Clostridium butyricum for treating pulping and papermaking wastewater, culture method and application thereof

Abstract

The present application relates to a Clostridium butyricum for treating pulping and papermaking wastewater and a culture method and application thereof. The Clostridium butyricum HF-B2022004 is preserved in the China General Microbiological Culture Collection Center on January 11, 2022, and the address is No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, and the strain number is CGMCC 24292. The present application also provides a culture method and application method of the Clostridium butyricum HF-B2022004. The present application first isolates the Clostridium butyricum HF-B2022004, which can effectively ferment and decompose cellulose, hemicellulose, miscellaneous fine bubbles, resin glue and starch polysaccharide substances in pulping and papermaking wastewater, can greatly reduce the treatment difficulty of complex substances in pulping and papermaking wastewater, and can significantly improve the treatment efficiency of pulping and papermaking wastewater.

Description

Technical Field

[0001] This invention relates to a strain of Clostridium butyricum for treating pulp and paper wastewater, its cultivation method and application, and belongs to the field of microbial technology. Background Technology

[0002] In recent years, with the gradual implementation of national environmental protection policies, industrial enterprises have gradually increased their investment in the construction of wastewater treatment systems.

[0003] The paper industry is one of the most energy- and material-intensive industries, causing significant environmental pollution. Its pollution characteristics include large volumes of wastewater, resulting in high levels of COD, suspended solids (SS), and severe color. Globally, papermaking wastewater is a major source of pollution; for example, the United States lists it as one of the six major environmental hazards, and Japan lists it as one of the five. The repeated recycling of waste paper fibers leads to increasingly lower raw material quality. During production, companies must add large amounts of chemicals to ensure paper quality. Furthermore, waste paper boxes come into contact with various substances during use and handling. Therefore, the effective treatment of this papermaking wastewater has become a major concern. Papermaking wastewater mainly originates from the pulping and papermaking processes in the paper industry. Pulping is the process of separating the fibers from plant materials to make pulp, which is then bleached. Papermaking is the process of diluting, shaping, pressing, and drying the pulp to make paper. The wastewater generated in these two processes contains a large amount of recalcitrant organic matter such as lignin, cellulose, and hemicellulose, as well as various added chemical agents and auxiliaries such as pigments, alcohols, ketones, and organic acids.

[0004] Currently, methods for treating pulp and paper wastewater include physical methods, chemical methods, physicochemical methods, and biological methods. Physical methods rely on physical processes. They primarily remove non-volatile suspended solids from the wastewater, while also removing some substances that contribute to COD and reducing color to some extent. Commonly used physical treatment methods include sedimentation, filtration, and centrifugation. Chemical methods involve adding chemicals to reduce COD and color, and remove some suspended solids. Chemical methods include oxidation, reduction, and neutralization.

[0005] Biological treatment has been widely used due to its cleanliness, practicality, and economy, and has become one of the main methods for secondary treatment of papermaking wastewater. The principle of biological treatment is to utilize the metabolic functions of microorganisms to degrade dissolved and colloidal organic pollutants in wastewater into harmless and stable substances, thereby purifying the wastewater. By artificially creating an environment suitable for the survival and reproduction of microorganisms, their proliferation is encouraged, thus improving their efficiency in oxidizing and decomposing organic matter. Based on the types of microorganisms involved and the oxygen supply, biological treatment is divided into three main categories: aerobic biological treatment, anaerobic biological treatment, and combined anaerobic-aerobic treatment. Among these, combined anaerobic-aerobic treatment is currently recognized as the most economical and efficient method.

[0006] However, in practical applications, papermaking wastewater contains complex pollutants, including a large amount of organic matter such as cellulose, hemicellulose, starch, and volatile acids. During production, various chemicals are added to ensure product quality, which strongly inhibit most microorganisms. This results in poor efficacy of general microbial degradation treatment of pulp and paper wastewater, or the need for frequent addition or replacement of microorganisms. In particular, conventional biochemical treatments struggle to degrade cellulose and hemicellulose in the wastewater, leading to extremely high subsequent treatment costs. Therefore, finding novel bacterial strains with good stability, high removal efficiency, strong reproductive and proliferative capabilities, and low sensitivity to the bacterial environment is of great significance for treating pulp and paper wastewater. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a strain of Clostridium butyricum for treating pulp and paper wastewater, along with its cultivation method and application.

[0008] The technical solution of the present invention is as follows: A strain of Clostridium butyricum ( Clostridium butyricum HF-B2022004 was deposited on January 11, 2022, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the strain number CGMCC 24292.

[0009] The cultivation method for Clostridium butyricum HF-B2022004 described above includes the following steps: (1) Take Clostridium butyricum HF-B2022004 and inoculate it into anaerobic RCM medium. Culture it in a shaker at 28~32℃ and 150~250rpm for 20~30h to obtain seed liquid; (2) The seeds obtained in step (1) are inoculated into anaerobic RCM medium at a volume percentage of 5-10% and cultured in a shaker at 28-32℃ and 150-250rpm for 20-30h to obtain Clostridium butyricum HF-B2022004 bacterial solution.

[0010] According to a preferred embodiment of the present invention, the composition per liter of the anaerobic RCM culture medium is as follows: 10g peptone, 10g beef powder, 3g yeast powder, 5g glucose, 1g soluble starch, 5g sodium chloride, 3g sodium acetate, 0.5g L-cysteine ​​hydrochloride.

[0011] The application of Clostridium butyricum HF-B2022004 in the treatment of pulp and paper wastewater.

[0012] According to a preferred embodiment of the present invention, the application comprises the following steps: The pulp and paper wastewater to be treated is fed into anaerobic sludge containing Clostridium butyricum HF-B2022004, stirred evenly, and subjected to anaerobic reaction treatment. Then, the anaerobic wastewater is separated into sludge and water. The separated sludge is retained in (or returned to) the system and mixed with the influent for further anaerobic treatment. Excess sludge is discharged from the anaerobic system through the sludge discharge pipe. The anaerobic effluent from the sludge-water separation is then subjected to subsequent aerobic biological treatment or further coagulation and oxidation treatment to obtain qualified clean water for discharge or reuse.

[0013] According to a preferred embodiment of the present invention, the anaerobic sludge is flocculent or a mixture of flocculent and granular anaerobic sludge.

[0014] According to a preferred embodiment of the present invention, the Clostridium butyricum HF-B2022004 is 0.05-15% of the mass of the anaerobic sludge.

[0015] According to a preferred embodiment of the present invention, the anaerobic reaction treatment temperature is controlled at 22~37℃.

[0016] The technical features and beneficial effects of this invention are as follows: 1. This invention is the first to isolate Clostridium butyricum (Clostridium butyricum) Clostridium butyricum HF-B2022004, this strain secretes a variety of pectinases, cellulases and glucans, which can effectively ferment and decompose cellulose, hemicellulose, impurities, resin gums and starches and other polysaccharides in pulp and paper wastewater. It can significantly reduce the difficulty of treating complex substances in pulp and paper wastewater and significantly improve the treatment efficiency of pulp and paper wastewater.

[0017] 2. The Clostridium butyricum HF-B2022004 provided by this invention has a strong survival ability and super tolerance in anaerobic wastewater treatment systems. It can grow stably in an environment with TDS of 12000~15000 mg / L. At the same time, it can convert various long-chain carbohydrates in wastewater into short-chain fatty acids, providing an energy source for other microorganisms in the anaerobic system. This is conducive to the efficient operation of other microorganisms, eliminating the need for frequent addition or replacement of microorganisms and reducing treatment costs.

[0018] 3. This invention specifically focuses on the anaerobic treatment of Clostridium butyricum in pulp and paper wastewater. Pulp and paper wastewater has a complex composition with highly variable pollutant components. Furthermore, the pulp and paper production process requires the addition of large amounts of bactericides and bacteriostatic chemicals, resulting in wastewater entering the wastewater treatment system containing these agents. Conventional bacteria struggle to survive stably in this system, and conventional Clostridium butyricum also has difficulty surviving and proliferating within the wastewater system. This invention addresses this issue through years of domestication, improvement, and research on Clostridium butyricum specifically for pulp and paper wastewater, cultivating a strain that has been successfully applied. Engineering practice has demonstrated that this strain exhibits good stability and high proliferation rate in the complex and variable pulp and paper wastewater, meeting the requirements of the production process.

[0019] 4. Wastewater treated by an anaerobic system containing Clostridium butyricum HF-B2022004 of this invention can significantly improve the operating environment of subsequent aerobic biological systems and maintain a high removal efficiency of the aerobic system. This strain can also be widely used in wastewater treatment in the food and fermentation industries. Attached Figure Description

[0020] Figure 1 Clostridium butyricum ( Clostridium butyricum Electron microscope image of HF-B2022004. Detailed Implementation

[0021] The specific implementation process of the present invention will be further described in detail below with reference to the embodiments. The described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Unless otherwise specified, the experimental techniques and scientific terms used in the embodiments have the same meaning as commonly understood by those skilled in the art.

[0022] Biomaterials: Clostridium butyricum ( Clostridium butyricum HF-B2022004 was deposited on January 11, 2022, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the strain number CGMCC 24292.

[0023] The composition of anaerobic RCM medium per liter is as follows: 10g peptone, 10g beef meal, 3g yeast powder, 5g glucose, 1g soluble starch, 5g sodium chloride, 3g sodium acetate, and 0.5g L-cysteine ​​hydrochloride.

[0024] The composition of each liter of anaerobic RCM solid medium is as follows: 10g peptone, 10g beef meal, 3g yeast powder, 5g glucose, 1g soluble starch, 5g sodium chloride, 3g sodium acetate, 0.5g L-cysteine ​​hydrochloride, and 0.5g agar.

[0025] Unless otherwise specified, all pharmaceuticals and reagents mentioned in the examples are commercially available products.

[0026] Example 1 Clostridium butyricum ( Clostridium butyricum Acquisition of HF-B2022004 1. Sample: Anaerobic sludge from the anaerobic treatment system of pulp and paper wastewater of a pulp and paper enterprise in Hebei Province. 100mL of anaerobic sludge was taken into a sterile bottle, sealed, and placed in a box with ice packs as a sample.

[0027] 2. Isolation: After mixing the sample, 1 mL was transferred to anaerobic basal medium and cultured on a shaker at 30℃ and 200 rpm for 7-10 days until the medium became turbid. Then, it was transferred to anaerobic RCM solid medium and cultured on a shaker at 30℃ and 200 rpm for 24 hours until the medium became turbid. 4 mL of the bacterial solution was then spread onto anaerobic RCM solid medium and cultured at 30℃ for 24 hours until colonies grew on the anaerobic RCM solid medium. Colonies were counted, and colonies with significant morphological differences were selected and repeatedly streaked onto anaerobic RCM solid medium until a single colony was obtained. The bacteria were repeatedly streaked on solid plates until a single colony grew and preserved as HF-B2022004.

[0028] 3. Strain identification: Gram staining was performed on the single colonies from step 2, and their morphology was then observed using an electron microscope. The results are as follows: Figure 1 As shown. This strain stains purple with Gram stain, indicating it is a Gram-positive bacterium, and its cells are round or oval.

[0029] Single colonies from step 2 were sent to BGI Genomics Co., Ltd., where DNA extraction, PCR amplification, and sequencing were performed. The sequencing results of the 16S rDNA are shown in SEQ ID NO.1, with a length of 1435bp.

[0030] A BLAST homology search was performed on the sequence in the GenBank database. Sequence comparison analysis revealed that HF-B2022004 is similar to... Clostridium butyricum strain LQ25, strain Ecaf-17 The 16S rDNA homology similarity of *Clostridium butyricum* was 99.0%. Based on morphological and physiological-biochemical characteristics, combined with the 16S rDNA sequence alignment results, this strain was identified as *Clostridium butyricum* and named... Clostridium butyricum HF-B2022004.

[0031] Example 2: Cultivation of Clostridium butyricum HF-B2022004 The culture method for Clostridium butyricum HF-B2022004 includes the following steps: (1) Take Clostridium butyricum HF-B2022004 and inoculate it into anaerobic RCM medium. Culture it in a shaker at 35℃ and 200rpm for 24h to obtain seed liquid; (2) The seeds obtained in step (1) were inoculated into anaerobic RCM medium at a volume percentage of 10% and cultured in a shaker at 30℃ and 200rpm for 24h to obtain Clostridium butyricum HF-B2022004 bacterial solution.

[0032] Example 3: Application of Clostridium butyricum HF-B2022004 in the treatment of pulp and paper wastewater The water quality conditions for pulp and paper wastewater are as follows: the wastewater generated from the production of packaging paper using OCC as raw material has the following indicators: COD concentration 4000~12000mg / L, total salt concentration 3500~10000mg / L, and SS (suspended solids) concentration 3000~6000mg / L.

[0033] The specific method is as follows: Pulping and papermaking wastewater is passed into anaerobic flocculent sludge containing Clostridium butyricum HF-B2022004, stirred evenly, and subjected to anaerobic reaction treatment at 35℃. Then, the anaerobic wastewater is separated into cement and water. The anaerobic effluent is then subjected to subsequent aerobic biological treatment or coagulation oxidation treatment to obtain qualified clean water for discharge or reuse. This is the experimental group.

[0034] The butyric acid bacillus HF-B2022004 is 3% of the mass of the anaerobic sludge.

[0035] Meanwhile, an existing Clostridium butyricum strain was set up to treat pulp and paper wastewater using the same method as a control group. The Clostridium butyricum strain is available from the China General Microbiological Culture Collection Center (CGMCC 1.5205).

[0036] The results of waste paper pulping and papermaking wastewater treatment in the experimental and control groups are shown in Table 1. The experimental conditions were waste paper pulping and papermaking wastewater with an influent COD concentration of 6500-8000 mg / L, total salt concentration of 5000-6500 mg / L, and SS concentration of 400-700 mg / L.

[0037] Table 1 As shown in Table 1, the *Clostridium butyricum* HF-B2022004 provided by this invention has a stronger biological environmental adaptability than other *Clostridium butyricum* species. It can survive in environments with TDS levels of 12,000 to 15,000, and secretes a variety of pectinases, cellulases, and glucans. The removal efficiency of cellulose and hemicellulose reaches over 45%, which is significantly better than existing *Clostridium butyricum* species. This can greatly reduce the difficulty of treating complex substances in pulp and paper wastewater and improve wastewater treatment efficiency. SEQUENCE LISTING <110> Peng Jicheng <120> A strain of Clostridium butyricum for treating pulp and paper wastewater, its culture method and application <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 1435 <212> DNA <213> Clostridium butyricum <400> 1 cggcgtgctt aacacatgca agtcgagcga tgaagtccct tcggggacgg attagcggcg 60 gacgggtgag taacacgtgg gcaacctgcc tcaaagtggg ggatagcctt ccgaaaggga 120 gattaatacc gcataaaatt agagtgtcac atgatacttt aatcaaagga gtaatccgct 180 ttgagatggg cccgcggcgc attagctagt tggtgaggta acggctcacc aaggcaacga 240 tgcgtagccg acctgagagg gtgatcggcc acattggaac tgagacacgg tccagactcc 300 tacgggaggc agcagtgggg aatattgcac aatgggggaa accctgatgc agcaacgccg 360 cgtgagtgat gaaggtcttc ggattgtaaa gctctgtctt ttgggacgat aatgacggta 420 ccaaaggagg aagccacggc taactacgtg ccagcagccg cggtaatacg taggtggcaa 480 gcgttgtccg gatttactgg gcgtaaaggg tgtgtaggcg gatatttaag tgagatgtga 540 aatccccgag cttaacttgg gtactgcatt tcaactgga tatctagagt gcaggagagg 600 agaatggaat tcctagtgta gcggtgaaat gcgtagagat taggaaac accagtggcg 660 720 ttagataccc tggtagtcca cgccgtaaac gatgaatact aggtgtagga ggtatcgacc 780 ccttctgtgc cgcagttaac acaataagta ttccgcctgg ggagtacggt cgcaagatta 840 aaactcaaag gaattgacgg gggcccgcac aagcagcgga gcatgtggtt taattcgaag 900 960 ccttcggggc agggagacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgtta 1020 ggttaagtcc tgcaacgagc gcaaccctta tcattagttg ctaccattaa gttgagcact 1080 ctagtgagac tgcccgggtt aaccgggagg aaggtgggga tgacgtcaaa tcatcatgcc 1140 ccttatgtcc agggcaacac acgtgctaca atggtgagta cagagagatg caataccgtg 1200 aggtggagcc aaactctaaa actcatccca gttcggattg taggctgaaa ctcgcctaca 1260 tgaagctgga gttgctagta atcgcgaatc agaatgtcgc ggtgaatacg ttcccgggcc 1320 ttgtacacac cgcccgtcac accatgagag ctggcaacac ccgaagtccg tgagataacc 1380 gtaaggagtc agcggccgaa ggtggggtta gtgattgggg tgaagtcgta aacaa 1435

Claims

1. A strain of Clostridium butyricum ( Clostridium butyricum HF-B2022004 was deposited on January 11, 2022, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the strain number CGMCC 24292.

2. The method for culturing Clostridium butyricum HF-B2022004 according to claim 1, characterized in that, The steps include the following: (1) Take Clostridium butyricum HF-B2022004 and inoculate it into anaerobic RCM medium. Culture it in a shaker at 28~32℃ and 150~250rpm for 20~30h to obtain seed liquid; (2) The seeds obtained in step (1) are inoculated into anaerobic RCM medium at a volume percentage of 5-10% and cultured in a shaker at 28-32℃ and 150-250rpm for 20-30h to obtain Clostridium butyricum HF-B2022004 bacterial solution.

3. The cultivation method as described in claim 2, characterized in that, The components per liter of the anaerobic RCM medium are as follows: 10g peptone, 10g beef powder, 3g yeast powder, 5g glucose, 1g soluble starch, 5g sodium chloride, 3g sodium acetate, 0.5g L-cysteine ​​hydrochloride.

4. The application of Clostridium butyricum HF-B2022004 as described in claim 1 in the treatment of pulp and paper wastewater, characterized in that, The steps are as follows: The pulp and paper wastewater to be treated is fed into anaerobic sludge containing Clostridium butyricum HF-B2022004, stirred evenly, and subjected to anaerobic reaction treatment. Then, the anaerobic wastewater is separated into mud and water. The separated sludge is retained in or returned to the system and mixed with the influent for further anaerobic treatment. Excess sludge is discharged from the anaerobic system through the sludge discharge pipe. The anaerobic effluent from mud-water separation is then subjected to subsequent aerobic biological treatment or further coagulation and oxidation treatment to obtain qualified clean water for discharge or reuse. Wherein, the anaerobic sludge is flocculent or a mixture of flocculent and granular anaerobic sludge; the Clostridium butyricum HF-B2022004 is 0.05~15% of the mass of the anaerobic sludge; and the anaerobic reaction treatment temperature is controlled at 22~37℃2.

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