content, and the straight bar 301, Bar 303 and bar 305 represent day 0, day 20, and day 60, respectively. As shown in Figure 3, compared with the 0th day (bar 301), on the 20th day (bar 303), the dsr A gene content all increased, and the increase was positively correlated with the sulfate concentration. However, on the 60th day (bar 305), the dsr A gene content of Preparation Example 1 to Preparation Example 3 did not increase but decreased, and the dsr A gene content of Preparation Example 2 was higher than that of Preparation Example 3.

Based on the above results, it can be inferred that higher sulfate concentration can promote the metabolism of sulfate-reducing bacteria, but excessive sulfate concentration will lead to excessive sulfide generation, thereby inhibiting the growth and metabolism of sulfate-reducing bacteria. Therefore, the sulfate concentration released from the gel composition needs to be greater than 100 mg/L and less than 500 mg/L, and 300 mg/L is the preferred concentration.

Example 4. Evaluating the efficiency of reducing hexavalent chromium with different substrates by column test

Please refer to FIG. 4 , which is a schematic diagram of an apparatus 400 for string testing according to an embodiment of the present invention. As shown in FIG. 4 , the device 400 includes a feed tank 410 , a peristaltic pump 420 , a first pipe string 430 , a second pipe string 440 , a third pipe string 450 and a water outlet tank connected in sequence. 460, wherein the first pipe string 430, the second pipe string 440 and the third pipe string 450 are three glass pipe strings with a diameter of 5cm and a length of 25cm, which are used to simulate the upstream area, midstream area and The downstream region, and the first string 430, the second string 440, and the third string 450 include sampling point 432, sampling point 442, and sampling point 452, respectively. The injection port 445 on the second column 440 is used to inject the matrix to simulate the water permeable bioreactor zone.

The first pipe string 430, the second pipe string 440 and the third pipe string 450 are respectively filled with local soil, and then a solution containing 80 mg/L hexavalent chromium is provided from the feeding tank 410, and the peristaltic pump 420 is used to provide power to carry out the process. Cyclic immersion until the concentration of hexavalent chromium in the first column 430, the second column 440 and the third column 450 reaches equilibrium (80mg/L), wherein the average pore size of the soil in situ is 178mL/column, and circulating The impregnation flow rate was 0.24 L/min, so the average residence time of the hexavalent chromium solution was 5.036 hours/column, and the total residence time was 15.11 hours. Considering the convenience of sampling and conversion, 2 Pore Volume (PV) was taken as 1 day. Next, in Comparative Example 1, Comparative Example 2 and Example 1, respectively inject the first matrix, the second matrix and the third matrix from the injection port 445, wherein the first matrix contains 3500 mg/L sodium sulfate, 5wt% emulsified oil, 0.4g/mL sodium bicarbonate and balance water, the second base comprises 5wt% emulsified oil, 40g/L carboxymethyl cellulose, 0.4g /mL of sodium bicarbonate and water in balance, and the third matrix contains 3500mg/L of sodium sulfate, 5wt% of emulsified oil, 40g/L of carboxymethyl cellulose, 0.4g/mL of sodium bicarbonate and a balanced amount of water.

From sampling point 432, sampling point 442 and sampling point 452, upstream samples, midstream samples and downstream samples can be obtained respectively to evaluate the effects of injecting different substrates in the midstream on hexavalent chromium concentration, sulfate concentration and native bacteria.

The content of hexavalent chromium was analyzed by the method described in Example 2, and the sulfate quantification step was carried out by the method published by the Environmental Inspection Institute No. NIEA W424.52A, followed by RNA extraction, 16S rRNA quantification, and 16S rRNA quantification. steps and dsrA quantification steps. The results are shown in FIGS. 5 to 12 .

5 is a line graph of time versus hexavalent chromium concentration in Comparative Example 1 according to an embodiment of the present invention, wherein the horizontal axis represents time, the vertical axis represents hexavalent chromium concentration, and broken lines 501 , 503 and 505 They represent the first upstream sample, the first midstream sample and the first downstream sample of Comparative Example 1, respectively. As shown in FIG. 5 , the hexavalent chromium concentration of the first upstream sample (broken line 501 ) was maintained at 80 mg/L, and the hexavalent chromium concentration of the first midstream sample and the first downstream sample was maintained before 34 PV (about 15 days). decreased, but after 34PV, the hexavalent chromium concentration was almost maintained at 17 mg/L to 19 mg/L.

FIG. 6 is a diagram of Comparative Example 1 described in relation to an embodiment of the present invention. A line graph of time versus sulfate concentration, wherein the horizontal axis represents time, the vertical axis represents hexavalent chromium concentration, and the line 601, line 603 and line 605 represent the first upstream sample, the first midstream sample and the first sample of Comparative Example 1, respectively downstream samples. As shown in FIG. 6 , the sulfate concentration of the first downstream sample measured at each time point is almost zero, indicating that the sulfate injected at the injection port 445 is local in the second column 440 and the third column 450 Used by microorganisms in the soil, or adsorbed on the soil. The initial sulfate concentration of the first midstream sample was 1314 mg/L, but the sulfate was rapidly lost from OPV to 4PV. From 4PV to 6PV, the decrease of sulfate concentration decreased, and the sulfate measured at this time may be the sulfate adsorbed on the soil particles with the emulsified oil. At 28PV, the sulfate concentration is only 4.39mg/L, which can be regarded as a complete loss.

7 is a line graph of time versus hexavalent chromium concentration in Comparative Example 2 according to an embodiment of the present invention, wherein the horizontal axis represents time, the vertical axis represents hexavalent chromium concentration, and broken lines 701 , 703 and 705 They represent the second upstream sample, the second midstream sample and the second downstream sample of Comparative Example 2, respectively. Since Matrix 2 only contains emulsified oil without sulfate, the sulfate-reducing bacteria of Comparative Example 2 do not have a better survival advantage than other native bacteria, so although the sulfate concentration of Comparative Example 2 can eventually be reduced to less than 0.5 mg /L, but requires more than 50PV of time (as shown by the broken line 703 and the broken line 705 in FIG. 7 ), which does not meet the time cost.

8 is a line graph of time versus sulfate concentration in Comparative Example 2 according to an embodiment of the present invention, wherein the horizontal axis represents time, the vertical axis represents hexavalent chromium concentration, and broken line 801 , broken line 803 and broken line 805 respectively generation The second upstream sample, the second midstream sample and the second downstream sample of Comparative Example 2 are shown in the table. As shown in FIG. 8 , because the second matrix is sulfate-free, the second upstream sample, the second midstream sample, and the second downstream sample are sulfate-free.

9 is a line graph of time versus hexavalent chromium concentration in Example 1 according to one embodiment of the present invention, wherein the horizontal axis represents time, the vertical axis represents hexavalent chromium concentration, and broken lines 901 , 903 and 905 They represent the third upstream sample, the third midstream sample and the third downstream sample of Example 1, respectively. As shown in Figure 9, the hexavalent chromium concentration of the third midstream sample and the third downstream sample rapidly decreased to 0.9 mg/L between 0PV and 22PV, and the hexavalent chromium concentration did not exceed 0.5 mg/L between 32PV and 60PV. L.

10 is a line graph of time versus sulfate concentration in Example 1 according to one embodiment of the present invention, wherein the horizontal axis represents time, the vertical axis represents hexavalent chromium concentration, and broken line 1001 , broken line 1003 and broken line 1005 are respectively Represents the third upstream sample, the third midstream sample and the third downstream sample of Example 1. Since the third matrix contains carboxymethyl cellulose and has a higher viscosity, it can delay the release of sulfate, wherein the sulfate concentration is between 300mg/L and 500mg/L at 10PV to 38PV, and At 16PV to 46PV, the sulfate concentration was between 200 mg/L and 400 mg/L.

Figure 11 is a bar graph of the drsA gene content of different substrates at different time points according to an embodiment of the present invention, wherein the horizontal axis represents the group, the vertical axis represents the gene content, and the bars 1101, 1103 and 1103 Bars 1105 represent the initial point (0PV), the middle point (30PV), and the final point (60PV), respectively. As shown in Figure 11, compared to the initial point (bar 1101), the ratio Comparative Example 1, Comparative Example 2 and Example 1 have higher gene content at the middle point (straight bar 1103), but because the second matrix does not contain sulfate and only has a carbon source, it is compared with Comparative Example 1 and Example 1. , the drsA gene content of Comparative Example 2 was lower at the midpoint (straight bar 1103). In addition, compared to the middle point (bar 1103), at the last point (bar 1105), the drsA gene content of Comparative Example 1 decreased, the drsA gene content of Comparative Example 2 was almost unchanged, and the drsA gene content of Example 1 content increased significantly.

12 is a bar graph of the 16S rRNA gene content of different substrates at different time points according to an embodiment of the present invention, wherein the horizontal axis represents the group, the vertical axis represents the gene content, and the bars 1201 and 1203 and bar 1205 represent the initial point (0PV), the middle point (30PV), and the final point (60PV), respectively. As shown in Figure 12, the 16S rRNA gene content of Comparative Example 2 and Example 1 increased with time, but the 16S rRNA gene content of Comparative Example 1 at the last point (bar 1205) was lower than that at the middle point (bar 1205). 1203) of the 16S rRNA gene content.

From the results in Figures 5 to 12, it can be speculated that the first matrix will release sulfate quickly (Figure 6) because there is no thickener (Figure 6), resulting in an excessively high concentration of sulfide, which in turn causes the protobacteria (straight bar 1205 in Figure 12). Death also caused the overall sulfate metabolism rate of sulfate-reducing bacteria to not increase but decrease (bar 1105 in Figure 11 ), so the effect of remediating hexavalent chromium was limited (Figure 5). The second matrix does not contain sulfate and only provides a carbon source. Although it can increase the number of native bacteria (Fig. 12), it cannot effectively promote the metabolism of sulfate by native sulfate-reducing bacteria (Fig. 11). Therefore, the use of the second matrix can only be slow. Reduction of hexavalent chromium (Figure 7). The viscosity of the third matrix is increased by adding carboxymethyl cellulose, so it can be released stably 300mg/L to 500mg/L of sulfate (Figure 10), in line with the preferred sulfate range (about 300mg/L) confirmed in Example 3, so the third matrix can stably promote the growth and metabolism of sulfate-reducing bacteria ( Fig. 11 bar 1105), thereby forming a water-permeable biological reaction zone, which can not only reduce hexavalent chromium rapidly and effectively (Fig. 9), but also continuously reduce hexavalent chromium above 40PV (Fig. 9, 20PV to 60PV).

As can be seen from the above examples, the gel composition of the present invention has the advantage of adjusting the viscosity of the gel composition by adding carboxymethyl cellulose, so as to control the release of an appropriate amount of sulfate, thereby promoting the growth of native sulfate-reducing bacteria. , thereby forming a water-permeable biological reaction zone that can effectively reduce hexavalent chromium. The effect of the above-mentioned water permeable bioreaction zone is long-lasting, thus reducing the frequency of applying the gel composition and reducing the required amount of the gel composition, thereby reducing the cost of bioremediation.

It should be understood that, although the present invention uses a specific administration method or a specific evaluation method as an example to illustrate the gel composition of the present invention and the method for on-site bioremediation of hexavalent chromium, it is not intended for anyone in the technical field to which the present invention pertains. Those skilled in the art can understand that the present invention is not limited to this, and the present invention can also be carried out using other administration methods or other evaluation methods without departing from the spirit and scope of the present invention.

Although the present invention has been disclosed above with several embodiments, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs, without departing from the spirit and scope of the present invention, can make various Therefore, the scope of protection of the present invention should be determined by the scope of the appended patent application.

<110> National Sun Yat-Sen University

<120> In situ bioremediation of hexavalent chromium gel composition and method

<140> TW 109128796

<141> 2020-08-24

<160> 4

<210> 1

<211> 22

<212> DNA

<213> Artificial sequences

<220>

<223> 16S rRNA upstream primer

<400> 1

<210> 2

<211> 16

<212> DNA

<213> Artificial sequences

<220>

<223> 16S rRNA downstream primer

<400> 2

<210> 3

<211> 20

<212> DNA

<213> Artificial sequences

<220>

<223> dsrA gene upstream primer

<400> 3

<210> 4

<211> 20

<212> DNA

<213> Artificial sequences

<220>

<223> Primer downstream of dsrA gene

<400> 4

901, 903, 905: Straight

Claims (8)

A gel composition for on-site bioremediation of hexavalent chromium, wherein the gel composition is 1L based on the gel composition, and the gel composition comprises: 30g to 50g of carboxymethyl cellulose (carboxymethyl ether of cellulose, CMC), wherein the One of the viscosity of carboxymethyl cellulose is 0.3 Pascal seconds (Pa.s) to 0.6Pa. s; an emulsified oil greater than 0.03L but not greater than 0.05L, based on 100 weight percent (wt %) of the emulsified oil, the emulsified oil comprising 50 wt % to 55 wt % of a vegetable oil, 15 wt % to 20 wt % of a surfactant and an equilibrium amount of water; and an equilibrium amount of a sulfate aqueous solution, and a concentration of the sulfate aqueous solution is 3.0g/L to 4.0g/L, and the gel composition is less than the 19th day to less than the 23rd day A concentration of sustained release sulfate is greater than 200 mg/L to 300 mg/L. The gel composition for in situ bioremediation of hexavalent chromium as claimed in claim 1, wherein a glucose polymerization degree of the carboxymethyl cellulose ranges from 100 to 2000. The gel composition for in situ bioremediation of hexavalent chromium according to claim 1, wherein a concentration of the sulfate aqueous solution is 3.5g/L. The gel composition for in situ bioremediation of hexavalent chromium according to claim 1, wherein the sulfate comprises alkali metal salts and/or alkaline earth metal salts kind. The gel composition for in situ bioremediation of hexavalent chromium according to claim 1, wherein the emulsified oil further comprises 0.001wt% to 0.002wt% of multivitamins. The gel composition for in situ bioremediation of hexavalent chromium according to claim 1, wherein the emulsified oil further comprises 0.4wt% to 0.5wt% of lactate. A method for on-site biological remediation of hexavalent chromium, comprising: applying a gel composition to the polluted area, so that the gel composition continues to release sulfate for a period of time, thereby promoting the growth of sulfate-reducing bacteria and decomposing hexavalent chromium Chromium pollutants, wherein the gel composition is as described in any one of claims 1 to 6, the contaminated area contains hexavalent chromium pollutants and the sulfate-reducing bacteria, and the gel composition is on the 19th day A concentration of the sulfate that sustained release to less than day 23 was greater than 200 mg/L to 300 mg/L. The method for on-site biological remediation of hexavalent chromium as claimed in claim 7, wherein the gel composition is administered for 16 to 30 days, and a concentration of the hexavalent chromium in the polluted area is less than or equal to 0.5 mg/L.

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