Preparation method of microbial agent for removing acesulfame potassium

Abstract

The invention belongs to the technical field of environmental pollutant water treatment, and discloses a preparation method of a microbial agent for removing acesulfame potassium. The microbial agent is prepared by compounding acesulfame potassium degrading flora enriched in a secondary sedimentation tank of a sewage treatment plant and electrochemical active bacterium Shewanella oneidensis MR-1, the Shewanella oneidensis MR-1 and the acesulfame potassium degrading flora can form a nano conduit for electron transfer, the activity of an electron transfer system in the whole system is improved, and the degradation effect of acesulfame potassium is improved. Therefore, the biodegradation of acesulfame potassium is accelerated. The microbial agent can realize efficient degradation and mineralization of acesulfame potassium, and has a good application prospect in rapid treatment of acesulfame potassium-containing wastewater.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental pollutant water treatment, utilizes microbial flora capable of degrading acesulfame potassium and electrochemically active bacteria Shewanella Oneida MR-1 to prepare a microbial agent, and uses the microbial agent to efficiently degrade acesulfame potassium. Background Art

[0002] In recent years, the high incidence of obesity, diabetes and other diseases caused by high-sugar and high-calorie diets has prompted the food industry to develop artificial sweeteners with special properties. Artificial sweeteners do not participate in human metabolism and are currently widely used in the food, beverage, pharmaceutical and personal care industries. Due to their high polarity and strong hydrophilicity, environmental water bodies have become their main destination. It has been reported that artificial sweeteners can produce ecotoxicity to aquatic organisms such as medaka, zebrafish, large fleas, and duckweed. Among the artificial sweeteners detected worldwide, acesulfame potassium is dominant, with the highest detected concentration reaching 2.5 mg / L, and it has been reported that acesulfame potassium can cause DNA damage in human peripheral blood lymphocytes. Therefore, how to effectively remove acesulfame potassium from environmental water bodies has attracted increasing attention.

[0003] In the current sewage treatment process, the removal effect of activated carbon adsorption, coagulation sedimentation, and chlorination disinfection on acesulfame potassium is very limited. The ozone advanced oxidation process can only partially remove artificial sweeteners under the dosage and contact time of the actual sewage treatment plant. Compared with the above-mentioned chemical method, the biological method has the advantages of being environmentally friendly and low cost. At present, the only acesulfame potassium degradation strains that have been isolated are Chelatococcus sp., Shinella spp., and Bosea sp., but the single bacteria are sensitive to environmental conditions and have poor adaptability. It has been reported that the bacterial community enriched with Phyllobacteriaceae, Methylophilaceae, Bradyrhizobiaceae and Pseudomonas genera can degrade 60% of the initial concentration of 125 μg / L acesulfame potassium within one month. However, its degradation process is slow. Therefore, it is necessary to enrich the bacterial community that efficiently degrades acesulfame potassium, and on this basis further improve the ability of the acesulfame potassium degradation bacterial community so as to be applied to the sewage treatment of acesulfame potassium.

[0004] Oneida Shewanella MR-1 is a facultative bacterium with the ability of extracellular electron transfer. Its transfer mechanism includes three types: direct electron transfer, nanowires, and electron shuttling of soluble redox mediators. However, Oneida Shewanella MR-1 has no degradation effect on acesulfame potassium. The present invention utilizes the good extracellular electron transfer ability of Oneida Shewanella MR-1 and compound it with acesulfame potassium degrading bacteria to form a bacterial agent. Nanoconduits can be formed between strain MR-1 and acesulfame potassium degrading bacteria in the bacterial agent to transfer electrons, thereby improving the activity of the electron transfer system in the entire system, so that the bacterial agent has the ability to efficiently degrade acesulfame potassium. Summary of the invention

[0005] The present invention compounds the bacterial flora that can degrade acesulfame potassium with the electrochemically active bacteria Oneida Shewanella MR-1, with the purpose of preparing a bacterial agent that can efficiently and quickly degrade acesulfame potassium in water. In this process, the electrochemically active bacteria Oneida Shewanella MR-1 can form nano-conduits with the enriched acesulfame potassium-degrading bacterial flora to carry out electron transfer and improve the activity of the electron transfer system in the entire system. At the same time, there are various low-concentration carbon sources in the wastewater, which can be used by the compounded bacterial agent to improve cell activity, thereby accelerating the biodegradation of acesulfame potassium. The bacterial agent can achieve efficient degradation and mineralization of 100 mg / L acesulfame potassium within 21 hours, and its degradation rate is increased by 72%, which has good application prospects in the rapid treatment of wastewater containing acesulfame potassium.

[0006] The technical solution of the present invention:

[0007] A method for preparing a microbial agent for removing acesulfame potassium comprises the following steps:

[0008] The composition of the microbial agent capable of degrading acesulfame potassium: microbial flora capable of degrading acesulfame potassium and Shewanella Oneida MR-1;

[0009] (1) Microbial flora enriched with acesulfame potassium degradation ability

[0010] The sludge obtained from the secondary sedimentation tank of the sewage treatment plant was inoculated into the nutrient medium 1 with acesulfame potassium as the only carbon source, and cultured in a shaker at 25-35°C and 150rpm. The nutrient medium was replaced every 5-7 days, and the concentration of acesulfame potassium in the medium was increased depending on the degradation of acesulfame potassium. After one month, a microbial flora capable of degrading acesulfame potassium was obtained;

[0011] The composition of the nutrient medium 1 is: acesulfame potassium 50-150 mg / L, Na2HPO4 5.23-5.63 g / L, KH2PO4 0.655-0.678 g / L, MgSO4·7H2O 0.2 g / L, NaNO3 0.02 g / L, CaCl2 0.02 g / L, FeCl3 5.6 mg / L, pH = 7.5-8.5;

[0012] (2) Activation of electrochemically active bacteria Shewanella Oneida MR-1

[0013] The nutrient medium 2 was used to activate the MR-1 strain of Shewanella oleracea. The strain was cultured in a shaker at 25-35°C and 150 rpm for 12 h. The strain was then washed with sterile saline and the OD was calculated. 600 A bacterial suspension of Shewanella Oneida MR-1 at 0.12-0.16;

[0014] The composition of nutrient medium 2 is: acid hydrolyzed casein 100-200 mg / L, Na2HPO4 5.23-5.63 g / L, KH2PO4 0.655-0.678 g / L, MgSO4·7H2O 0.2 g / L, NaNO3 0.02 g / L, CaCl2 0.02 g / L, FeCl3 5.6 mg / L, pH = 7.5-8.5;

[0015] (3) Preparation of microbial agents

[0016] The microbial flora that can degrade acesulfame potassium obtained by enrichment in step (1) is activated using nutrient medium 3. During activation, it is placed in a shaker at 25-35°C and 150 rpm for 12 hours, and then the bacteria are washed with sterile saline and prepared into OD 600 A bacterial suspension of 0.02-0.05 acesulfame potassium-degrading bacteria;

[0017] The bacterial suspension of Shewanella Oneida MR-1 and acesulfame potassium-degrading bacteria was mixed to obtain a microbial agent to control OD 600 The ratio is 1:2-10.

[0018] Among them, the ingredients of nutrient medium 3 are: acesulfame potassium 50-150 mg / L, acid hydrolyzed casein 100-200 mg / L, Na2HPO4 5.23-5.63 g / L, KH2PO4 0.655-0.678 g / L, MgSO4·7H2O 0.2 g / L, NaNO3 0.02 g / L, CaCl2 0.02 g / L, FeCl3 5.6 mg / L, pH = 7.5-8.5.

[0019] The beneficial effects of the present invention are as follows: the present invention utilizes the enriched bacterial community that can degrade acesulfame potassium under aerobic conditions, and compounding the bacterial community with the electrochemically active bacteria Shewanella Oneida MR-1 to prepare a bacterial agent, and Shewanella MR-1 can enhance the activity of the electron transfer system in the entire system and accelerate the biodegradation of acesulfame potassium. The bacterial agent can achieve efficient degradation and mineralization of 100 mg / L acesulfame potassium within 21 hours, and has good application prospects in the rapid treatment of wastewater containing acesulfame potassium. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the degradation effect of acesulfame potassium by the electrochemically active bacteria Shewanella Oneida MR-1.

[0021] Figure 2 This is a diagram showing the effect of acesulfame potassium degradation by acesulfame potassium-degrading bacteria.

[0022] Figure 3 This is a graph showing the degradation effect of acesulfame potassium-degrading bacteria on acesulfame potassium at different concentrations.

[0023] Figure 4 This is a diagram showing the effects of various bacterial agents (adding different proportions of electrochemically active bacteria Shewanella Oneida MR-1 to the acesulfame-degrading bacterial community) on the degradation of acesulfame-potassium. DETAILED DESCRIPTION

[0024] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.

[0025] Comparative Example 1

[0026] The preserved electrochemically active bacteria Shewanella Oneida MR-1 were activated using a nutrient medium (which did not contain acesulfame potassium). During activation, the bacteria were cultured in a shaker at 30°C and 150 rpm for 12 h. The bacteria were then washed with sterile saline and prepared into OD 600 It is 0.15±0.01 of bacterial suspension;

[0027] The bacterial suspension was added to the reaction system and reacted in a shaker at 30°C and 150 rpm.

[0028] The culture medium used in the reaction system was 100 mg / L acesulfame potassium, 200 mg / L acid hydrolyzed casein, 5.53 g / L Na2HPO4, 0.675 g / L KH2PO4, 0.2 g / L MgSO4·7H2O, 0.02 g / L NaNO3, 0.02 g / L CaCl2, 5.6 mg / L FeCl3, pH = 8.0.

[0029] The electrochemically active bacteria Shewanella Oneida MR-1 cannot degrade acesulfame potassium.

[0030] Comparative Example 2

[0031] The sludge obtained from the secondary sedimentation tank of the Lingshui River sewage treatment plant was inoculated into a culture medium with acesulfame potassium as the sole carbon source, and cultured in a shaker at 30°C and 150rpm. The culture medium was replaced every 5 days, and the acesulfame potassium concentration in the culture medium was increased depending on the degradation of acesulfame potassium. After one month, a bacterial community that can degrade acesulfame potassium was obtained.

[0032] The 16S rRNA gene high-throughput sequencing of the bacterial community was performed by Sangon Biotech (Shanghai) Co., Ltd. It was determined that the bacterial genera with a higher proportion of the bacterial community were Pseudochrobactrum, Aeromonas, Castellaniella, and Stenotrophomonas, accounting for 30.4%, 13.7%, 11.7%, and 7.7%, respectively.

[0033] The acesulfame potassium-degrading bacteria were activated using nutrient medium 3. During activation, they were placed in a shaker at 30°C and 150 rpm for 12 h, and then the bacteria were washed with sterile saline and prepared into OD 600 It is 0.03±0.01 of bacterial suspension;

[0034] The bacterial suspension was added to the reaction system and reacted in a shaker at 30°C and 150 rpm.

[0035] The culture medium used in the reaction system was acesulfame potassium 100 mg / L, Na2HPO4 5.53 g / L, KH2PO4 0.675 g / L, MgSO4·7H2O 0.2 g / L, NaNO3 0.02 g / L, CaCl2 0.02 g / L, FeCl3 5.6 mg / L, pH = 8.0.

[0036] Example 1

[0037] The acesulfame potassium-degrading bacteria were activated using nutrient medium 3. During activation, they were placed in a shaker at 30°C and 150 rpm for 12 h, and then the bacteria were washed with sterile saline and prepared into OD 600 It is 0.03±0.01 of bacterial suspension;

[0038] The bacterial suspension was added to each reaction system and reacted in a shaker at 30°C and 150 rpm.

[0039] The culture medium used in the reaction system was acesulfame potassium 50, 100, 150 mg / L, Na2HPO4 5.53 g / L, KH2PO4 0.675 g / L, MgSO4·7H2O 0.2 g / L, NaNO3 0.02 g / L, CaCl2 0.02 g / L, FeCl3 5.6 mg / L, pH = 8.0.

[0040] Acesulfame potassium-degrading bacteria can degrade acesulfame potassium at different concentrations under aerobic conditions.

[0041] Example 2

[0042] The acesulfame potassium-degrading bacteria were activated using nutrient medium 3. During activation, they were placed in a shaker at 30°C and 150 rpm for 12 h, and then the bacteria were washed with sterile saline and prepared into OD600 It is 0.03±0.01 of bacterial suspension;

[0043] Shewanella Oneida MR-1 was activated using nutrient medium 2. During activation, the bacteria were cultured in a shaker at 30°C and 150 rpm for 12 h. The bacteria were then washed with sterile saline and the OD 600 for bacterial suspensions of 0.06 ± 0.01 and 0.3 ± 0.01;

[0044] The bacterial suspension of Shewanella Oneida MR-1 and acesulfame potassium-degrading bacteria was mixed to obtain a microbial agent. 600 The ratios are 1:2 and 1:10.

[0045] The bacterial agent was added into the reaction system and reacted in a shaker at 30°C and 150 rpm.

[0046] The culture medium used in the reaction system was 100 mg / L acesulfame potassium, 200 mg / L acid hydrolyzed casein, 5.53 g / L Na2HPO4, 0.675 g / L KH2PO4, 0.2 g / L MgSO4·7H2O, 0.02 g / L NaNO3, 0.02 g / L CaCl2, 5.6 mg / L FeCl3, pH = 8.0.

[0047] Comparative Example 1 shows that the electrochemically active bacteria Oneida Shewanella MR-1 has no degradation effect on acesulfame potassium. The Oneida Shewanella MR-1 added to the microbial agent has electrochemical activity, and can form nano-conduits with the acesulfame potassium-degrading bacterial community to carry out interspecies electron transfer, thereby improving the activity of the electron transfer system in the entire acesulfame potassium degradation system. At the same time, the composite microbial agent can utilize the acid hydrolysis of casein in the system to improve cell activity and further accelerate the biodegradation of acesulfame potassium. Taking Comparative Example 2 as the control group, the microbial agent compounded with the electrochemically active bacteria Oneida Shewanella MR-1 and the enriched acesulfame potassium-degrading bacterial community can achieve efficient degradation and mineralization of 100 mg / L acesulfame potassium within 21 hours, and its degradation rate is increased by 72%, which greatly improves the degradation efficiency of acesulfame potassium and shortens its degradation time.

Claims

1. A method for preparing a microbial agent for removing acesulfame potassium, characterized in that: The microbial agent used to remove acesulfame potassium comprises: microbial flora capable of degrading acesulfame potassium and Shewanella Oneida MR-1; The specific steps are as follows: (1) Microbial flora enriched with acesulfame potassium degradation ability The sludge obtained from the secondary sedimentation tank of the sewage treatment plant was inoculated into the nutrient medium 1 with acesulfame potassium as the only carbon source, and cultured in a shaker at 25-35°C and 150rpm. The nutrient medium was replaced every 5-7 days, and the concentration of acesulfame potassium in the medium was increased depending on the degradation of acesulfame potassium. After one month, a microbial flora capable of degrading acesulfame potassium was obtained; (2) Activation of electrochemically active bacteria Shewanella Oneida MR-1 The nutrient medium 2 was used to activate the Shewanella MR-1 on Oneida, and the culture was placed in a shaker at 25-35°C and 150 rpm for 12 h. The bacteria were then washed with sterile saline and the OD was adjusted to 600 A bacterial suspension of Shewanella Oneida MR-1 at 0.12-0.16; (3) Preparation of microbial agents The microbial flora that can degrade acesulfame potassium obtained by enrichment in step (1) is activated using nutrient medium 3. During activation, it is placed in a shaker at 25-35°C and 150 rpm for 12 hours, and then the bacteria are washed with sterile saline and prepared into OD 600 A bacterial suspension of 0.02-0.05 acesulfame potassium-degrading bacteria; The bacterial suspension of Shewanella Oneida MR-1 and acesulfame potassium-degrading bacteria was mixed to obtain a microbial agent to control OD 600 The ratio is 1:2-10.

2. The preparation method according to claim 1, characterized in that: The ingredients of the nutrient medium 1 are: acesulfame potassium 50-150 mg / L, Na2HPO4 5.23-5.63 g / L, KH2PO4 0.655-0.678 g / L, MgSO4·7H2O 0.2 g / L, NaNO3 0.02 g / L, CaCl2 0.02 g / L, FeCl3 5.6 mg / L, pH = 7.5-8.

5.

3. The preparation method according to claim 1, characterized in that: The ingredients of the nutrient medium 2 are: acid hydrolyzed casein 100-200 mg / L, Na2HPO4 5.23-5.63 g / L, KH2PO4 0.655-0.678 g / L, MgSO4·7H2O 0.2 g / L, NaNO3 0.02 g / L, CaCl2 0.02 g / L, FeCl3 5.6 mg / L, pH=7.5-8.

5.

4. The preparation method according to claim 1, characterized in that: The ingredients of the nutrient medium 3 are: acesulfame potassium 50-150 mg / L, acid hydrolyzed casein 100-200 mg / L, Na2HPO4 5.23-5.63 g / L, KH2PO4 0.655-0.678 g / L, MgSO4·7H2O 0.2 g / L, NaNO3 0.02 g / L, CaCl2 0.02 g / L, FeCl3 5.6 mg / L, pH = 7.5-8.5.

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