Complex microbial inoculant for treating phenolic substances in high-acid wastewater and application of complex microbial inoculant

Abstract

The invention belongs to the technical field of wastewater treatment and microorganism application, and particularly relates to a complex microbial inoculant for treating phenolic substances in high-acid wastewater and application of the complex microbial inoculant. The complex microbial inoculant is prepared from lactobacillus acidophilus, Ralstonia piceatrii, bacillus modestus and bacillus mucilaginosus; wherein in the complex microbial inoculant, the weight ratio of the lactobacillus acidophilus to the Ralstonia piceatrii to the deposited halobacillus to the silt bacillus is (1-20): (1-10): (1-10): (1-10): (1-10). In the complex microbial inoculant provided by the invention, lactobacillus acidophilus, Ralstonia piceatrii, Mesohalobacillus sp. And Bacillus mucilaginosus are specifically matched and combined, and the tolerance of the microbial inoculant to an acid environment can be enhanced by utilizing the mutual synergistic effect of microbial strains; the effect of treating phenolic substances in the high-acid wastewater is obviously improved.

Description

Technical Field

[0001] The present application belongs to the field of wastewater treatment and microbial application technology, and in particular relates to a composite bacterial agent for treating phenolic substances in high-acid wastewater and its application. Background Art

[0002] Highly acidic, phenol-rich industrial wastewater primarily originates from industries such as pharmaceuticals, pulp and paper, and traditional plant-based brewing. During the production processes of these industries, complex components such as lignin and tannins within plant tissues undergo a series of chemical and biological degradation processes, releasing a wide variety of phenolic compounds that are directly incorporated into the resulting wastewater. This wastewater exhibits significant biotoxicity, with its harmful substances tending to persist in the environment for long periods. Combined with their high resistance to degradation, it poses a serious threat to aquatic ecosystems, hindering the maintenance of ecological balance and further exacerbating the degradation of water quality and the overall health of the aquatic environment.

[0003] At present, the mainstream treatment technologies for wastewater rich in phenolic substances include chemical oxidation, adsorption and biological methods. However, traditional physical and chemical treatment methods, such as the use of adsorption materials or oxidants, although they have played a treatment role to a certain extent, have exposed many limitations in practical applications. The primary problem is the high treatment cost, including material procurement and operation and maintenance costs, which undoubtedly increases the economic burden of enterprises. Secondly, such methods are often accompanied by secondary pollution problems, that is, new harmful by-products may be produced during the treatment process, resulting in the transfer of environmental pollution rather than elimination. Furthermore, in the face of complex pollutants in wastewater, especially high molecular weight, structurally stable phenolic substances, the treatment efficiency and thoroughness are often unsatisfactory, and cannot meet the increasingly stringent environmental protection standards and the public's expectations for clean production.

[0004] The microbial bioprocess involves the biodegradation of phenols in phenolic-rich wastewater using microbial communities under aerobic or anaerobic conditions. In practical applications, a variety of microbial species are known to be capable of degrading phenols. These include Pseudomonas species, which can convert phenols into more biodegradable intermediates through a series of enzymatic reactions (such as monooxygenases, dioxygenases, and dehydrogenases), which ultimately mineralize into carbon dioxide and water. Bacillus species have a high tolerance to phenols and can secrete extracellular enzymes to enhance the bioconversion of phenols. White-rot fungi possess powerful ligninolytic enzyme systems (such as manganese peroxidase and laccase), which can also act on phenolic compounds, oxidizing them into more biodegradable forms. However, the application of these microbial treatment systems is limited by their strict requirements for the environment in which they survive. For example, Pseudomonas and Bacillus species generally require a neutral to slightly alkaline environment (pH 6.5-8.5) and a phenolic concentration below 500 mg / L; white-rot fungi are best suited to growing in a neutral to slightly acidic environment (pH 4.0-7.0). Although wastewater can be pretreated in production by actively adjusting the pH or diluting the phenolic concentration, these methods undoubtedly further increase the economic and labor costs of wastewater treatment.

[0005] Therefore, the present application aims to provide a more economical, efficient and environmentally friendly wastewater treatment solution based on biological methods for phenolic substances or high-content phenolic substances in high-acid wastewater / sewage. Summary of the Invention

[0006] The purpose of this application is to provide a composite bacterial agent for treating phenolic substances in high-acid wastewater and its application.

[0007] According to the first aspect of the present application, the present application provides a composite bacterial agent for treating phenolic substances in high-acid wastewater and its application, wherein the composite bacterial agent includes: Lactobacillus jinshanaceticus, Ralstonia pekinensis, Bacillus halogenids in sedimentation, and Bacillus oceanicus in silt; wherein, in the composite bacterial agent, the weight ratio between Lactobacillus jinshanaceticus and Ralstonia pekinensis, Bacillus halogenids in sedimentation, and Bacillus oceanicus in silt is 1-20:1-10:1-10:1-10:1-10:1-10.

[0008] Specifically, the "high-acid wastewater" in this application mainly refers to wastewater with high acidity, wherein "high acidity" refers to an acidity range of 1-3% or a pH range of 2.0-5.0; the content of phenolic substances in the "high-acid wastewater" is 0.5-2 g / L or higher than 2.0 g / L; phenolic substances include: at least one of protocatechuic acid, gallic acid, caffeic acid, coumaric acid, catechol and phenol.

[0009] As described in the background technology section, the treatment of phenolic substances in wastewater based on microbial degradation disclosed in the prior art is mostly limited by the strict requirements of microorganisms for the living environment. For example, the microorganisms disclosed in the prior art that can be used for the degradation or treatment of phenolic substances in wastewater generally need to grow in a neutral to slightly alkaline (pH 6.5-8.5) environment, and the concentration of the corresponding phenolic substances in the wastewater is required not to be too high, such as less than 500 mg / L. In actual application, this type of microbial degradation technology needs to be actively adjusted by the pH of the wastewater or by dilution to adjust the concentration of the phenolic substances in the wastewater in order to achieve the treatment of phenolic substances in the wastewater. Based on this, the present application provides a composite bacterial agent that can efficiently treat high-content phenolic substances in high-acid wastewater. Through research, the present application found that by specifically combining Jinshan acetate lactobacillus with Ralstonia pinellia, sedimentary salt bacillus and silt oceanic bacillus, and utilizing the mutual synergistic effect between microbial species, the tolerance of the bacterial agent to the acidic environment can be enhanced, and the treatment capacity of phenolic substances in high-acid wastewater can be significantly improved. The composite bacterial agent provided by the present application can play a synergistic role in the treatment effect of high-content phenolic substances in high-acid wastewater.

[0010] In some embodiments of the present application, the weight ratio of Lactobacillus jinshanensis to Ralstonia pinnatifida, Bacillus salt in sediment and Bacillus oceanicus in silt is 1-20:1-6:1-6:1-6:1-6:1-6; preferably, the weight ratio of Lactobacillus jinshanensis to Ralstonia pinnatifida, Bacillus salt in sediment and Bacillus oceanicus in silt is 1-15:1-5:1-5:1-5:1-5:1-5; preferably, the weight ratio of Lactobacillus jinshanensis to Ralstonia pinnatifida, Bacillus salt in sediment and Bacillus oceanicus in silt is 1-15:1-2:1-2:1-2:1-2.

[0011] In some embodiments of the present application, the cell concentration of the composite bacterial agent, Lactobacillus jinshanensis, Ralstonia pekinensis, Bacillus salinarum and Bacillus silt oceanicus is (0.5-10)×10 9 Specifically, the cell concentration of Lactobacillus jinshanensis, Ralstonia pekinensis, Bacillus halogenides in sediments and Bacillus oceanica in silt can be 0.5×10 9 cells / ml, 1.0×10 9 cells / ml、1.5×10 9 cells / ml, 2.0×10 9 cells / ml、2.5×10 9 cells / ml, 3.0×10 9 cells / ml、3.5×10 9cells / ml, 4.0×10 9 cells / ml、4.5×10 9 cells / ml、5.0×10 9 cells / ml、5.5×10 9 cells / ml、6.0×10 9 cells / ml、6.5×10 9 cells / ml、7.0×10 9 cells / ml、7.5×10 9 cells / ml、8.0×10 9 cells / ml、8.5×10 9 cells / ml、9.0×10 9 cells / ml、9.5×10 9 cells / ml、10×10 9 cells / ml or a range consisting of any two of the above values.

[0012] In some embodiments of the present application, the deposition number of Lactobacillus jinshanensis is CICC 6269; the deposition number of Ralstonia pekinensis is CGMCC 1.10820; the deposition number of Bacillus halobacterium is CICC 24779; and the deposition number of Bacillus oceanica is CICC 24563.

[0013] According to the second aspect of the present application, the present application provides a method for preparing the composite bacterial agent as described in any one of the first aspects of the present application, the preparation method comprising the following steps:

[0014] (1) Lactobacillus jinshanensis, Ralstonia pekinensis, Bacillus halogenides of sediments, and Bacillus oceanica of silt were respectively prepared into corresponding bacterial solutions;

[0015] (2) The prepared corresponding bacterial solutions are mixed according to a certain proportion to obtain the composite bacterial agent.

[0016] In some embodiments of the present application, the cell concentration corresponding to the bacterial solution is (0.5-10)×10 9 cells / ml. Specifically, the cell concentration of the bacterial solution can be 0.5×10 9 cells / ml, 1.0×10 9 cells / ml、1.5×10 9 cells / ml, 2.0×10 9 cells / ml、2.5×10 9 cells / ml, 3.0×10 9cells / ml、3.5×10 9 cells / ml, 4.0×10 9 cells / ml、4.5×10 9 cells / ml、5.0×10 9 cells / ml、5.5×10 9 cells / ml、6.0×10 9 cells / ml、6.5×10 9 cells / ml、7.0×10 9 cells / ml、7.5×10 9 cells / ml、8.0×10 9 cells / ml、8.5×10 9 cells / ml、9.0×10 9 cells / ml、9.5×10 9 cells / ml、10×10 9 cells / ml or a range consisting of any two of the above values.

[0017] In some embodiments of the present application, the preparation method of the Jinshan Lactobacillus acetate liquid comprises: inoculating Jinshan Lactobacillus acetate into the MRS culture medium of the fermented mash extract, and culturing the culture in an anaerobic incubator at 28-35°C; preferably, the preparation method of the Ralstonia pinnatifida liquid, the sedimentary salt bacillus liquid and the silt oceanic bacillus liquid comprises: inoculating Ralstonia pinnatifida, the sedimentary salt bacillus liquid and the silt oceanic bacillus liquid into NB culture medium, respectively, and culturing the culture in a shaker at 100-200 rpm at 28-35°C. Specifically, in the preparation method of the Jinshan Lactobacillus acetate liquid, the culture temperature can be 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C or a range consisting of any two of the above values. Specifically, in the preparation methods of the Ralstonia pekinensis liquid, the sedimentary Bacillus salinarum liquid, and the silt oceanic Bacillus liquid, the culture temperature can be 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or a range consisting of any two of the above values. Specifically, the rotation speed of the shaking culture can be 100 rpm, 110 rpm, 115 rpm, 120 rpm, 125 rpm, 130 rpm, 135 rpm, 140 rpm, 145 rpm, 150 rpm, 155 rpm, 160 rpm, 165 rpm, 170 rpm, 175 rpm, 180 rpm, 185 rpm, 190 rpm, 195 rpm, 200 rpm, or a range consisting of any two of the above values.

[0018] According to the third aspect of the present application, the present application also provides an application of the composite bacterial agent as described in any one of the first aspects of the present application in the field of wastewater treatment.

[0019] In some embodiments of the present application, the wastewater comprises highly acidic wastewater containing phenols, or highly acidic wastewater containing a high content of phenols; and the application comprises treating phenols in the highly acidic wastewater. Specifically, "highly acidic" wastewater refers to wastewater with an acidity range of 1-3% or a pH range of 2.0-5.0; "high content of phenols" refers to wastewater with a phenolic content of 0.5-2 g / L or greater than 2.0 g / L.

[0020] In some embodiments of the present application, the phenolic substance includes at least one of gallic acid, caffeic acid, p-coumaric acid, protocatechuic acid and phenol.

[0021] According to the fourth aspect of the present application, the present application also provides a method for degrading phenolic substances, the method comprising:

[0022] The composite bacterial agent as described in any one of the first aspects of the present application is added to a fermentation system containing phenolic substances, and fermentation culture is carried out to degrade the phenolic substances in the fermentation system during the fermentation process.

[0023] In some embodiments of the present application, the inoculation amount of the composite bacterial agent relative to the fermentation system is 5% to 15%. Specifically, the inoculation amount of the composite bacterial agent can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or a range consisting of any two of the above values.

[0024] In some embodiments of the present application, the dissolved oxygen content in the fermentation system is 2 to 5 ppm. Specifically, the dissolved oxygen content in the fermentation system can be 2 ppm, 2.2 ppm, 2.4 ppm, 2.5 ppm, 2.6 ppm, 2.8 ppm, 2.9 ppm, 3.0 ppm, 3.2 ppm, 3.4 ppm, 3.5 ppm, 3.6 ppm, 3.8 ppm, 3.9 ppm, 4.0 ppm, 4.2 ppm, 4.4 ppm, 4.5 ppm, 4.6 ppm, 4.8 ppm, 4.9 ppm, 5 ppm, or a range consisting of any two of the above values.

[0025] In some embodiments of the present application, the acid content in the fermentation system is 1-3% or the pH is 2-5. Specifically, the acid content in the fermentation system can be 1%, 1.2%, 1.4%, 1.5%, 1.6%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 2.9%, 3%, or a range consisting of any two of the above values. Specifically, "acid content" refers to the content of the corresponding acid in the fermentation system, and can also refer to the acidity in the fermentation system, such as the acidity in the fermentation system is 1-3%. Specifically, the pH in the fermentation system can be 2, 2.2, 2.4, 2.5, 2.6, 2.8, 3.0, 3.2, 3.4, 3.5, 3.6, 3.8, 4.0, 4.2, 4.4, 4.5, 4.6, 4.8, 5, or a range consisting of any two of the above values.

[0026] In some embodiments of the present application, the content of phenolic substances in the fermentation system is 0.5-2 g / L or higher than 2 g / L. Specifically, the content of phenolic substances in the fermentation system can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2 g / L, or a range consisting of any two of the above values, or higher than 2 g / L.

[0027] In some embodiments of the present application, the fermentation temperature is 25-35°C; the fermentation time is 5-15 days. Specifically, the fermentation temperature can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or a range consisting of any two of the above values. Specifically, the fermentation time can be 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or a range consisting of any two of the above values.

[0028] In some embodiments of the present application, the fermentation system containing phenolic substances includes a culture medium containing phenolic substances; preferably, the culture medium is a fermented mash extract MRS culture medium.

[0029] In some embodiments of the present application, the phenolic substance includes at least one of gallic acid, caffeic acid, p-coumaric acid, protocatechuic acid and phenol.

[0030] According to a fourth aspect of the present application, the present application further provides a method for treating phenolic substances in high-acid wastewater, the method comprising:

[0031] The composite bacterial agent described in any one of the first aspects of the present application is added to the high-acid wastewater for fermentation, and the phenolic substances in the high-acid wastewater are degraded during the fermentation process.

[0032] In some embodiments of the present application, the acidity of the high-acid wastewater is 1-3% or the pH is 2.0-5.0; in some embodiments of the present application, the content of phenolic substances in the fermentation system is 0.5-2 g / L or higher than 2 g / L; the COD in the high-acid wastewater is 450-500 mg / L; the ammonia nitrogen concentration in the high-acid wastewater is 30-40 mg / L; the total nitrogen in the high-acid wastewater is 100-150 mg / L. Specifically, the phenolic content in the fermentation system may be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2 g / L, or a range consisting of any two of the above values, or higher than 2 g / L. Specifically, the acid content in the fermentation system may be 1%, 1.2%, 1.4%, 1.5%, 1.6%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 2.9%, 3%, or a range consisting of any two of the above values. Specifically, "acid content" refers to the content of the corresponding acid in the fermentation system, and may also refer to the acidity in the fermentation system, such as the acidity in the fermentation system being 1-3%. Specifically, the pH in the fermentation system may be 2, 2.2, 2.4, 2.5, 2.6, 2.8, 3.0, 3.2, 3.4, 3.5, 3.6, 3.8, 4.0, 4.2, 4.4, 4.5, 4.6, 4.8, 5, or a range consisting of any two of the above values.

[0033] In some embodiments of the present application, the inoculation amount of the composite bacterial agent relative to the fermentation system is 5% to 15%. Specifically, the inoculation amount of the composite bacterial agent can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or a range consisting of any two of the above values.

[0034] In some embodiments of the present application, the dissolved oxygen content in the fermentation system is 2 to 5 ppm. Specifically, the dissolved oxygen content in the fermentation system can be 2 ppm, 2.2 ppm, 2.4 ppm, 2.5 ppm, 2.6 ppm, 2.8 ppm, 2.9 ppm, 3.0 ppm, 3.2 ppm, 3.4 ppm, 3.5 ppm, 3.6 ppm, 3.8 ppm, 3.9 ppm, 4.0 ppm, 4.2 ppm, 4.4 ppm, 4.5 ppm, 4.6 ppm, 4.8 ppm, 4.9 ppm, 5 ppm, or a range consisting of any two of the above values.

[0035] In some embodiments of the present application, the temperature of the fermentation process is 25-35°C, and the fermentation time is 5-15 days. Specifically, the fermentation temperature can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or a range consisting of any two of the above values. Specifically, the fermentation time can be 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or a range consisting of any two of the above values.

[0036] In some embodiments of the present application, the phenolic substance includes at least one of gallic acid, caffeic acid, p-coumaric acid, protocatechuic acid and phenol.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present application provides a composite bacterial agent that can efficiently treat high-content phenolic substances in high-acid wastewater. The present application has found that by combining Jinshan Lactobacillus aceticus with Ralstonia pituitarius, Bacillus salinarum, and Bacillus oceanicus in silt, and utilizing the mutual synergistic effect between the microbial strains, the tolerance of the bacterial agent to acidic environments can be enhanced, and the treatment capacity of phenolic substances in high-acid wastewater can be significantly improved. The composite bacterial agent provided by the present application can play a synergistic role in the treatment effect of high-content phenolic substances in high-acid wastewater. The composite bacterial agent provided by the present application can efficiently treat high-content phenolic substances in high-acid wastewater, and also provides a new idea for wastewater treatment in the traditional fermentation industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0040] Figure 1The comparison results of the growth of the composite bacteria in Example 2 of this application under different acidic environments are shown below. Figure 1 -The left picture shows the pH of the culture medium with different lactic acid addition amounts. Figure 1 -The right figure shows the growth of composite bacteria in culture medium with different lactic acid addition amounts;

[0041] Figure 2 The results are a comparison of the degradation ability of the composite bacteria in Example 3 of the present application on different phenolic substances;

[0042] Figure 3 The results are the comparative results of the effects of different dissolved oxygen environments on the growth of composite bacteria and the degradation ability of phenolic substances in the comparative examples of this application; wherein, Figure 3 -The left picture shows the growth changes of composite bacteria under different dissolved oxygen conditions. Figure 3 -The right figure shows the degradation ability of composite bacteria on different phenolic substances under different dissolved oxygen conditions;

[0043] Figure 4 This is the comparative result of the degradation ability of the single bacterial agent on phenolic substances in Comparative Example 2 of this application; wherein, Figure 4 -The left figure shows the degradation of catechol by single bacterial agents of Lactobacillus aceticus Jinshan, Ralstonia pinelliae, Bacillus halogenides in sediments and Bacillus oceanica in silt. Figure 4 -The right figure shows the degradation of phenol by the corresponding single bacterial agents of Lactobacillus aceticus Jinshan, Ralstonia pinelliae, Bacillus halogenides in sediments, and Bacillus oceanica in silt;

[0044] Figure 5 The results are the comparison of the degradation ability of the composite bacterial agent obtained by any bacterial combination in Comparative Example 2 of this application on phenolic substances; wherein, Figure 5 -The left figure shows the degradation of catechol by composite bacterial agents corresponding to Ralstonia pitei + Bacillus halogenis in sediment + Bacillus sludge oceanicus, Lactobacillus aceticus from Jinshan + Bacillus halogenis in sediment + Bacillus sludge oceanicus, Lactobacillus aceticus from Jinshan + Ralstonia pitei + Bacillus sludge oceanicus, and Lactobacillus aceticus from Jinshan + Ralstonia pitei + Bacillus sludge oceanicus. Figure 5 -The right figure shows the degradation of phenol by composite bacterial agents corresponding to Ralstonia pitei + Bacillus halogenis in sediment + Bacillus oceanicus in silt, Lactobacillus aceticus from Jinshan + Bacillus halogenis in sediment + Bacillus oceanicus in silt, Lactobacillus aceticus from Jinshan + Ralstonia pitei + Bacillus oceanicus in silt, and Lactobacillus aceticus from Jinshan + Ralstonia pitei + Bacillus oceanicus in silt;

[0045] Figure 6 The results are the comparative results of the effects of the composite bacterial agents prepared by combining different proportions of bacterial liquids in Comparative Example 3 of this application on the degradation ability of phenolic substances; wherein, Figure 6 -The left figure shows the degradation of catechol by Lactobacillus jinshanensis mixed with Ralstonia pinelliae, Bacillus halogenis in sediments, and Bacillus oceanica in silts at weight ratios of 7:1:1:1, 1:1:1:1, 1:7:1:1, and 1:1:1:7, respectively. Figure 6 -The right figure shows the degradation of phenol by the bacterial agents obtained by mixing Lactobacillus jinshanensis with Ralstonia pinellia, Bacillus halogenis in sediments, and Bacillus oceanica in silts in weight ratios of 7:1:1:1, 1:1:1:1, 1:7:1:1, and 1:1:1:7, respectively. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field based on this application are within the scope of protection of this application.

[0047] 1. The Jinshan Lactobacillus acetate, Ralstonia pekinensis, Bacillus halogenis, and Bacillus oceanicus used in the examples of this application are all commercial strains and can be purchased directly from the CICC (China Industrial Microbiological Culture Collection Center) or the CGMCC (China General Microbiological Culture Collection Center); among them, the Jinshan Lactobacillus acetate strain has a collection number of CICC 6269, and its strain information on the CICC is: Lactobacillus sp. The strain deposit number of Ralstonia pickettii is CGMCC 1.10820, its scientific name is "Ralstonia pickettii", and its Chinese translation is "Ralstonia pickettii". The strain deposit number of Bacillus sedimentaris is CICC24779, and its species information in "CICC" is Aquibacillus sp. The deposit number of the strain of Oceanobacillus caeni is CICC 24563, and its strain information in "CICC" is: Oceanobacillus caeni

[0048] 2. The culture medium information used in the examples of this application is as follows:

[0049] The preparation formula of NB culture medium is as follows: beef extract powder, 3g; sodium chloride, 5g; peptone, 10g; sterile deionized water, 1000ml.

[0050] The preparation formula of MRS culture medium for wine mash extract is as follows: beef extract, 10g; peptone, 10g; yeast extract, 5g; glucose, 20g; diammonium hydrogen citrate, 2.5g; Tween 80, 0.5mL; acetic acid, 5ml; sodium acetate, 5g; dipotassium hydrogen phosphate, 2g; magnesium sulfate, 0.2g; manganese sulfate, 0.05g; wine mash extract, 200ml; sterile deionized water, 800ml.

[0051] 3. The method for detecting the content of phenolic substances (protocatechuic acid, gallic acid, caffeic acid, coumaric acid, catechol, phenol) in the examples of this application is as follows:

[0052] Accurately measure 2 ml of sample, add 40 mL (1:20) 80% ethanol, and stir magnetically at 75°C for 30 minutes. After extraction, filter the liquid with a Buchner funnel, then centrifuge at 1260×g for 10 minutes, remove the supernatant and save; repeat this step three times, place the supernatant in a rotary evaporator at 37°C; evaporate the organic reagent under vacuum to obtain a higher concentration of aqueous liquid; after rotary evaporation, add an appropriate amount of methanol to the extract, filter with a 0.22μm filter membrane, and inject 10μL of sample into a reverse phase C-18 column (ODS Chromatographic separation was performed on a high performance liquid chromatography system using a C-18 column; wherein the chromatographic separation conditions were as follows: gradient elution using two mobile phases, mobile phase A comprising a 0.1% formic acid solution, and mobile phase B comprising pure acetonitrile; the specific elution program was as follows: 95-85% A, 0-5 min; 85-50% A, 5-45 min; 50-30% A, 45-47 min; 30%-0% A, 47-48 min; 0% A, 48-55 min; 0-95% A, 55-56 min; 95 % A, 56-65 minutes; the flow rate was 1.0 mL / min, the column temperature was 25°C, and the detection wavelengths were 288 nm and 330 nm, respectively; standard solutions with concentrations of 0.01, 0.05, 0.1, 0.2, 0.4, and 0.8 mg / mL were prepared, respectively (the standard substances used included protocatechuic acid, gallic acid, caffeic acid, coumaric acid, catechol, and phenol), and a standard curve was drawn according to the concentration and the corresponding peak area, and the content of the corresponding phenolic substances in the sample was calculated based on the drawn standard curve.

[0053] Example 1

[0054] This embodiment, based on the invention content of this application, provides a composite bacterial agent for treating phenolic substances in high-acid wastewater and a preparation method thereof, comprising the following steps:

[0055] Step 1: Lactobacillus jinshanensis was inoculated into MRS medium of fermented grains extract, and static culture was carried out in an anaerobic incubator at 30°C; Ralstonia pekinensis, Bacillus sedimentaris and Bacillus silt were inoculated into NB medium respectively, and cultured at 30°C in a shaker at 150 rpm; the cell concentration in the culture medium was monitored until the cell concentration in each culture medium reached 10 9 cells / ml, namely, the Jinshan Lactobacillus aceticus solution, the Pittsburgh Ralstonia solution, the sedimentary Bacillus salt solution and the silt oceanic Bacillus solution were obtained respectively;

[0056] Step 2: Mix the four bacterial solutions in a ratio of 70% by weight of Lactobacillus jinshanensis, 10% by weight of Ralstonia pinelliae, 10% by weight of Bacillus halogenides in sediment, and 10% by weight of Bacillus oceanica sludge to obtain a composite bacterial agent.

[0057] Example 2: Growth ability test of composite bacteria in acidic environment

[0058] This example is based on the composite bacterial agent prepared in Example 1, and further tests the growth ability of the composite bacterial agent in an acidic environment, as follows:

[0059] The composite strain prepared in Example 1 was inoculated into the test culture medium at a rate of 10% and cultured at a temperature of 30°C for 10 days. After the culture was completed, the pH and OD values ​​of the culture medium were measured. 600 The initial pH and OD of the culture medium were tested. 600 The values ​​were detected and recorded; the detection culture medium used was as follows: lactic acid was added to the MRS culture medium of the fermented mash extract to adjust the pH of the culture medium, and the amount of lactic acid added was set to 0%, 1%, 2%, 3%, 4% and 5% respectively.

[0060] Test results such as Figure 1 As shown, according to Figure 1 The results shown in the figure show that the composite bacterial agent prepared by Example 1 of the present application can grow well in an environment with a lactic acid addition amount of 1-3% (pH of 3.4-4.0), further indicating that the composite bacterial agent provided in Example 1 of the present application can grow and metabolize in a wide spectrum of high-acid environments, meeting the treatment requirements of wastewater discharged by traditional fermentation industries.

[0061] Example 3 Determination of the ability of composite bacteria to degrade different phenolic substances

[0062] This embodiment is based on Example 1, and the composite bacterial agent prepared in Example 1 is inoculated into the detection culture medium at a 10% inoculation rate for culture. The culture temperature is 30°C, the culture time is 10 days, and the dissolved oxygen content of the culture medium is controlled to 4 ppm. After the culture, the residual phenolic substances in the culture medium are quantitatively detected, and the degradation rate of different phenolic substances is calculated; wherein, the detection culture medium used is as follows: 2% lactic acid is added to the MRS culture medium of the fermented mash extract, and phenolic substance mother liquors (phenolic substances include: phenol, gallic acid, caffeic acid, p-coumaric acid, and protocatechuic acid) with a corresponding phenolic substance content of 1 g / L are added in a ratio (1:9). The phenolic substance mother liquor is processed as follows: 1 g / L of phenolic substance is added to deionized water, ultrasonically vibrated for 30 minutes, and passed through a 0.22 μm PES hydrophilic membrane to remove undissolved phenolic substances and microorganisms to obtain mother liquors corresponding to the corresponding phenolic substances.

[0063] Test results such as Figure 2 As shown, according to Figure 2 The results shown in the figure show that the composite bacterial agent prepared by Example 1 of the present application has a strong degradation ability for phenolic substances having a catechol structure with two ortho-hydroxyl groups, such as protocatechuic acid and gallic acid having a trihydroxybenzoic acid structure, among which the degradation rate of protocatechuic acid is as high as about 90%, and the degradation rate of gallic acid is about 85%. However, the degradation ability of caffeic acid and p-coumaric acid with non-catechol structures is weaker, among which the degradation rate of caffeic acid is about 32%, the degradation rate of p-coumaric acid is 30%, and the degradation rate of phenol can reach about 82%.

[0064] Example 4

[0065] This embodiment, based on Example 1, provides a method for degrading high-acid wastewater using a composite bacterial strain, and the specific steps are as follows:

[0066] The composite bacterial agent prepared in Example 1 was inoculated into high-acid, high-phenol content wastewater at an inoculum rate of 10%, the dissolved oxygen content of the culture medium system was controlled at 4 ppm, the culture temperature was 30° C., and the culture time was 10 days. After the culture, the high-acid, high-phenol content wastewater was tested for water quality, and the residual phenolic substances were quantitatively detected, and the degradation rate of the phenolic substances was calculated. The high-acid, high-phenol content wastewater used in this example was taken from the distillation pot water in the production workshop of a certain sauce-flavor liquor company. The pH of the high-acid, high-phenol content wastewater was 4.8, and the phenolic substance content was 0.65 g / L.

[0067] The results of the degradation treatment of high-acid, high-phenol content wastewater using the above method are as follows: before adding the composite bacterial agent, the COD of the high-acid, high-phenol content wastewater was 470 mg / L, the ammonia nitrogen concentration was 35 mg / L, and the total nitrogen was 120 mg / L. After adding the composite bacterial agent for degradation treatment, the degradation rate of phenolic substances in the treated high-acid, high-phenol content wastewater was as high as 90%, the COD was stabilized at around 80 mg / L, the ammonia nitrogen effluent concentration was around 7 mg / L, and the total nitrogen effluent concentration was around 60 mg / L. The above degradation treatment results further demonstrate that the composite bacterial agent provided in this application can achieve efficient treatment of high-acid, high-phenol content wastewater, especially the degradation of phenolic substances in wastewater.

[0068] Comparative Example 1

[0069] Based on Example 1, this comparative example further explored the effect of dissolved oxygen on the ability of the composite bacterial agent to degrade phenolic substances, as follows:

[0070] The composite bacterial agent prepared in Example 1 was inoculated into the detection culture medium at an inoculum size of 10%, and the culture temperature was 30° C. The culture time was 10 days. After the culture, the residual phenolic substances in the culture medium were quantitatively detected, and the degradation rate of the phenolic substances was calculated; wherein, the detection culture medium used in this comparative example is as follows: sterile air was introduced into the MRS culture medium of the fermented grains extract, the air flux was controlled, and the dissolved oxygen content of the culture medium was maintained at 0 ppm (anaerobic), 2 ppm, 4 ppm, 7 ppm, and 10 ppm, respectively. At the same time, based on the same method as in Example 3, a phenolic substance mother liquor with a phenolic substance content of 1 g / L was added to the culture medium (phenolic substances include: phenol, gallic acid, caffeic acid, p-coumaric acid, and protocatechuic acid).

[0071] The test results are as follows Figure 3 As shown, according to Figure 3The results show that the oxygen demand in the system when the composite bacterial agent degrades phenolic substances will have a great influence on the degradation effect of phenolic substances: for example, when the dissolved oxygen content is 0 ppm, the composite bacterial agent has almost no degradation ability on phenolic substances; when the dissolved oxygen content is 2 ppm, the composite bacterial agent's degradation ability on phenolic substances is significantly improved, for example, the composite bacterial agent has a degradation rate of 60% for protocatechuic acid, a degradation rate of about 45% for gallic acid, a degradation rate of about 32% for caffeic acid, and a degradation rate of 20% for coumaric acid. When the dissolved oxygen content is 4 ppm, the degradation rate of the composite bacterial agent on protocatechuic acid is increased to about 90%, the degradation rate on gallic acid is increased to about 85%, and the degradation rate on phenol is increased to about 82%; when the dissolved oxygen content is 7 ppm, the degradation rate of the composite bacterial agent on phenolic substances is significantly reduced; when the dissolved oxygen content increases to 10 ppm, the degradation ability of the composite bacterial agent on phenolic substances is significantly reduced, for example, the degradation rate of phenol is reduced to about 20%.

[0072] Comparative Example 2

[0073] Based on Example 1, this comparative example further explored the effects of bacterial agents prepared by different bacterial strain combinations on the degradation ability of phenolic substances, as follows:

[0074] 1. Prepare microbial agents and set up the following groups:

[0075] Group 1 - Jinshan Lactobacillus acetate inoculant: Jinshan Lactobacillus acetate was inoculated into MRS medium of fermented mash extract and cultured in an anaerobic incubator at 30°C. The cell concentration in the culture medium was monitored until the cell concentration in the culture medium reached 10 9 cells / ml, that is, Jinshan Lactobacillus acetate agent is obtained;

[0076] Group 2 - Ralstonia pekinensis inoculum: Ralstonia pekinensis was inoculated into NB medium and cultured at 30°C with shaking at 150 rpm. The cell concentration in the medium was monitored until the cell concentration in the medium reached 10 9 cells / ml, that is, the Ralstonia pekinensis agent was obtained;

[0077] Group 3 - Sedimentary Bacillus salt bacteria: Sedimentary Bacillus salt bacteria and silt oceanic Bacillus were inoculated into NB medium, cultured at 30℃, shaken at 150rpm, and the cell concentration in the culture medium was monitored until the cell concentration in the culture medium reached 10 9 cells / ml, that is, the sedimentary medium salt bacillus agent is obtained;

[0078] Group 4 - Bacillus sludge Oceanus agent: Inoculate Bacillus sludge Oceanus into NB medium and culture at 30℃ with shaking at 150rpm. Monitor the cell concentration in the medium until the cell concentration reaches 10 9 cells / ml, that is, the sludge oceanic bacillus agent is obtained;

[0079] Group 5 - Composite bacterial agent (Ralstonia pekinensis + Bacillus salinarum + Bacillus sludge oceanicus): Ralstonia pekinensis, Bacillus salinarum, and Bacillus sludge oceanicus were inoculated into NB medium respectively, cultured at 30°C and shaken at 150 rpm; the cell concentration in the culture medium was monitored until the cell concentration in each culture medium reached 10 9 cells / ml, namely, obtaining Ralstonia pinnatifida solution, Bacillus halogenis in sediment solution, and Bacillus oceanicus in silt solution respectively; mixing the three bacterial solutions in a weight ratio of 1:1:1 between Ralstonia pinnatifida, Bacillus halogenis in sediment, and Bacillus oceanicus in silt to obtain a composite bacterial agent;

[0080] Group 6 - Composite bacterial agent (Lactobacillus aceticus from Jinshan + Bacillus halogenus from sediment + Bacillus oceanicus from silt): Lactobacillus aceticus from Jinshan was inoculated into MRS medium of fermented mash extract and cultured in an anaerobic incubator at 30°C; Bacillus halogenus from sediment and Bacillus oceanicus from silt were inoculated into NB medium respectively and cultured at 30°C in a shaker at 150 rpm; the cell concentration in the culture medium was monitored until the cell concentration in each culture medium reached 10 9 cells / ml, namely, obtaining Jinshan Lactobacillus acetate liquid, sedimentary halobacillus liquid, and silt oceanic Bacillus liquid respectively; mixing the three bacterial liquids in a weight ratio of 7:1:1 between Jinshan Lactobacillus acetate liquid, sedimentary halobacillus liquid, and silt oceanic Bacillus liquid to obtain a composite bacterial agent;

[0081] Group 7 - Composite bacterial agent (Lactobacillus aceticus Jinshan + Ralstonia pitei + Bacillus sludge oceanicus): Lactobacillus aceticus Jinshan was inoculated into MRS medium of fermented mash extract, and cultured in an anaerobic incubator at 30°C; Ralstonia pitei and Bacillus sludge oceanicus were inoculated into NB medium respectively, and cultured in a shaker at 150 rpm at 30°C; the cell concentration in the culture medium was monitored until the cell concentration in each culture medium reached 10 9cells / ml, namely, obtaining Jinshan Lactobacillus acetate liquid, Ralstonia pekinensis and Oceania sludge Bacillus liquid respectively; mixing the three bacterial liquids in a weight ratio of 7:1:1 between Jinshan Lactobacillus acetate liquid, Ralstonia pekinensis and Oceania sludge Bacillus liquid to obtain a composite bacterial agent;

[0082] Group 8 - Composite bacterial agent (Lactobacillus aceticus from Jinshan + Ralstonia pinnatifida + Bacillus salinarum): Lactobacillus aceticus from Jinshan was inoculated into MRS medium of fermented mash extract and cultured in an anaerobic incubator at 30°C; Ralstonia pinnatifida and Bacillus salinarum were inoculated into NB medium respectively and cultured at 30°C in a shaker at 150 rpm; the cell concentration in the culture medium was monitored until the cell concentration in each culture medium reached 10 9 cells / ml, that is, Jinshan Lactobacillus acetate liquid, Ralstonia pinnatifida and sedimentary salt Bacillus were obtained respectively; the three bacterial liquids were mixed according to the weight ratio of Jinshan Lactobacillus acetate liquid, Ralstonia pinnatifida and sedimentary salt Bacillus of 7:1:1 to obtain a composite bacterial agent.

[0083] 2. Detection of the degradation ability of phenolic substances: The composite bacterial agent prepared above was inoculated into the detection culture medium at a 10% inoculation rate and cultured at a temperature of 30°C for 10 days. The dissolved oxygen content of the culture medium was controlled to 4 ppm. After the culture, the residual phenolic substances in each culture medium were quantitatively detected, and the degradation rates of different phenolic substances were calculated. The detection culture medium used was as follows: 2% lactic acid was added to the MRS culture medium of the mash extract, and catechol and phenol mother liquor were added in a ratio of (1:9).

[0084] Test results such as Figure 4 、 Figure 5 As shown, according to Figure 4 、 Figure 5The results shown in the figure show that the degradation rate of catechol by using group 1, Lactobacillus aceticus from Jinshan, is about 75%. The degradation rate of catechol by using group 2, Ralstonia pituitarius, is close to 60%. The degradation rate of catechol by using group 3, Bacillus salti from sediment, is close to 50%. The degradation rate of catechol by using group 4, Bacillus oceanica from silt, is close to 55%. Group 5-composite bacterial agent (Ralstonia pituitii + sedimentary halobacillus + silt oceanic bacillus) is used to degrade catechol, and its degradation rate is about 70%. Group 6-composite bacterial agent (Lactobacillus acetic acid from Jinshan + sedimentary halobacillus + silt oceanic bacillus) is used to degrade catechol, and its degradation rate is about 85%. Group 7-composite bacterial agent (Lactobacillus acetic acid from Jinshan + Ralstonia pituitii + silt oceanic bacillus) is used to degrade catechol, and its degradation rate is about 75%. Group 8-composite bacterial agent (Lactobacillus acetic acid from Jinshan + Ralstonia pituitii + sedimentary halobacillus) is used to degrade catechol, and its degradation rate is about 72%.

[0085] As can be seen above, when group 1-Lactobacillus aceticus from Jinshan is used to degrade catechol, its degradation rate can reach about 75%. However, when it is combined with any two of Bacillus salt in sediment, Bacillus oceanicus in sludge, and Ralstonia pinelliae as a composite bacterial agent to degrade catechol, the corresponding degradation rate is not significantly improved. When it is combined with Ralstonia pinelliae, Bacillus oceanicus in sludge, or Ralstonia pinelliae and Bacillus salt in sediment, the corresponding degradation rate is not significantly improved. The rate does not increase but decreases or remains unchanged. As in Example 3 of the present application, when Lactobacillus jinshanensis is specifically combined with Bacillus halogenids in sediment, Bacillus silt oceanicus and Ralstonia pinellia, the degradation rate of polyphenols with a catechol structure can be as high as about 90%. It can be seen that the specific combination of Lactobacillus jinshanensis with Bacillus halogenids in sediment, Bacillus silt oceanicus and Ralstonia pinellia in the present application has achieved a synergistic effect in degrading polyphenols.

[0086] At the same time, according to Figure 4 、 Figure 5The results shown in the figure show that the degradation rate of phenol by using group 1 - Lactobacillus aceticus from Jinshan was about 2.5%, the degradation rate of phenol by using group 2 - Ralstonia pekinensis was about 12%, the degradation rate of phenol by using group 3 - Bacillus halogenids from sediment was about 7.5%, the degradation rate of phenol by using group 4 - Bacillus sulphurid was about 13%; the degradation rate of phenol by using group 5 - composite bacterial agent (Ralstonia pekinensis + Bacillus halogenids from sediment + Bacillus sulphurid) was about 13%. ) was used to degrade phenol, with a degradation rate of about 45%. Group 6-composite bacterial agent (Lactobacillus aceticus from Jinshan + Bacillus halogenids from sediment + Bacillus oceanicus from silt) was used to degrade phenol, with a degradation rate of about 63%. Group 7-composite bacterial agent (Lactobacillus aceticus from Jinshan + Ralstonia pitei + Bacillus oceanicus from silt) was used to degrade phenol, with a degradation rate of about 72%. Group 8-composite bacterial agent (Lactobacillus aceticus from Jinshan + Ralstonia pitei + Bacillus oceanicus from sediment) was used to degrade phenol, with a degradation rate of about 70%.

[0087] As can be seen from the above analysis, when a single bacterial agent (such as Group 1, Group 2, Group 3, and Group 4) is used to degrade phenol, the corresponding degradation rate is relatively low (about 2.5% to 13%). For example, when Group 1-Jinshan Lactobacillus acetate single bacterial agent is used to degrade phenol, its degradation rate is only 2.5%. It can be seen that Jinshan Lactobacillus acetate single bacterial agent has almost no degradation ability for phenol. However, the present application unexpectedly discovered that when Jinshan Lactobacillus acetate is used in combination with different bacteria, its effect on phenol degradation ability is different. For example, when Jinshan Lactobacillus acetate is used in combination with Ralstonia pituitarius, Bacillus siltii, or with Ralstonia pituitarius and Bacillus salinarum, the degradation rate of phenol can reach about 70%. When Jinshan Lactobacillus acetate is used in combination with Bacillus salinarum and Bacillus siltii, the degradation rate of phenol is only about 63%. Further referring to the results shown in Example 3 of the present application, when Lactobacillus jinshanensis was used in a specific combination with Bacillus halogenus in sediment, Bacillus oceanicus in silt, and Ralstonia pekinensis, the degradation rate of phenol was as high as 82%. It can be seen that the combination of Lactobacillus jinshanensis with Bacillus halogenus in sediment, Bacillus oceanicus in silt, and Ralstonia pekinensis achieved a synergistic effect in degrading phenol substances. At the same time, the above data further demonstrate that the combination of Lactobacillus jinshanensis with different bacteria has a significantly different effect on the degradation ability of phenol.

[0088] Comparative Example 3

[0089] Based on Example 1, this comparative example further explored the effects of bacterial agents prepared by combining bacterial solutions in different proportions on the degradation ability of phenolic substances, as follows:

[0090] 1. Preparation of composite bacterial agents with different proportions:

[0091] Lactobacillus jinshanensis was inoculated into MRS medium of fermented mash extract and cultured in an anaerobic incubator at 30°C. Ralstonia pinnatifida, Bacillus salinarum and Bacillus sludge were inoculated into NB medium respectively and cultured at 30°C in a shaker at 150 rpm. The cell concentration in the culture medium was monitored until the cell concentration in each culture medium reached 10 9 cells / ml, namely, Jinshan Lactobacillus acetate liquid, Pieri Ralstonia liquid, sedimentary halobacillus liquid and silt oceanic bacillus liquid were obtained respectively; the prepared Jinshan Lactobacillus acetate liquid, Pieri Ralstonia liquid, sedimentary halobacillus liquid and silt oceanic bacillus liquid were mixed in different ratios of 7:1:1:1, 1:1:1:1, 1:7:1:1, 1:1:7:1 and 1:1:1:7 to obtain a composite bacterial agent;

[0092] 2. Detection of the degradation ability of phenolic substances: The composite bacterial agent prepared above was inoculated into the detection culture medium at a 10% inoculation rate and cultured at a temperature of 30°C for 10 days. The dissolved oxygen content of the culture medium was controlled to 4 ppm. After the culture, the residual phenolic substances in each culture medium were quantitatively detected, and the degradation rates of different phenolic substances were calculated. The detection culture medium used was as follows: 2% lactic acid was added to the MRS culture medium of the mash extract, and catechol and phenol mother liquor were added in a ratio of (1:9).

[0093] Test results such as Figure 6 As shown, according to Figure 6The results shown in the figure show that in the preparation of the composite bacterial agent, the mixing ratio between Jinshan Lactobacillus acetate, Pierrotei Ralstonia, Sedimentary Halobacillus, and Sludge Oceanic Bacillus also has a significant effect on the degradation ability of phenolic substances. For example, when Jinshan Lactobacillus acetate liquid is mixed with Pierrotei Ralstonia, Sedimentary Halobacillus, and Sludge Oceanic Bacillus in a ratio of 7:1:1:1, the degradation rate of the prepared composite bacterial agent for catechol is as high as about 90%. When Jinshan Lactobacillus acetate liquid is mixed with Pierrotei Ralstonia, Sedimentary Halobacillus, and Sludge Oceanic Bacillus in a ratio of 1:1:1:1, 1:7:1:1, 1:1:7:1, and 1:1:1:7, the degradation rate of the prepared composite bacterial agent for catechol is significantly reduced. The degradation rate of catechol is low, for example, when the ratio of 1:1:7:1 is mixed, the degradation rate of catechol is reduced to about 60%; at the same time, when the Jinshan Lactobacillus acetate liquid is mixed with the Pierrotoni Ralstonia liquid, the sedimentary salt Bacillus liquid, and the silt oceanic Bacillus liquid in a ratio of 7:1:1:1, the degradation rate of phenol by the prepared composite bacterial agent is about 82%, and when the Jinshan Lactobacillus acetate liquid is mixed with the Pierrotoni Ralstonia liquid, the sedimentary salt Bacillus liquid, and the silt oceanic Bacillus liquid in ratios such as 1:1:1:1, 1:7:1:1, 1:1:7:1, and 1:1:1:7, the degradation rate of phenol by the prepared composite bacterial agent is significantly reduced, for example, when the mixture is mixed in a ratio of 1:7:1:1, the degradation rate of phenol is reduced to below 50%.

[0094] It is understood that the present application is described by way of some embodiments, and it is understood by those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present application. In addition, under the guidance of the present application, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the scope of the present application. Therefore, the present application is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of the present application are within the scope of protection of the present application.

Claims

1. A composite bacterial agent for treating phenolic substances in high-acid wastewater and its application, characterized in that: The composite bacterial agent comprises: Lactobacillus jinshanaceticus, Ralstonia pinnatifida, Bacillus halogenids in sediment and Bacillus oceanicus in silt; wherein, in the composite bacterial agent, the weight ratio of Lactobacillus jinshanaceticus to Ralstonia pinnatifida, Bacillus halogenids in sediment and Bacillus oceanicus in silt is 1-20:1-10:1-10:1-10:1-10:1-10.

2. The composite bacterial agent according to claim 1, characterized in that The weight ratio of the Jinshan Lactobacillus acetate to Ralstonia pinnatifida, Bacillus salt in sediment and Bacillus oceanicus in silt is 1-20:1-6:1-6:1-6:1-6; preferably, the weight ratio of the Jinshan Lactobacillus acetate to Ralstonia pinnatifida, Bacillus salt in sediment and Bacillus oceanicus in silt is 1-15:1-5:1-5:1-5:1-5:1-5; preferably, the weight ratio of the Jinshan Lactobacillus acetate to Ralstonia pinnatifida, Bacillus salt in sediment and Bacillus oceanicus in silt is 1-15:1-2:1-2:1-2:1-2.

3. The composite bacterial agent according to claim 1, characterized in that In the composite bacterial agent, the cell concentration of Lactobacillus jinshanensis, Ralstonia pekinensis, Bacillus salinarum and Bacillus sludge oceanicus is (0.5-10)×10 9 cells / ml.

4. The composite bacterial agent according to claim 1, characterized in that The deposit number of the Lactobacillus jinshanensis is CICC 6269; the deposit number of the Ralstonia pekinensis is CGMCC 1.10820; the deposit number of the sedimentary halobacillus is CICC 24779; and the deposit number of the silt oceanic bacillus is CICC 24563.

5. A method for preparing the composite bacterial agent according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: (1) Lactobacillus jinshanensis, Ralstonia pekinensis, Bacillus halogenides of sediments, and Bacillus oceanica of silt were respectively prepared into corresponding bacterial solutions; (2) mixing the prepared corresponding bacterial solutions according to a certain proportion to obtain the composite bacterial agent; Preferably, the cell concentration of the bacterial solution is (0.5-10)×10 9 cells / ml; Preferably, the preparation method of the Jinshan Lactobacillus acetate liquid comprises: inoculating Jinshan Lactobacillus acetate into an MRS culture medium of a fermented mash extract, and performing static culture in an anaerobic incubator under culture conditions of 28 to 35° C.; Preferably, the preparation method of the Ralstonia pinnatifida liquid, the sedimentary salt Bacillus liquid and the silt oceanic Bacillus liquid comprises: inoculating Ralstonia pinnatifida, the sedimentary salt Bacillus liquid and the silt oceanic Bacillus liquid respectively into NB culture medium, and performing shaking culture at 28 to 35° C. on a shaker at 100 to 200 rpm.

6. An application of the composite bacterial agent according to any one of claims 1 to 4 in the field of wastewater treatment; preferably, the wastewater comprises highly acidic wastewater containing phenolic substances or highly acidic wastewater containing a high content of phenolic substances; the application comprises treating phenolic substances in highly acidic wastewater.

7. A method for degrading phenolic substances, characterized in that: The method comprises: Adding the composite bacterial agent according to any one of claims 1 to 4 to a fermentation system containing phenolic substances, performing fermentation culture, and degrading the phenolic substances in the fermentation system during the fermentation process; Preferably, the inoculation amount of the composite bacterial agent relative to the fermentation system is 5% to 15%; Preferably, the dissolved oxygen content in the fermentation system is 2 to 5 ppm; Preferably, the acid content in the fermentation system is 1-3% or the pH is 2-5; Preferably, the fermentation temperature is 25-35°C and the fermentation time is 5-15 days; Preferably, the fermentation system containing phenolic substances comprises a culture medium containing phenolic substances; preferably, the culture medium is a fermented grains extract MRS culture medium; Preferably, the phenolic substance includes at least one of gallic acid, caffeic acid, p-coumaric acid, protocatechuic acid and phenol.

8. A method for treating phenolic substances in high-acid wastewater, characterized in that: The method comprises: The composite bacterial agent according to any one of claims 1 to 4 is added to the high-acid wastewater for fermentation, and phenolic substances in the high-acid wastewater are degraded during the fermentation process.

9. The method according to claim 8, characterized in that The acidity of the high-acid wastewater is 1-3% or the pH is 2.0-5.0; the content of phenolic substances in the high-acid wastewater is 0.5-2.0 g / L or higher than 2.0 g / L; the COD of the high-acid wastewater is 450-500 mg / L; the ammonia nitrogen concentration of the high-acid wastewater is 30-40 mg / L; and the total nitrogen of the high-acid wastewater is 100-150 mg / L.

10. The method according to claim 8, characterized in that The inoculation amount of the composite bacterial agent relative to the fermentation system is 5% to 15%; Preferably, the dissolved oxygen content in the fermentation system is 2 to 5 ppm; Preferably, the temperature of the fermentation process is 25-35°C and the fermentation time is 5-15 days; Preferably, the phenolic substance includes at least one of gallic acid, caffeic acid, p-coumaric acid, protocatechuic acid and phenol.

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