Lactobacillus fermentum for treating pulp and papermaking wastewater and culture method and application thereof

Abstract

The present application relates to a lactobacillus fermentum for treating pulping and papermaking wastewater and a culture method and application thereof. Lactobacillus fermentum ) HF-F2021003, preserved in China General Microbiological Culture Collection Center on January 11, 2022, address: No. 1, Xibahe Road, Yard 3, Chaoyang District, Beijing, strain number: CGMCC NO. 24291. The lactobacillus fermentum HF-F2021003 is isolated for the first time, which can effectively ferment and decompose polysaccharide substances such as cellulose, hemicellulose, miscellaneous fine bubbles and starch in pulping and papermaking wastewater, and can effectively remove potassium ions and sodium ions in the pulping and papermaking wastewater, can greatly reduce the treatment difficulty of complex substances in the pulping and papermaking wastewater, reduce the total salt content in the wastewater, and significantly improve the treatment efficiency of the pulping and papermaking wastewater.

Description

Technical Field

[0001] This invention relates to a fermenting lactobacillus strain 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] Papermaking wastewater mainly comes from the pulping and papermaking processes in the papermaking industry. Pulping involves separating the fibers from plant materials to make pulp, which is then bleached. Papermaking involves diluting, shaping, pressing, and drying the pulp to make paper. The wastewater generated from 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, converting them into green energy sources such as biogas and hydrogen, or other 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 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, due to the complex composition of pollutants in papermaking wastewater, which contains a large amount of volatile acids and sugary organic matter, and the addition of various chemicals during the production process to ensure product quality, most microorganisms are strongly inhibited. This results in poor effectiveness of general microbial degradation treatment of pulp and papermaking wastewater, or the need for frequent addition or replacement of microorganisms, leading to extremely high treatment costs. Therefore, it is of great significance to find new strains of bacteria that have good stability, high removal efficiency, strong reproductive and proliferation capabilities, and low sensitivity to bacterial environment for treating pulp and papermaking wastewater. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a fermentation lactobacillus strain 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 fermenting lactobacillus ( Lactobacillus fermentum HF-F2021003 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 NO.24291.

[0009] The cultivation method for the above-mentioned Lactobacillus fermentum HF-F2021003 includes the following steps: (1) Take Lactobacillus fermentum HF-F2021003 and inoculate it into anaerobic RCM medium. Culture it in a shaker at 30~32℃ and 150~250rpm for 20~30h to obtain seed liquid; (2) The seed liquid obtained in step (1) is inoculated into anaerobic RCM medium at a volume percentage of 5-10% and cultured in a shaker at 30-32℃ and 150-250rpm for 20-30h to obtain Lactobacillus fermentum HF-F2021003 bacterial liquid.

[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 and 0.5g L-cysteine ​​hydrochloride, pH 6.8.

[0011] The application of the above-mentioned Lactobacillus fermentum HF-F2021003 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 passed into anaerobic sludge containing Lactobacillus fermentum HF-F2021003, stirred evenly, and subjected to anaerobic reaction treatment. Then, the anaerobic wastewater is separated into sludge 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.

[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 fermenting lactobacillus HF-F2021003 is 0.01~2% of the mass of the anaerobic sludge.

[0015] The technical features and beneficial effects of this invention are as follows: 1. This invention is the first to isolate and obtain Lactobacillus fermentum (… Lactobacillus fermentum HF-F2021003 is a facultative or obligate anaerobic bacillus that is a heterologous fermentation strain. After fermentation, it produces lactic acid, ethanol, and CO2. It can effectively ferment and decompose cellulose, hemicellulose, impurities, and starch and other polysaccharides in pulp and paper wastewater. It can also effectively remove potassium and sodium ions from pulp and paper wastewater, which can significantly reduce the difficulty of treating complex substances in pulp and paper wastewater, reduce the total salt content in wastewater, and significantly improve the treatment efficiency of pulp and paper wastewater.

[0016] 2. The Lactobacillus fermentum HF-F2021003 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 a TDS of 12,000 to 15,000. At the same time, it can convert various long-chain carbohydrates in wastewater into short-chain substances, 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, improving the stability of system operation, and reducing treatment costs.

[0017] 3. The fermented lactobacillus HF-F2021003 provided by this invention, after treating pulp and paper wastewater, absorbs potassium, nitrogen, phosphorus and other substances during bacterial growth, which can be used as fertilizer by plants through the sludge discharge system.

[0018] 4. This invention specifically focuses on the anaerobic treatment of pulp and paper wastewater using *Lactobacillus fermentationus*. 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 bactericidal and bacteriostatic chemicals, resulting in wastewater entering the wastewater treatment system containing these agents. Conventional bacteria struggle to survive stably in this system for extended periods, and conventional *Lactobacillus* used in emulsion fermentation also finds it difficult to survive and proliferate within the wastewater system. This invention addresses this issue through years of domestication, improvement, and research on *Lactobacillus fermentationus* 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. Wastewater treated by an anaerobic system containing *Lactobacillus fermentationus* HF-F2021003 of this invention significantly improves the operating environment of subsequent aerobic biological systems, maintaining high removal efficiency. This strain can also be widely applied to wastewater treatment in the food and fermentation industries. Attached Figure Description

[0019] Figure 1 Lactobacillus fermentum ( Lactobacillus fermentum Electron microscope image of HF-F2021003. Detailed Implementation

[0020] 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.

[0021] Biomaterials: Lactobacillus fermentum ( Lactobacillus fermentum HF-F2021003 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 NO.24291.

[0022] The composition of each liter of anaerobic RCM medium is as follows: 10g peptone, 10g beef meal, 3g yeast extract, 5g glucose, 1g soluble starch, 5g sodium chloride, 3g sodium acetate and 0.5g L-cysteine ​​hydrochloride, with a pH of 6.8.

[0023] 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.

[0024] Unless otherwise specified, all pharmaceuticals and reagents mentioned in the examples are commercially available products.

[0025] Example 1: Lactobacillus fermentation ( Lactobacillus fermentum Acquisition of HF-F2021003 1. Sample: Anaerobic sludge from the integrated anaerobic treatment system of pulp and paper wastewater of a pulp and paper enterprise in Hebei Province. 100mL of anaerobic sludge was taken in a sterile bottle, sealed, and placed in a box with ice packs as a sample.

[0026] 2. Isolation: After mixing the sample, 1 mL was transferred to anaerobic RCM 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 enhanced RCM medium and cultured on a shaker at 30℃ and 200 rpm for 24 hours until the medium became turbid. 4 mL of the bacterial culture 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-F2021003.

[0027] 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 rod-shaped.

[0028] 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 1476 bp.

[0029] A BLAST homology search was performed on the sequence in the GenBank database. Sequence comparison analysis revealed that HF-B2022004 is similar to... Lactobacillus fermentum strain 6879, 6020 The 16S rDNA homology similarity of *Lactobacillus fermentum* was 99.0%. Based on morphological and physiological-biochemical characteristics, combined with the 16S rDNA sequence alignment results, this strain was identified as *Lactobacillus fermentum* and named... Lactobacillus fermentumHF-F2021003 .

[0030] Example 2: Cultivation of Lactobacillus fermentum HF-F2021003 The cultivation method for Lactobacillus fermentum HF-F2021003 includes the following steps: (1) Take Lactobacillus fermentum HF-F2021003 and inoculate it into anaerobic RCM medium. Under the conditions of 30℃ and 200rpm, culture it in a shaker for 24h to obtain seed liquid; (2) The seed liquid obtained in step (1) is inoculated into anaerobic RCM medium at a volume percentage of 5-10% and cultured in a shaker at 30℃ and 200rpm for 24h to obtain Lactobacillus fermentum HF-F2021003 bacterial liquid.

[0031] Example 3: Application of Lactobacillus fermentum HF-F2021003 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.

[0032] The specific method is as follows: The pretreated pulp and paper wastewater is added to an anaerobic system containing anaerobic flocculent sludge or a mixture of flocculent and granular sludge containing Lactobacillus fermentum HF-F2021003. The mixture is stirred evenly and subjected to anaerobic reaction treatment. The anaerobic wastewater is then 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 a sludge discharge pipe. The anaerobic effluent after mud-water separation is then subjected to subsequent aerobic biological treatment and coagulation oxidation to obtain qualified clean water for discharge or reuse. This is the experimental group.

[0033] The fermenting lactobacillus HF-F2021003 is 0.3% of the mass of the anaerobic sludge.

[0034] Meanwhile, an existing Lactobacillus fermentation strain was used to treat pulp and paper wastewater using the same method as a control group. The existing Lactobacillus fermentation strain is available at the China General Microbiological Culture Collection Center (CGMCC) under the code CGMCC 1.15608.

[0035] 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.

[0036] Table 1

[0037] As shown in Table 1, the *Lactobacillus fermentatus* HF-F2021003 provided by this invention has a stronger biological environmental adaptability than other *Lactobacillus fermentatus* species. It can survive in environments with TDS levels of 12,000 to 15,000. After fermentation, it produces lactic acid and a relatively large amount of acetic acid, ethanol, and CO2. It can effectively ferment and decompose polysaccharides such as cellulose, hemicellulose, impurities, and starch in pulp and paper wastewater. Furthermore, its removal efficiency for potassium and sodium ions in pulp and paper wastewater reaches over 23%, which is significantly better than existing *Lactobacillus fermentatus* species. This can greatly reduce the difficulty of treating complex substances in pulp and paper wastewater and significantly improve the treatment efficiency of pulp and paper wastewater. SEQUENCE LISTING <110> Shandong Huanfa Technology Development Co., Ltd. <120> A strain of *Lactobacillus fermentatus* for treating pulp and paper wastewater, its culture method and application <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 1476 <212> DNA <213> Lactobacillus fermentum <400> 1 cgggcgctgc tataatgcag tcgaacgcgt tggcccaatt gattgatggt gcttgcacct 60 gattgatttt ggtcgccaac gagtggcgga cgggtgagta acacgtaggt aacctgccca 120 gaagcggggg acaacatttg gaaacagatg ctaataccgc ataacagcgt tgttcgcatg 180 aacaacgctt aaaagatggc ttctcgctat cacttctgga tggacctgcg gtgcattagc 240 ttgttggtgg ggtaacggcc taccaaggcg atgatgcata gccgagttga gagactgatc 300 ggccacaatg ggactgagac acggcccata ctcctacggg aggcagcagt agggaatctt 360 ccacaatggg cgcaagcctg atggagcaac accgcgtgag tgaagaaggg tttcggctcg 420 taaagctctg ttgttaaaga agaacacgta tgagagtaac tgttcatacg ttgacggtat 480 ttaaccagaa agtcacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540 cgttatccgg atttattggg cgtaaagaga gtgcaggcgg ttttctaagt ctgatgtgaa 600 agccttcggc ttaaccggag aagtgcatcg gaaactggat aacttgagtg cagaagaggg 660 tagtggaact ccatgtgtag cggtggaatg cgtagatata tggaagaaca ccagtggcga 720 aggcggctac ctggtctgca actgacgctg agactcgaaa gcatgggtag cgaacaggat 780 tagataccct ggtagtccat gccgtaaacg atgagtgcta ggtgttggag ggtttccgcc 840 cttcagtgcc ggagctaacg cattaagcac tccgcctggg gagtacgacc gcaaggttga 900 aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 960 tacgcgaaga accttaccag gtcttgacat cttgcgccaa ccctagagat agggcgtttc 1020 cttcgggaac gcaatgacag gtggtgcatg gtcgtcgtca gctcgtgtcg tgagatgttg 1080 ggttaagtcc cgcaacgagc gcaacccttg ttactagttg ccagcattaa gttgggcact 1140 ctagtgagac tgccggtgac aaaccggagg aaggtgggga cgacgtcaga tcatcatgcc 1200 ccttatgacc tgggctacac acgtgctaca atggacggta caacgagtcg cgaactcgcg 1260 agggcaagca aatctcttaa aaccgttctc agttcggact gcaggctgca actcgcctgc 1320 acgaagtcgg aatcgctagt aatcgcggat cagcatgccg cggtgaatac gttcccgggc 1380 cttgtacaca ccgcccgtca caccatgaga gtttgtaaca cccaaagtcg gtggggtaac 1440 cttttaggag ccagccgcct aaggtgactg agaatt 1476

Claims

1. A strain of Lactobacillus fermentum ( Lactobacillus fermentum HF-F2021003 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 NO.24291.

2. The method for culturing Lactobacillus fermentum HF-F2021003 according to claim 1, characterized in that, The steps include the following: (1) Take Lactobacillus fermentum HF-F2021003 and inoculate it into anaerobic RCM medium. Culture it in a shaker at 30~32℃ and 150~250rpm for 20~30h to obtain seed liquid; (2) The seed liquid obtained in step (1) is inoculated into anaerobic RCM medium at a volume percentage of 5-10% and cultured in a shaker at 30-32℃ and 150-250rpm for 20-30h to obtain Lactobacillus fermentum HF-F2021003 bacterial liquid.

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 and 0.5g L-cysteine ​​hydrochloride, pH 6.

8.

4. The application of Lactobacillus fermentum HF-F2021003 as described in claim 1 in the treatment of pulp and paper wastewater, characterized in that, The application steps are as follows: Pulping and papermaking wastewater to be treated is passed into anaerobic sludge containing *Lactobacillus fermentum* HF-F2021003, stirred evenly, and subjected to anaerobic reaction treatment. The anaerobic-treated wastewater is then separated into sludge and water. The anaerobic effluent undergoes subsequent aerobic biological treatment or further coagulation and oxidation treatment to obtain compliant clean water for discharge or reuse. The anaerobic sludge is flocculent or a mixture of flocculent and granular anaerobic sludge; the *Lactobacillus fermentum* HF-F2021003 constitutes 0.01~2% of the mass of the anaerobic sludge.

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