Long-acting repairing biological stimulating agent for petroleum contaminated groundwater and preparation method thereof

Abstract

The application relates to the field of biological stimulation medicaments, and discloses a long-acting biological stimulation medicament for repairing oil-polluted underground water and a preparation method, wherein the content of each component is as follows: KNO3 5-10 parts, FeCl2 5-10 parts, glycerol disodium phosphate 1-5 parts, corn peptide 5-10 parts, humic acid 1-5 parts and sorghum powder fermentation liquor 20-50 parts. The method comprises the following steps: first, preparing the sorghum powder fermentation liquor; and second, preparing a multifunctional composite sustained-release agent to obtain the multifunctional composite sustained-release agent. The application has the beneficial effect of significantly improving the repair efficiency and effectively avoiding ecological adaptability problems caused by rapid migration or excessive instant addition, and provides reliable materials and theoretical support for the engineering application of the underground water biological repair technology.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of biological stimulation medicaments and preparation method, in particular to a kind of long-acting repair of oil-contaminated groundwater biological stimulation medicaments and preparation method. BACKGROUND

[0002] In recent years, with the popularization of green sustainable repair concept, microbial remediation technology has become an important means for the treatment of groundwater oil pollution with its low cost, environmental friendliness and sustainability, effectively inhibiting the rebound of pollutants. Among them, in-situ biological stimulation technology based on indigenous microorganisms has become a key direction for the sustainable remediation of oil-contaminated groundwater. However, the effect of this technology is limited by low biological availability of pollutants, complex chemical composition, and low temperature, oxygen deficiency, and nutrient deficiency in groundwater environment. By adding biological stimulants such as nutrient ingredients, electron donors / acceptors and growth factors, the growth environment of microorganisms can be improved, their metabolic activity can be enhanced, and oil degradation can be promoted. However, the current in-situ biological stimulation technology still has problems such as insufficient sustainability of biological stimulants, poor migration, difficult to accurately control the dosage (excessive dosage may lead to secondary pollution, and insufficient dosage may affect the removal efficiency) and unclear mechanism, which restricts the remediation effect. Therefore, developing an economic and efficient microbial remediation technology has become an important research direction for the treatment of oil-contaminated groundwater.

[0003] Sustained / controlled release technology can maintain the effective concentration in the system and improve the utilization efficiency by regulating the release rate of active substances, and is widely used in the field of environmental remediation due to its long-acting and environmental friendliness. In the biological remediation of groundwater organic pollution, sustained / controlled release biological stimulants can be divided into three categories according to the active components: oxygen-releasing type, substrate-releasing type and pH-adjusting type. Among them, substrate-releasing type stimulants have the advantages of low cost, long-acting and good environmental compatibility, showing good application prospect. However, the existing plant fibers, organic acids (alcohols) and oils have problems such as poor stability, short time efficiency and low microbial uptake efficiency. Current researches mainly focus on single active substance, and there is still a lack of systematic exploration of composite stimulants that can play a synergistic effect. Traditional sustained / controlled release agents are usually coated with inert substrates such as clay, biochar, paraffin and high molecular polymer, but they generally have the problem of high initial release rate and insufficient release in the later stage, which may lead to waste of active ingredients and secondary pollution risk, and some difficult-to-degrade substrates may also have adverse effects on the permeability of aquifer. SUMMARY

[0004] The main purpose of the present application is to solve the problems of single component, short-acting, high cost and secondary pollution caused by existing agents in microbial remediation technology, and to provide a long-acting biological stimulation agent for repairing oil-contaminated groundwater and a preparation method thereof.

[0005] The long-acting biological stimulating agent for repairing oil-polluted groundwater provided by the present application comprises KNO3, FeCl2, disodium glycerophosphate, corn peptide, humic acid and sorghum powder fermentation liquor, and the content of each component is as follows: KNO3 5-10 parts, FeCl2 5-10 parts, disodium glycerophosphate 1-5 parts, corn peptide 5-10 parts, humic acid 1-5 parts and sorghum powder fermentation liquor 20-50 parts.

[0006] The present application provides a preparation method of the long-acting biological stimulating agent for repairing oil-polluted groundwater, which comprises the following steps:

[0007] The first step is to prepare the sorghum powder fermentation liquor:

[0008] The sorghum powder is weighed and soaked in ultrapure water at a solid-liquid mass ratio of 1:50-1:100, the soaking time is 8-24h, and 10mL of indigenous microbial bacteria liquid of oil hydrocarbon-polluted groundwater is inoculated into the sorghum powder matrix, and the closed fermentation is carried out under constant temperature oscillation conditions, the temperature of the closed fermentation is 20-30℃, the time is 7-10d, and the oscillation speed is 1000-2000rpm;

[0009] The second step is to prepare the multifunctional composite slow-release agent:

[0010] The KNO3 5-10 parts, FeCl2 5-10 parts, disodium glycerophosphate 1-5 parts, corn peptide 5-10 parts, humic acid 1-5 parts and sorghum powder fermentation liquor 20-50 parts prepared in the first step are weighed according to the weight parts, and the above components are mixed and stirred uniformly by a homogenizer, the stirring speed of the homogenizer is greater than 2000-4000rpm, and the multifunctional composite slow-release agent is obtained.

[0011] The present application has the following beneficial effects:

[0012] The multi-component composite biological stimulating agent provided by the present application can provide balanced and multifunctional nutrient substrates, and more comprehensively support the metabolic activity and degradation function of indigenous microbial communities, thereby significantly improving the repair efficiency.

[0013] The present application takes crop food as the core component of biomass-based nutritional medicament, and develops a kind of multi-cereal fermentation base composite slow-release agent which can efficiently stimulate the activity of petroleum hydrocarbon degrading bacteria by compounding other functional components.The preparation process of the preparation is less, the cost is low, the stimulation effect is good and the action time is long, etc.Under the action of the preparation, the microbial biomass in the contaminated groundwater presents a typical logarithmic growth-stable period dynamic change: from 0.07±0.02mg / L to 12.46±0.32mg / L rapidly in the early culture period (0-5d), and then tends to be stable (10.94-11.26mg / L); Correspondingly, the petroleum hydrocarbon degradation rate increases from 36.49±2.44% to 80.69±7.04% within 3 days.The slow-release agent can realize the sustained and stable release of the nutritional components, and the continuous and efficient action time is more than 2 months, which effectively avoids the ecological adaptability problem caused by rapid migration or instantaneous addition of excessive amount, significantly enhances the actual degradation efficiency of petroleum hydrocarbon, and provides reliable materials and theoretical support for the engineering application of groundwater bioremediation technology. DETAILED DESCRIPTION

[0014] The long-acting biological stimulating agent for repairing petroleum contaminated groundwater provided by the present application comprises KNO3, FeCl2, disodium glycerophosphate, corn peptide, humic acid and sorghum powder fermentation liquor, and the content of each component is 5-10 parts of KNO3, 5-10 parts of FeCl2, 1-5 parts of disodium glycerophosphate, 5-10 parts of corn peptide, 1-5 parts of humic acid and 20-50 parts of sorghum powder fermentation liquor.

[0015] The preparation method of the long-acting biological stimulating agent for repairing petroleum contaminated groundwater provided by the present application comprises the following steps:

[0016] First step, preparation of sorghum powder fermentation liquor:

[0017] Sorghum powder is weighed and soaked in ultrapure water at a solid-liquid mass ratio of 1:50-1:100, the soaking time is 8-24h, and 10mL of indigenous microbial bacteria liquid of petroleum hydrocarbon contaminated groundwater is inoculated into the sorghum powder matrix, and the closed fermentation is carried out under constant temperature oscillation conditions, the temperature of the closed fermentation is 20-30℃, the time is 7-10d, and the oscillation speed is 1000-2000rpm;

[0018] Second step, preparation of multifunctional composite slow-release agent:

[0019] According to the weight, 5-10 parts of KNO3, 5-10 parts of FeCl2, 1-5 parts of disodium glycerophosphate, 5-10 parts of corn peptide, 1-5 parts of humic acid and 20-50 parts of the sorghum powder fermentation liquor prepared in the first step are weighed, the above components are mixed and stirred uniformly by a homogenizer, the stirring speed of the homogenizer is greater than 2000-4000rpm, and the multifunctional composite slow-release agent is obtained.

[0020] The specific implementation is as follows:

[0021] Example 1,

[0022] The nutritional component allocation ratio of the composite slow-release agent of fermented miscellaneous grains is as follows: 20.00 mL of sorghum fermentation liquor, 5.00 mg of KNO3, 25.00-10.00 mg of FeCl2, 1.00-5.00 mg of glycerophosphate disodium, 5.00-10 mg of corn peptide, and 1.00-5.00 mg of humic acid. 10.00 g of sorghum powder is placed in 1 L of water, and fermentation is carried out at 25°C in a sealed state for 7 days.

[0023] An indoor microcosm simulation experiment is adopted, and a gradient exposure system is set. A certain volume of underground water (initial petroleum hydrocarbon concentration gradient: 1, 5, 10, 20, 40 mg / L) is placed in a 100 mL brown glass bottle, and the composite slow-release agent of fermented miscellaneous grains is added in a volume fraction (0.1, 0.5%, 1%, 2%, 5%, 10%), and 100 mL of petroleum hydrocarbon contaminated underground water is formed. The sterilization group (composite slow-release agent of fermented miscellaneous grains + petroleum hydrocarbon) and the blank group (indigenous bacteria + composite slow-release agent of fermented miscellaneous grains) are set as controls. All treatments are set in triplicate, and are cultured at 10°C, 120 rpm in the dark. Sampling is carried out at 0.5-7.0 days (at intervals of 0.5 days), and microbial biomass and residual concentration of petroleum hydrocarbon are determined.

[0024] The results showed that the composite slow-release agent of fermented grains significantly promoted the growth and reproduction of indigenous microorganisms in petroleum-contaminated groundwater and improved the degradation efficiency. Under the stimulation of the composite slow-release agent of fermented grains, the microbial biomass in the petroleum-contaminated groundwater showed a typical dynamic characteristic of logarithmic growth-stationary phase: it rapidly increased from 0.07 ± 0.02 mg / L to 12.46 ± 0.32 mg / L in the early stage of culture (0-5 days) and then tended to be stable (10.94-11.26 mg / L). Correspondingly, the petroleum hydrocarbon degradation rate increased from 36.49 ± 2.44% to 80.69 ± 7.04% within 3 days, and then decreased due to the consumption of effective carbon sources, but still maintained a high degradation level (up to 90.24 ± 2.65% within 7 days). The concentration gradient experiment further revealed that the composite slow-release agent of fermented grains showed good biological stimulation effect on petroleum-contaminated groundwater with different concentrations. After 3 days of treatment with 1% composite slow-release agent of fermented grains, the microbial biomass decreased in a dose-dependent manner with the increase of petroleum hydrocarbon concentration (1-40 mg / L) (9.32 ± 0.89, 8.11 ± 0.69, 7.64 ± 0.31, 6.76 ± 0.92, and 6.49 ± 0.56 mg / L, respectively), and the corresponding petroleum hydrocarbon removal rates were 72.68 ± 4.11%, 75.94 ± 1.68%, 70.89 ± 4.47%, 57.64 ± 6.33%, and 42.49 ± 3.13%, respectively. After extending the treatment time to 7 days, the microbial biomass (7.40-11.21 mg / L) and removal rate (74.97-95.31%) in each concentration group (1-50 mg / L) were significantly improved, confirming that the composite slow-release agent of fermented grains as a biological stimulant could effectively activate the metabolic activity of microorganisms.

[0025] The dosage of the composite slow-release agent based on fermented coarse grains showed a significant dose-effect relationship with microbial growth and petroleum hydrocarbon degradation efficiency. The biological stimulation effect of the composite slow-release agent based on fermented coarse grains had obvious concentration-dependent characteristics. Under low concentration (1 mg / L) conditions, when the dosage of the composite slow-release agent based on fermented coarse grains increased from 0.1% to 10%, the microbial amount increased from 6.86 ± 0.55 to 11.06 ± 0.53 mg / L within 3 days, and the removal rate increased from 62.73 ± 4.03% to 84.71 ± 3.71% (2% dosage reached the peak value). After 7 days, the microbial amount increased to 16.81 ± 0.50 mg / L, and the removal rate was > 92%. Similarly, under 5 mg / L conditions, the 2% dosage reached the best effect within 3 days (microbial amount: 10.69 ± 0.48 mg / L, removal rate: 85.59 ± 4.76%), and the 5% dosage reached a removal rate of 96.77 ± 2.49% after 7 days. Notably, as the initial petroleum hydrocarbon concentration increased (10-40 mg / L), the optimal dosage of the composite slow-release agent based on fermented coarse grains showed an increasing trend. The 2% dosage reached a removal rate of 90.24 ± 2.65% after 7 days at 10 mg / L, the 10% dosage reached a removal rate of 89.36 ± 3.96% after 7 days at 20 mg / L, and the 5% dosage reached a removal rate of 85.39 ± 2.69% after 7 days at 40 mg / L. The effect of the composite slow-release agent based on fermented coarse grains showed a significant time accumulation effect. For example, under 10 mg / L conditions, the 2% dosage reached a removal rate of 90.24 ± 2.65% after 7 days, which was significantly higher than that after 3 days (80.69 ± 7.04%). The growth of microbial amount and the improvement of degradation efficiency showed a certain lag under high concentration (20-40 mg / L) conditions, which might be related to the initial toxic inhibition of high-concentration petroleum hydrocarbons. The optimal dosage of the composite slow-release agent based on fermented coarse grains depended on the concentration of pollutants, and high-dose addition could effectively alleviate the toxicity of petroleum hydrocarbons and enhance long-term remediation effect. Under low concentration (1-5 mg / L) conditions, 1-2% dosage could achieve ideal effect, while under high concentration (20-40 mg / L) conditions, higher dosage (5-10%) was needed to overcome the initial inhibition and obtain the best stimulation effect.

[0026] Example 2,

[0027] The nutrient component allocation ratio of the composite slow-release agent based on fermented coarse grains was as follows: 20.00 mL of fermented sorghum liquid, 5.00 mg of KNO3, 5.00-10.00 mg of FeCl2, 1.00-5.00 mg of glycerol disodium phosphate, 5.00-10 mg of corn peptide, and 1.00-5.00 mg of humic acid. 10.00 g of sorghum powder was placed in 1 L of water, and the mixture was sealed and fermented at 25 °C for 7 days.

[0028] Combined with the hydrogeological conditions of petroleum contaminated sites, the in-situ injection of fermented grain-based composite slow-release agent was carried out to remediate the simulated groundwater contaminated by petroleum hydrocarbons. The device included a water supply device, a peristaltic pump, a plexiglass simulation tank, and a waste liquid collection device. The size of the plexiglass simulation tank was 60 cm (length) x 40 cm (width) x 50 cm (height), the top was sealed with a plexiglass plate, and two ends were provided with a length of 5 cm water storage pool as water distribution area, and the water inlet and outlet holes were opened respectively. The simulation tank was filled with egg gravel, coarse sand, medium sand, fine sand and silty clay from bottom to top to simulate the lithological structure of the actual aquifer. The groundwater hydraulic gradient was controlled by the height difference between the inlet and outlet water storage pools, which was about 5‰. The water level of the inlet was controlled by the peristaltic pump, and the water level of the outlet was adjusted through the sampling hole on one side. Four rows of sampling holes with an inner diameter of 1.0 cm were arranged in the main body of the plexiglass tank, which were labeled as X, Z, C, and L rows from top to bottom. Each row had five sampling points, labeled 1 to 5, for sample collection and monitoring at different lithological layers and horizontal positions.

[0029] Firstly, the simulated groundwater solution was prepared by diluting the actual contaminated groundwater sample, and the petroleum hydrocarbon concentration was adjusted to 5 mg / L. The migration test was conducted for 20 days, and samples were collected regularly to analyze the migration rule of petroleum hydrocarbons in groundwater. After the migration test, indigenous degrading bacteria were added to wells #1 and #2, and the simulated groundwater solution was continuously injected for 20 days. The change of petroleum hydrocarbon concentration was monitored regularly to evaluate the effect of biological enhanced degradation. Subsequently, according to the “Guidelines for In-situ Injection Remediation of Contaminated Groundwater” (T / GIA02-2019), 1 L of biomass-based nutrient agent was added to well 1# under simulated actual engineering injection conditions, and an in-situ injection remediation test was conducted for 40 days. During the test, samples were taken regularly to determine the petroleum hydrocarbon concentration and microbial biomass, and the remediation efficiency of the agent and the microbial response characteristics were evaluated. The results showed that during the period without injection of the agent (0-20 days), the degradation effect of indigenous microorganisms on petroleum hydrocarbons was poor due to the limitation of the oligotrophic environment of groundwater, and there was no significant difference in degradation efficiency among different lithology media. The degradation efficiency tended to be stable after 10 days, and the petroleum hydrocarbon removal rate remained between 38.12-42.32%. After the injection of biomass-based nutrient agent, the nutrients required for microbial growth in the underground environment were rapidly supplemented, and the degradation performance of the bacterial community was significantly improved. Consistent with the dynamic release of nutrients, the petroleum hydrocarbon degradation rate reached a peak in each lithology layer about 10 days after the injection of the agent, with the maximum degradation rates in fine sand, medium sand, coarse sand and gravel layers being 82.21%, 87.21%, 88.61% and 84.83%, respectively. Subsequently, the degradation effect decreased due to the gradual consumption of nutrient components. After 40 days of injection, the petroleum hydrocarbon degradation rates in each lithology medium were maintained in the following ranges: fine sand layer 73.05-77.12%, medium sand layer 69.00-75.60%, coarse sand layer 74.00-77.12%, and gravel layer 73.00-78.60%, indicating that the agent has effectiveness and stability in continuously enhancing biological degradation.

[0030] After the addition of the composite slow-release agent of fermented miscellaneous grains, the microbial community showed a significant response to the environmental conditions conducive to petroleum hydrocarbon degradation, which was manifested by the general increase in microbial biomass and the obvious evolution of community structure. The abundance of Proteobacteria, the dominant bacteria before stimulation, decreased from 76.01–97.62% to 27.50–60.69%, while Verrucomicrobiota, Patescibacteria, and Bacteroidota were newly added as dominant phyla, with their abundance ranges being 3.10–24.89%, 0.60–14.52%, and 9.39–16.94%, respectively. These phyla had high nutritional adaptability. Although the community structure changed, the total abundance of petroleum hydrocarbon-degrading functional bacteria remained dominant. In addition, the injection of biomass-based nutritional agents enhanced the ecological association between dominant bacterial genera, significantly improved the degradation efficiency of petroleum hydrocarbons by strengthening the intergeneric co-metabolic mechanism.

[0031] Example 3,

[0032] The nutritional component allocation ratio of the composite slow-release agent of fermented miscellaneous grains was as follows: 20.00 mL of sorghum fermentation broth, 5.00 mg of KNO3, 5.00–10.00 mg of FeCl2, 1.00–5.00 mg of glycerophosphate disodium, 5.00–10 mg of corn peptide, and 1.00–5.00 mg of humic acid. 10.00 g of sorghum powder was placed in 1 L of water, and the mixture was sealed and fermented at 25°C for 7 days.

[0033] Based on the actual situation of petroleum hydrocarbon contaminated site, the performance of the grain fermentation-based composite slow-release agent and the benchmark agent was compared by the idea of partitioned block verification. Before the injection of the agents, the concentration of petroleum hydrocarbon (C10–C40) in groundwater ranged from 2.306 to 5.403 mg / L, in which the concentration in the demonstration area was 2.519–5.291 mg / L, and the concentration in the benchmark area was 2.306–5.403 mg / L, both of which exceeded the third class of groundwater quality standard (0.3 mg / L). After the injection of the grain fermentation-based composite slow-release agent, the concentration of petroleum hydrocarbon (C10–C40) in groundwater at each monitoring point decreased to different degrees. In the demonstration area, the concentration of petroleum hydrocarbon (C10–C40) in groundwater was 1.531–3.120, 0.678–2.229, 0.235–0.799, undetected–0.294 and undetected–0.101 mg / L, respectively, after the injection of the grain fermentation-based composite slow-release agent for 30, 78, 101, 124 and 167 days. In the benchmark area, the concentration of petroleum hydrocarbon (C10–C40) in groundwater was 1.492–4.779, 1.892–4.888, 1.035–3.588, 0.476–1.935 and 0.478–1.884 mg / L, respectively, after the injection of the benchmark agent for 30, 78, 101, 124 and 167 days. The concentration of petroleum hydrocarbon (C10–C40) in groundwater decreased after the injection of the benchmark agent, but it did not reach the remediation target value. After the injection of the grain fermentation-based composite slow-release agent for 3 months, the concentration of petroleum hydrocarbon (C10–C40) in groundwater at some monitoring points reached the remediation target value (0.3 mg / L), and after 4 months, the concentration of petroleum hydrocarbon (C10–C40) in groundwater at all monitoring points reached the remediation target value, and there was no rebound phenomenon of petroleum hydrocarbon pollutants.

[0034] The degradation effect of petroleum hydrocarbon (C10–C40) by microorganisms under the stimulation of slow-release agents was evaluated, with the concentration of petroleum hydrocarbon (C10–C40) in groundwater before the injection of the agents as the initial value. In the demonstration area, the biodegradation rates of petroleum hydrocarbon (C10–C40) were 39.05–47.87%, 46.29–74.45%, 68.27–94.48%, 88.33–100.00% and 96.00–100.00%, respectively, after the injection of the grain fermentation-based composite slow-release agent for 30, 78, 101, 124 and 167 days. In the benchmark area, the biodegradation rates were –0.42–35.30%, 7.06–48.36%, 33.59–64.87%, 64.18–87.16% and 49.16–80.33%, respectively, after the injection of the benchmark agent for the corresponding time. The results showed that the stimulation effect of the grain fermentation-based composite slow-release agent on the degradation of petroleum hydrocarbon by indigenous microorganisms was significantly better than that of the benchmark agent, and the biodegradation rate could be increased by up to 46.84%. Moreover, the cost of the grain fermentation-based composite slow-release agent was more than 40% lower than that of the foreign similar agent, and it could significantly improve the activity of the indigenous microorganism action network and the activity of alkane hydroxylase.

Claims

1. A method for preparing a long-acting biostimulant for remediating petroleum-contaminated groundwater, characterized in that: The method includes the following steps: Step 1: Preparation of sorghum flour fermentation liquid: Weigh out sorghum powder and soak it in ultrapure water at a solid-liquid mass ratio of 1:50-1:100 for 8-24 hours. Inoculate the sorghum powder matrix with 10 mL of indigenous microbial culture solution from petroleum hydrocarbon-contaminated groundwater. Carry out closed fermentation under constant temperature and shaking conditions. The temperature of closed fermentation is 20-30℃, the time is 7-10 days, and the shaking speed is 1000-2000 rpm. Step 2: Preparation of biostimulant: Weigh out 5-10 parts by weight of KNO3, 5-10 parts by weight of FeCl2, 1-5 parts by weight of disodium glycerophosphate, 5-10 parts by weight of corn peptide, 1-5 parts by weight of humic acid, and 20-50 parts by weight of sorghum flour fermentation liquid obtained in the first step. Mix them together and stir evenly using a homogenizer. The stirring speed of the homogenizer should be greater than 2000-4000 rpm to obtain a multifunctional compound slow-release agent.

2. The application of the long-acting biostimulant for remediating petroleum-contaminated groundwater as described in claim 1, characterized in that: Groundwater used for long-term remediation of oil-contaminated water.