An anaerobic treatment method for heparin sodium wastewater

Abstract

The application discloses an anaerobic treatment method of heparin sodium wastewater, and belongs to the field of water pollution prevention and control. The application provides a strain of Arthrobacter protophormiae A1, which is preserved in the China Center for Type Culture Collection on May 23, 2024, and the preservation number is CCTCC NO: M20241041. The Arthrobacter protophormiae provided by the application can tolerate 8.0% salinity, and can still effectively secrete protease under high-salt stress. The bacteria can provide effective strain resources for the anaerobic biological treatment of high-salt and high-protein wastewater such as heparin sodium wastewater, so that the treatment cost is significantly reduced, and the bacteria have important application value.

Description

Technical Field

[0001] The invention belongs to the field of water pollution prevention and control, and particularly relates to an anaerobic treatment method for sodium heparin wastewater. Background Art

[0002] High-salt organic wastewater mainly comes from chemical, pharmaceutical, food and pesticide related industries. In addition to organic pollutants, this type of wastewater also contains high concentrations of soluble inorganic ions (Cl - , SO4 2- , Na + , Ca 2+ Currently, the main treatment methods for high-salinity wastewater include biological methods, membrane separation methods, and high-efficiency evaporation methods. Biological methods rely on microorganisms to remove pollutants and are mainly divided into anaerobic biological treatment and aerobic biological treatment.

[0003] High-salt, high-protein wastewater primarily originates from the food industry, such as meat curing and sausage casing production, as well as pharmaceutical industries like sodium heparin production. For example, sodium heparin production wastewater is a typical example of high-salt, high-protein wastewater, with salinity typically exceeding 3.6% and a high COD content (primarily protein-derived organic matter). Anaerobic biological treatment is relatively suitable for treating high-concentration organic wastewater, but improving treatment capacity in high-salt environments is crucial.

[0004] To improve the anaerobic treatment efficiency of high-salt, high-protein wastewater, such as sodium heparin wastewater, one feasible approach, based on the concept of bioaugmentation, is to construct a highly salt-tolerant, protein-degrading bacterial community. Bioaugmentation technology, which requires no changes to the treatment process and offers low cost, demonstrates strong potential for wastewater treatment and pollutant biodegradation. Summary of the Invention

[0005] Purpose of the Invention: This patent provides a method for the anaerobic treatment of sodium heparin wastewater. Specifically, it utilizes a highly salt-tolerant, protease-producing strain of Arthrobacter protothecoides, which can tolerate salinity levels of 8.0% and effectively secrete protease even under high-salinity stress. This strain can effectively enhance the anaerobic biological treatment of high-salt, high-protein wastewater, such as sodium heparin wastewater, thereby significantly reducing treatment costs and possessing significant application value.

[0006] To achieve the above object, the technical solution of the present invention is:

[0007] The first object of the present invention is to provide an anaerobic treatment method for sodium heparin wastewater, the method comprising: using highly salt-tolerant and protease-producing Arthrobacter protophormiae A1 to strengthen the anaerobic treatment of sodium heparin wastewater, wherein the highly salt-tolerant and protease-producing Arthrobacter protophormiae A1 was deposited in the China Center for Type Culture Collection on May 23, 2024, in Wuhan, China, with a deposit number of CCTCC NO: M 20241041.

[0008] Optionally, in some embodiments of the present invention, the 16SrDNA sequence of the original Arthrobacterium vitae A1 is shown as SEQ ID NO.1.

[0009] Optionally, in some embodiments of the present invention, the method specifically includes constructing a heparin sodium wastewater-degrading bacterial community for anaerobic treatment of heparin sodium wastewater, and fermenting for 10-15 days at an inoculation amount of 5%-15%, a pH of 6.5-7.5, and 30-38°C, wherein the heparin sodium wastewater anaerobic degrading bacterial community includes salt-tolerant methanogenic sludge and Protothecobacterium glabratum A1, and the salt-tolerant methanogenic sludge is methanogenic sludge with a salinity tolerance of 3.0%-3.6%.

[0010] Optionally, in some embodiments of the present invention, in the sodium heparin wastewater anaerobic degradation bacterial community, the volume ratio of the salt-tolerant methanogenic activated sludge to the proto-glassae bacillus A1 activation solution is 5:1-15:1, preferably 10:1.

[0011] Optionally, in some embodiments of the present invention, the salt-tolerant methanogenic sludge is methanogenic sludge having a salinity tolerance of 3.0%-3.2%.

[0012] Optionally, in some embodiments of the present invention, the method for cultivating salt-tolerant methanogenic sludge includes using anaerobic methanogenic sludge as the initial inoculum and peptone as the sole carbon source and nitrogen source, and acclimating salt-tolerant methanogenic sludge capable of tolerating a salinity of more than 3.0% by gradiently increasing salt stress pressure.

[0013] Optionally, in some embodiments of the present invention, the Arthrobacterium vitae produces protease under anaerobic conditions and has a salinity tolerance of 4.0% or more.

[0014] Further optionally, in some embodiments of the present invention, the salinity tolerance of the original Arthrobacterium vitae A1 is up to 8.0%.

[0015] Further optionally, in some embodiments of the present invention, the salinity tolerance of the original Arthrobacterium vitae A1 is 4.0%-8.0%.

[0016] Optionally, in some embodiments of the present invention, the enzymatic activity of the protease reaches above 25 U / mL.

[0017] Further optionally, in some embodiments of the present invention, when the salt tolerance of the Arthrobacterium vitae A1 is 4.0%-6.0%, the enzymatic activity of the protease reaches above 35 U / mL.

[0018] Further optionally, in some embodiments of the present invention, when the salt tolerance of the Arthrobacterium vitae A1 is 6.0%-8.0%, the enzymatic activity of the protease reaches above 25 U / mL.

[0019] Optionally, in some embodiments of the present invention, the salinity of the sodium heparin wastewater reaches above 4.0%.

[0020] Optionally, in some embodiments of the present invention, the protein degradation rate of the heparin sodium wastewater is above 70%, and the COD removal rate is above 50%.

[0021] Further optionally, in some embodiments of the present invention, the protein degradation rate of the heparin sodium wastewater is above 80%, and the COD removal rate is above 60%.

[0022] Further optionally, in some embodiments of the present invention, the protein degradation rate of the heparin sodium wastewater is above 90%, and the COD removal rate is above 70%.

[0023] Optionally, in some embodiments of the present invention, the methane production reaches 150 mL / g-COD or more.

[0024] The second object of the present invention is to provide an anaerobic bacterial community for degrading sodium heparin wastewater, comprising salt-tolerant methane-producing sludge and any of the above-mentioned highly salt-tolerant protease-producing Arthrobacterium vitae.

[0025] Optionally, in some embodiments of the present invention, the volume ratio of the salt-tolerant methanogenic sludge bacteria liquid to the activated Arthrobacter glabratus liquid is 5:1-15:1, preferably 10:1.

[0026] Beneficial Effects: Anaerobic biological treatment of high-salt organic wastewater, such as sodium heparin, has always been a difficult problem in the field of environmental protection. Compared with existing technologies, the method disclosed in this invention can construct a protein-degrading bacterial community that can tolerate salinity levels above 4.5%. When treating actual sodium heparin wastewater with a salinity of 4.3%, the COD removal rate and protein degradation rate reached 76.3% and 92.5%, respectively. Therefore, the technical method described in this patent can provide an effective bacterial strain resource for strengthening the anaerobic treatment of high-salt, high-protein wastewater such as sodium heparin wastewater, thereby significantly reducing treatment costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : The process of strain screening and salt-tolerant degradation bacterial community construction of the present invention.

[0028] Figure 2 : Changes in COD removal efficiency during adaptive evolution.

[0029] Figure 3 : Changes in protein degradation rates during adaptive evolution.

[0030] Figure 4 : Colony morphology of strain A1.

[0031] Figure 5 : Salt tolerance of the strain.

[0032] Figure 6 : Effect of salinity on the growth of Arthrobacterium truncatum A1.

[0033] Figure 7 : Effect of salinity on protease secretion from Arthrobacterium truncatum A1.

[0034] Figure 8 : Effect of salinity on protein degradation by Arthrobacterium protothecoides A1.

[0035] Figure 9 : Construction of anaerobic degradation bacterial community for heparin sodium wastewater.

[0036] Figure 10 : Application effect of anaerobic degradation bacteria in heparin sodium wastewater. DETAILED DESCRIPTION

[0037] On the one hand, the present invention discloses an anaerobic treatment method for heparin sodium wastewater, which belongs to the field of water pollution prevention and control. Figure 1 The following is a process flow for strain screening and salt-tolerant degradation bacterial community construction. This method utilizes a top-down adaptive evolution strategy for anaerobic bacterial communities combined with targeted screening for highly salt-tolerant protease-producing bacteria to construct an anaerobic heparin sodium wastewater degradation bacterial community. The main steps include:

[0038] (1) First, a "top-down" adaptive evolution strategy was adopted to domesticate salt-tolerant methanogenic sludge that could tolerate salinity exceeding 3.0% by gradiently increasing salt stress;

[0039] (2) To address the problem of protein hydrolysis inhibition under high salt stress, the above-mentioned salt-tolerant methane-producing sludge was used as the bacterial strain source to screen out highly salt-tolerant protease-producing strains;

[0040] (3) The selected highly salt-tolerant protease-producing strains were compounded with salt-tolerant methane-producing sludge to further construct a highly salt-tolerant protein-degrading bacterial community (tolerant to salinity above 4.5%), thereby effectively improving the anaerobic treatment effect of sodium heparin wastewater.

[0041] As a preferred improvement of the present invention, the salt-tolerance adaptive evolution of the anaerobic bacterial community is carried out in a continuous-flow fully mixed anaerobic reactor, with the initial inoculum being anaerobic methanogenic sludge at an inoculum size of 25-35 g TS / L. Peptone is used as the sole carbon and nitrogen source, and the influent concentration is 6.0-10.0 g / L.

[0042] As a preferred improvement of the present invention, the salt tolerance adaptive evolution conditions are: temperature 30-38°C, pH 6.5-7.5, and hydraulic retention time 5-10 days. Based on a steady-state COD removal rate of >70%, salt stress is gradually increased to obtain salt-tolerant methanogenic sludge that can tolerate salinity exceeding 3.0%.

[0043] As a preferred improvement of the present invention, highly salt-tolerant protease-producing bacteria are isolated and screened from salt-tolerant methane-producing sludge. The isolation and screening process includes: separation and purification using 4.0% salinity peptone solid culture medium, primary screening for protease production ability at 4.0% salinity, and secondary screening for salt tolerance at 8.0% salinity.

[0044] On the other hand, according to the above screening method, the present invention provides a highly salt-tolerant protease-producing Protoglassae A1, whose preservation number is CCTCC NO: M 20241041, the preservation time is May 23, 2024, and the preservation location is China Center for Type Culture Collection.

[0045] As a preferred improvement of the present invention, the 16S rDNA of the proto-Glasnobacterium has a nucleotide sequence as shown in SEQ ID NO.1.

[0046] As a preferred improvement of the present invention, the salt tolerance of the original Arthrobacter glabraensis under anaerobic conditions can reach above 4.0%, even above 6.0%, even up to 8.0%, and it secretes protease.

[0047] As a preferred improvement of the present invention, the original Arthrobacter glabratus is isolated and screened from anaerobic methanogenic sludge that has undergone salt tolerance adaptive evolution. The adaptive evolution is carried out in a continuous flow fermentation mode with peptone as the sole carbon and nitrogen source, and the salt stress pressure gradient is increased to above 3.0%.

[0048] As a preferred improvement of the present invention, the isolation and screening process of the original glass fly Arthrobacter includes: dilution and separation culture in 4.0% salinity peptone solid culture medium, primary screening of protease production ability at 4.0% salinity, and secondary screening of salt tolerance at 8.0% salinity.

[0049] The present invention also provides an application of the Arthrobacter protovitamin Bacillus in anaerobic treatment of sodium heparin wastewater.

[0050] The application of the present invention in the anaerobic treatment of sodium heparin wastewater includes: after the activation and cultivation of the original glass fly Arthrobacter A1, it is compounded with salt-tolerant adaptively evolved methanogenic sludge (also referred to as salt-tolerant methanogenic sludge in the following examples) as an inoculum (volume ratio of 1:10); under the conditions of an inoculum amount of 10%, a pH of 7.0, and a temperature of 36.0°C, actual sodium heparin wastewater with a salinity of 4.3% is treated, and the COD removal rate and protein degradation rate reach 76.3% and 92.5%, respectively.

[0051] The present invention will be further explained below with reference to specific examples. However, it will be readily understood by those skilled in the art that the specific process conditions and results described in the examples are merely illustrative of the present invention and should not and will not limit the present invention described in detail in the claims.

[0052] Example:

[0053] The high salt-tolerant protease producing bacteria separation and fermentation medium involved in the following examples are as follows:

[0054] ①4.0% salinity peptone solid medium: 10.0 g / L tryptone, 20.0 g / L agar, 40 g / L sodium chloride, 0.2 g / L magnesium sulfate heptahydrate, 1.0 g / L potassium dihydrogen phosphate, 1.5 g / L dipotassium hydrogen phosphate, 0.25 g / L L-cysteine ​​hydrochloride monohydrate. In addition, add 1.0 mL of vitamin solution and 1.0 mL of trace element solution per unit volume (L) to adjust the pH to 7.0.

[0055] ②4.0% salinity clear zone medium: 16.0 g / L casein, 30.0 g / L sucrose, 20.0 g / L agar, 40 g / L sodium chloride, 1.0 g / L disodium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 2.0 g / L sodium nitrate, 0.01 g / L ferrous sulfate. Adjust pH to 7.0.

[0056] ③4.0% salinity gelatin liquefaction medium: beef extract 3.0 g / L, gelatin 200.0 g / L, trypsin 5.0 g / L, sodium chloride 40 g / L, adjust the pH to 7.0.

[0057] ④LB medium: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L sodium chloride, adjust the pH to 7.0.

[0058] ⑤ Salt-tolerance rescreening medium: Tryptone 10 g / L, sodium chloride 80 g / L, magnesium sulfate heptahydrate 0.2 g / L, potassium dihydrogen phosphate 1.0 g / L, dipotassium hydrogen phosphate 1.5 g / L, L-cysteine ​​hydrochloride monohydrate 0.25 g / L. In addition, add 1.0 mL of vitamin solution and 1.0 mL of trace element solution per unit volume (L) to adjust the pH to 7.0.

[0059] ⑥Basic fermentation medium: 6.5 g / L tryptone, 0.2 g / L magnesium sulfate heptahydrate, 1.0 g / L potassium dihydrogen phosphate, 1.5 g / L dipotassium hydrogen phosphate, 0.25 g / L L-cysteine ​​hydrochloride monohydrate. In addition, add 1 mL of vitamin solution and 1 mL of trace element solution per unit volume (L). Sodium chloride is added in varying proportions to adjust the pH to 7.0.

[0060] Example 1 Adaptive evolution strategy to cultivate salt-tolerant methanogenic sludge

[0061] This example employs an adaptive evolution strategy for microbial flora to cultivate salt-tolerant methanogenic sludge. Adaptive evolution was conducted in a fully mixed anaerobic reactor using a continuous flow fermentation mode, with the initial inoculum consisting of anaerobic methanogenic activated sludge. The reactor influent composition is shown in Table 1. Peptone was used as the sole carbon and nitrogen source, the initial inoculum sludge concentration was 33.0 g-TS / L, the agitation speed was 100 rpm, the fermentation temperature was 36°C, and the pH was controlled at 7.0. The initial influent salinity was 1.5%, and the hydraulic retention time was 10 days.

[0062] Table 1 Influent components of adaptive evolution reactor

[0063]

[0064] (1) Changes in COD removal rate during adaptive evolution

[0065] During the adaptive evolution process under salt stress, COD removal efficiency was used to characterize changes in the salt tolerance of anaerobic bacterial communities. Based on a steady-state COD removal efficiency exceeding 70%, the reactor influent salinity was gradually increased to allow for salt tolerance in the anaerobic methanogenic sludge, resulting in a methanogenic sludge that could tolerate salinity exceeding 3.0%.

[0066] like Figure 2 As shown, the inoculated anaerobic methanogenic sludge was able to adapt quickly under 1.5% (1-6 days) and 2.0% (7-14 days) salt stress, with the effluent COD ranging from 1280 to 1920 mg / L and the COD removal rate maintained above 80%.

[0067] When the influent salinity was increased to 2.6% (15-21 days), the removal rate dropped to 64.0% and then quickly recovered to 77.6%.

[0068] When the influent salinity was further increased to 2.9% (22-51d), under this stress, COD removal first decreased to 58.1%, and then gradually recovered to 72.3%, but the tolerance response time was significantly prolonged.

[0069] When the salinity was further increased to 3.2% (52nd-61st day), the effluent COD concentration did not change significantly, and the COD removal rate remained stable between 74.4% and 84.0%.

[0070] This suggests that salt-tolerant microorganisms have become dominant through salinity gradient acclimation. However, when salinity increased to 3.6% (62-116 days), the COD removal rate gradually decreased to around 55.0% and was difficult to recover. Therefore, a "top-down" adaptive evolution strategy for anaerobic bacterial communities can initially construct methanogenic sludge that tolerates 3.2% salinity.

[0071] (2) Changes in protein degradation rates during adaptive evolution

[0072] The degradation rate of proteins during adaptive evolution is as follows Figure 3 As shown in the figure, under 1.5% low salt stress, the average protein degradation rate can reach 93.2%.

[0073] As the influent salinity gradient increased and the anaerobic bacteria gradually adapted to the salt stress, protein degradation first decreased and then gradually recovered. Finally, under 3.2% salt stress, the protein degradation rate recovered to about 90%.

[0074] However, when the inlet salinity was further increased to 3.6%, the effluent protein concentration increased significantly, the protein degradation rate decreased to about 71.2%, and it was difficult to recover.

[0075] This indicates that the anaerobic bacteria have insufficient protease secretion capacity under this salt stress, and protein hydrolysis is inhibited. Therefore, this example uses salt-tolerant methanogenic sludge obtained under 3.2% salt stress as a source to screen for highly salt-tolerant protease-producing bacteria.

[0076] Example 2 Directed screening of high salt-tolerant protease-producing bacteria

[0077] (1) Isolation and purification of protease-producing bacteria and initial screening of enzyme production capacity

[0078] Although a top-down adaptive evolution strategy initially established a methanogenic bacterial community tolerant to 3.2% salinity, under higher salt stress, insufficient protease secretion led to inhibition of protein hydrolysis. Therefore, to enhance protease production under high salt stress, salt-tolerant enzyme-producing microorganisms were screened from salt-tolerant methanogenic sludge.

[0079] The isolation and screening process involves placing anaerobic bacteria from salt-tolerant methanogenic sludge obtained under 3.2% salt stress in an anaerobic tube, adding glass beads, and vortexing to disperse them. The suspension is then diluted in a gradient using saline. The diluted suspension is then spread onto a 4.0% salinity peptone solid medium plate and incubated upside down in a 36°C anaerobic incubator for 48 hours. The resulting single colonies are then purified using the plate streak method, and the purified strains are then stored on a 4.0% salinity solid slant medium.

[0080] A total of 16 strains (designated A1-A16) were isolated and purified at 4.0% salinity. These strains were inoculated onto 4.0% salinity clear zone medium and 4.0% salinity gelatin liquefaction medium using the three-point method and puncture inoculation, respectively. The ratio of clear zone diameter to colony diameter (>1.5) and the degree of gelatin liquefaction were used as evaluation criteria to screen for salt-tolerant strains with strong protease production. As shown in Table 2, 56.25% of the strains had a clear zone diameter / colony diameter ratio of 1.5 < 2. Nine strains (A1, A2, A3, A4, A5, A7, A8, A10, and A13) with a ratio >1.5 and high gelatin liquefaction were selected for further analysis of their salt tolerance.

[0081] Table 2 Preliminary screening of enzyme production capacity of protease-producing bacteria

[0082]

[0083] (2) Rescreening of salt tolerance of protease-producing bacteria

[0084] The protease-producing bacteria screened were subjected to salt tolerance rescreening. The specific process included: after the screened strains were cultured in LB liquid medium for 48 hours, the diluted bacterial suspensions were respectively applied to 8.0% salinity salt tolerance rescreening solid medium, and then the protease-producing bacteria that could tolerate 8.0% salinity were screened (such as Figure 4 shown).

[0085] like Figure 5 As shown in the figure, after 48 h of batch fermentation in 8.0% salt-tolerant secondary screening medium, the biomass of the fermentation broth of strain A1 was significantly higher than that of other strains, and the OD 600 The value of the salt tolerance test was 0.95. Therefore, strain A1 had a better salt tolerance.

[0086] (3) Identification of bacterial species

[0087] The 16S rDNA sequence of strain A1 obtained by screening using bacterial universal primers was amplified and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the 16S rDNA sequence was compared on GenBank. The results showed that the strain sequence length was 1437bp, and the similarity with the 16S rDNA sequence of Arthrobacter protophormiae (GenBank Number: GQ395612.1) was as high as 99.72%. Therefore, the highly salt-tolerant protease-producing strain was identified as Arthrobacter protophormiae, and was deposited in the China Center for Type Culture Collection on May 23, 2024. Its preservation number is CCTCC NO: M 20241041, and it was named Arthrobacter protophormiae A1. Its 16S rDNA sequence is shown in SEQ ID NO.01. Example 3 Salt tolerance of Arthrobacter protophormiae A1

[0088] (1) Effect of salinity on the growth of Arthrobacterium truncatum A1

[0089] The original glass fly Arthrobacter A1 was transferred to the basic culture medium for batch fermentation to investigate the effects of salt stress on its growth, protease secretion and protein degradation. The growth curves under different salinity (2.0%, 4.0%, 6.0%, 8.0%) are shown in Figure 2. Figure 6 As shown, it can grow under salinity conditions of 2.0%-8.0%. When the salinity reaches 4.0%, 6.0% and 8.0%, the strain has the highest biomass (OD 600 ) were 1.68, 1.32 and 0.99, respectively, compared with 2.0% salinity (the highest OD 600 =1.76) decreased by 4.5%, 25.0%, and 43.7%, respectively. Accordingly, it can be concluded that at high salinity levels of 4.0%, 6.0%, and 8.0%, the maximum biomass retention rates reached 95.5%, 75.0%, and 56.3%, respectively. This shows that Arthrobacter protocis A1 has a high salt tolerance. Although high salinity can inhibit its growth efficiency to a certain extent, it can still maintain more than half of its maximum biomass at a high salinity of 8.0%.

[0090] (2) Effect of salinity on protease secretion by Arthrobacterium truncatum A1

[0091] Effects of salinity on the secretion of protease from Arthrobacterium truncatum A1 Figure 7As shown, the protease activity in the fermentation broth under different salt stresses reached its maximum value during the bacterial growth stabilization period (the second day). At salinity levels of 2.0%, 4.0%, 6.0%, and 8.0%, the maximum enzyme activity values ​​were 55.2 U / mL, 52.6 U / mL, 35.9 U / mL, and 25.5 U / mL, respectively. It can be concluded that when the salinity reached 4.0%, 6.0%, and 8.0%, the enzyme activity remained at 95.3%, 65.0%, and 46.2% compared to that at 2.0%. This indicates that Arthrobacter protophyte A1 can still effectively grow and secrete proteases under high salt stress.

[0092] (3) Effect of salinity on protein degradation by Arthrobacterium truncatum A1

[0093] Effects of salinity on protein degradation by Arthrobacterium protothecoides A1 Figure 8 As shown in Figure 3, after 3 days of anaerobic batch fermentation at salinity levels of 2.0%, 4.0%, 6.0%, and 8.0%, the protein degradation rates reached 61.1%, 55.1%, 48.9%, and 41.4%, respectively. This indicates that Arthrobacter protocis A1 can effectively hydrolyze proteins under high salt stress.

[0094] Example 4 Construction and application effect of anaerobic degradation bacterial community of heparin sodium wastewater

[0095] (1) Construction of anaerobic bacterial community for heparin sodium wastewater degradation

[0096] The screened original glass fly Arthrobacter A1 was activated in LB culture medium and then compounded with salt-tolerant methanogenic sludge in a volume ratio of 1:10, and the salt stress acclimation pressure was further increased.

[0097] like Figure 9 As shown, compared with salt-tolerant methanogenic sludge, the COD removal rate and protein degradation rate of the composite bacterial consortium increased to 76.5% and 93.2%, respectively, at 3.6% salinity. When the salinity increased to 4.0% (days 36-45), the COD removal rate remained above 75%. When the salinity was further increased to 4.6% (days 46-67), after a short period of adaptation, the COD removal rate of the bacterial consortium still reached 72.6%. However, when the salinity increased to 5.2% (days 68-161), the COD removal rate dropped significantly to around 39.0% and was difficult to recover. Throughout the entire salinity increase period, the protein removal rate remained above 90%. In addition, the VFA concentration increased significantly to above 4.0 g / L under 5.2% salinity stress. This shows that protein hydrolysis was not affected under 5.2% salinity stress, and the accumulation of VFAs was the main reason for the decrease in COD removal rate. By combining Arthrobacterium glabratum A1 and salt-tolerant methane-producing sludge, we successfully constructed an anaerobic degradation bacterial community that can tolerate a salinity of 4.6% and used it for anaerobic treatment of sodium heparin wastewater.

[0098] (2) Application effect of anaerobic degradation bacteria in sodium heparin wastewater

[0099] The anaerobic degrading bacteria of sodium heparin wastewater (tolerant to 4.6% salinity), salt-tolerant methanogenic sludge (tolerant to 3.2% salinity) and original methanogenic sludge were used to treat actual sodium heparin wastewater with a salinity of 4.3%, respectively.

[0100] like Figure 10 As shown, the COD removal rate, protein degradation rate, and methane production of the original methanogenic activated sludge were only 28.7%, 32.8%, and 67.3 mL / g-COD, respectively; while the COD removal rate, protein degradation rate, and methane production of the salt-tolerant methanogenic sludge were 39.1%, 45.7%, and 101.4 mL / g-COD, respectively. In comparison, the COD removal rate, protein degradation rate, and methane production of the anaerobic degrading bacterial community in sodium heparin wastewater reached 76.3%, 92.5%, and 201.6 mL / g-COD, respectively. This shows that the method of the present invention can effectively enhance the anaerobic treatment of high-salt, high-protein wastewater such as sodium heparin wastewater.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An anaerobic treatment method for heparin sodium wastewater, characterized by: The method includes using highly salt-tolerant and protease-producing Arthrobacter protophormiaeA1 to strengthen the anaerobic treatment of heparin sodium wastewater. The Arthrobacter protophormiaeA1 was deposited in the China Center for Type Culture Collection on May 23, 2024, with a deposit number of CCTCC NO: M20241041.

2. The method according to claim 1, wherein: The method specifically includes constructing a heparin sodium wastewater-degrading bacterial community for anaerobic treatment of heparin sodium wastewater, and performing anaerobic fermentation for 10-15 days at an inoculation amount of 5%-15%, a pH of 6.5-7.5, and 30-38°C, wherein the heparin sodium wastewater-degrading bacterial community includes salt-tolerant methanogenic sludge and Protothecobacterium glabratum A1, and the salt-tolerant methanogenic sludge is methanogenic sludge with a salinity tolerance of 3.0%-3.6%.

3. The method according to claim 2, wherein: In the sodium heparin wastewater-degrading bacterial community, the volume ratio of the salt-tolerant methanogenic sludge bacterial liquid to the activated liquid of the original glassworm Arthrobacter A1 is 5:1-15:

1.

4. The method according to claim 2, wherein: The salt-tolerant methane-producing sludge is methane-producing sludge with a salinity tolerance of 3.0%-3.2%.

5. The method according to claim 4, characterized in that: The method for cultivating salt-tolerant methanogenic sludge comprises the following steps: using anaerobic methanogenic sludge as initial inoculum, using peptone as sole carbon source and nitrogen source, and acclimating salt-tolerant methanogenic sludge capable of tolerating salinity above 3.0% by gradiently increasing salt stress pressure.

6. The method according to claim 1, wherein: The original Arthrobacterium glabratum A1 produces protease under anaerobic conditions and has a salt tolerance of more than 4.0%.

7. The method according to claim 6, characterized in that: The salt tolerance of the original Arthrobacterium vitae A1 is 4.0%-8.0%.

8. The method according to claim 6, wherein: The enzymatic activity of the protease reaches above 25 U / mL.

9. The method according to claim 8, characterized in that: When the salt tolerance of the original Arthrobacterium vitae A1 is 4.0%-6.0%, the enzyme activity of the protease reaches above 35 U / mL.

10. The method according to claim 8, characterized in that: When the salt tolerance of the original Arthrobacterium vitae A1 is 6.0%-8.0%, the enzyme activity of the protease reaches above 25 U / mL.

11. The method according to claim 1, wherein: The salinity of the heparin sodium wastewater reaches above 4.0%.

12. The method according to claim 1, wherein: The protein degradation rate of the sodium heparin wastewater is above 70%, and the COD removal rate is above 50%.

13. The method according to claim 12, wherein: The protein degradation rate of the heparin sodium wastewater is above 80%, and the COD removal rate is above 60%.

14. The method according to claim 12, wherein: The protein degradation rate of the sodium heparin wastewater is above 90%, and the COD removal rate is above 70%.

15. The method according to claim 12, wherein: The methane production reaches above 150mL / g-COD.

16. A bacterial community for anaerobic degradation of sodium heparin wastewater, characterized by: It includes salt-tolerant methanogenic activated sludge and original glass fly Arthrobacter A1. The salt-tolerant methanogenic activated sludge is methanogenic sludge with a salinity tolerance of 3.0%-3.6%. The original glass fly Arthrobacter A1 was deposited in the China Center for Type Culture Collection on May 23, 2024, with a preservation number of CCTCC NO: M20241041.

17. The anaerobic heparin sodium wastewater-degrading bacterial community according to claim 16, characterized in that: The volume ratio of the salt-tolerant methanogenic activated sludge bacterial solution to the activated solution of Arthrobacter glabratus A1 is 5:1-15:1.

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