A composite microbial flora for chemical pulp wastewater treatment, a microbial agent and application thereof

Abstract

The application discloses a kind of composite microbial flora for chemical pulp wastewater treatment, microbial inoculant and application thereof, belong to wastewater, sewage treatment and microbial field, solve how to realize low cost and without producing secondary pollution or solid waste under the condition, remove COD in chemical pulp wastewater problem, the application discloses that the effective viable count ratio of Calidifermentibacillus hisashii, thermophilic fat soil bacillus, ordinary high-temperature actinomycete, thermophilic lactic acid pale bacillus in thermophilic denitrifying geobacillus is (10~15%): (20~30%): (20~30%): (10~30%): (10~25%), and the composite microbial flora and microbial inoculant are formed, are applied to biochemical system, remove COD in chemical pulp wastewater from source, improve removal efficiency, reduce operating cost, solve the problem of large investment and environmental pollution.

Description

Technical Field

[0001] This invention relates to the fields of wastewater, sewage treatment and microbial technology, and in particular to a composite microbial community, microbial agent and its application for the treatment of chemical pulp wastewater. Background Technology

[0002] Currently, a large number of pulp and paper mills in China produce chemimechanical pulp. The wastewater from this pulping process, specifically chemimechanical pulp, refers to the wastewater generated during the pulping process using chemical mechanical methods (such as CTMP and APMP), and has the following significant characteristics:

[0003] 1. High pollutant concentration and large load:

[0004] Chemical oxygen demand (COD) concentration: usually between 10,000 and 30,000 mg / L, and in high-concentration wastewater it can even reach over 50,000 mg / L; BOD5 concentration: approximately 3,000 to 10,000 mg / L, B / C ratio is generally between 0.2 and 0.35, and biodegradability is poor; suspended solids content: SS concentration can reach 1,000 to 5,000 mg / L, mainly consisting of fine fibers, bark chips, etc.

[0005] 2. Complex composition, containing many recalcitrant substances:

[0006] Lignin and its derivatives: accounting for 30%-50% of total organic matter, with stable structure and difficult to biodegrade; hemicellulose, sugars, and resin acids: partially biodegradable, but easily forming complex organic mixtures; inorganic salts: containing large amounts of cations such as Na+, K+, and Ca2+, as well as anions such as SO42- and Cl-; toxic substances: containing small amounts of terpenes, phenols, and other substances that are toxic to microorganisms.

[0007] 3. High color intensity, high water temperature:

[0008] The color intensity can reach 1000-5000 times, mainly caused by lignin and its derivatives; the water temperature is usually between 40-60℃, which is not conducive to the growth and reproduction of conventional microorganisms.

[0009] 4. Large fluctuations in water quality and quantity:

[0010] Differences between different production stages: The wastewater discharged from pulping, washing, screening and other stages varies significantly; Impact of production cycle: Intermittent production leads to drastic fluctuations in wastewater discharge and pollutant concentration, with fluctuation ranges reaching ±50%.

[0011] Of all pulping wastewaters, chemimechanical pulping wastewater is the most difficult to treat. The main difficulties, current treatment technologies, and problems are as follows:

[0012] 1. Poor biodegradability, resulting in poor effectiveness of direct biological treatment:

[0013] Causes: The wastewater contains a large amount of lignin and its derivatives that are difficult to degrade, and the B / C ratio is low (0.2-0.35); Current treatment status: Direct aerobic biological treatment only achieves a COD removal rate of 40%-60%, which is difficult to meet emission standards. Therefore, the current process route adds relatively high-investment and high-operating-cost physicochemical and advanced oxidation technologies after biological treatment; Problems: The biological treatment efficiency is low, the cost of deep treatment is high, and the amount of sludge generated is large.

[0014] 2. Toxic substances inhibit microbial activity:

[0015] Inhibiting factors: Terpenes, phenols and other substances have a destructive effect on microbial cell membranes and enzyme systems; Existing treatment methods: High-concentration wastewater directly enters the biological system, which will lead to microbial poisoning and inactivation, and a sharp drop in treatment efficiency; Problems: Poor stability of the biological system, large fluctuations in effluent COD.

[0016] 3. Difficulty in removing color:

[0017] Causes: Lignin and its derivatives are the main color-producing substances, with stable structures that are difficult to remove by conventional methods; Current treatment status: After conventional biological treatment, the effluent color can still reach hundreds of times higher, failing to meet discharge standards; Existing problems: High effluent color leads to excessive use of downstream deep treatment agents.

[0018] Regarding the current status of chemical pulp wastewater treatment:

[0019] Currently, the treatment of chemical pulp wastewater typically employs a combined process of "pretreatment - biological treatment - advanced treatment":

[0020] 1. Pretreatment stage: Physical treatment: using screens, sieves, and air flotation to remove suspended solids and fibers; Chemical treatment: using acid and alkali to adjust pH value and adding coagulants to remove some organic matter and color; Biological pretreatment: using pre-acidification and anaerobic hydrolysis to improve the biodegradability of wastewater.

[0021] 2. Biochemical treatment stage:

[0022] Anaerobic treatment: High-efficiency anaerobic reactors such as IC reactors and UASB reactors are used to remove most of the organic matter; Aerobic treatment: Oxidation ditches, biological contact oxidation and other processes are used to further degrade the residual organic matter.

[0023] 3. Deep processing stage:

[0024] Physicochemical deep treatment: using coagulation sedimentation, filtration, activated carbon adsorption and other methods to remove suspended solids and residual organic matter; advanced oxidation treatment: using Fenton oxidation, ozone oxidation and other methods to remove recalcitrant organic matter and color.

[0025] In particular, the current efficiency of biological treatment is relatively low. Anaerobic treatment efficiency is typically between 30% and 50%, while aerobic treatment efficiency is around 40%. The COD of the anaerobic influent is between 7000 and 10000 mg / L, and the COD of the anaerobic effluent is between 3500 and 6000 mg / L. After aerobic treatment, the COD is between 2000 and 4000 mg / L, with high ammonia nitrogen and color, far exceeding discharge standards. Therefore, after biological treatment, multi-stage advanced treatment is required to meet discharge standards. This presents significant challenges to the treatment of chemimechanical slurry wastewater: First, the treatment cost is extremely high, especially the end-stage advanced treatment cost, which can reach 20 yuan per ton of water. The main cost sources are hydrogen peroxide, ferrous sulfate, and PAM. Second, the sludge production is large, containing a large amount of recalcitrant organic matter and inorganic salts generated through downstream chemical treatment. This sludge has poor dewatering performance, is difficult to dispose of, and easily causes secondary pollution. Summary of the Invention

[0026] This invention addresses the technical challenge of improving COD removal efficiency in the biochemical treatment stage of chemimechanical pulp (CMP) wastewater by preparing composite microbial communities or agents for CMP wastewater treatment, without generating secondary pollution or solid waste, while maintaining low cost. The invention provides a composite microbial community, agent, and its application for CMP wastewater treatment. By applying microbial technology to a biochemical system, it enhances COD removal from CMP wastewater at the source of the treatment process. This not only improves removal efficiency and reduces operating costs but also solves the problems of high investment and secondary pollution, achieving a low-cost, harmless technological breakthrough. It has broad application prospects in the treatment of CMP wastewater and sewage.

[0027] The objective of this invention is specifically achieved through the following technical solutions:

[0028] The first aspect of this invention discloses a composite microbial community for treating chemical pulp wastewater, comprising: *Geobacillus thermodenitrificans* (CGMCC No. 31848); *Calidifermentibacillus hisashii* (CGMCC No. 31843); *Geobacillus stearothermophilus* (CGMCC No. 31849); *Thermoactinomyces vulgaris* (CGMCC No. 31846); and *Pallidibacillus thermolactis* (CGMCC No. 31846). Number 31847; The deposit date for all entries is September 3, 2024; the depositary institution for all entries is the China General Microbiological Culture Collection Center (CGMCC); the address for all entries is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0029] The effective viable count ratio of *Geobacillus thermodenitrificans*, *Calidifermentibacillus hisashii*, *Geobacillus stearothermophilus*, *Thermoactinomyces vulgaris*, and *Pallidibacillus thermolactis* is (10~15%): (20~30%): (20~30%): (10~30%): (10~25%).

[0030] Furthermore, the optimal strain ratio of *Geobacillus thermodenitrificans*, *Calidifermentibacillus hisashii*, *Geobacillus stearothermophilus*, *Thermoactinomyces vulgaris*, and *Pallidibacillus thermolactis* is 15%:25%:20%:15%:15%.

[0031] Furthermore, the optimal strain ratio of *Geobacillus thermodenitrificans*, *Calidifermentibacillus hisashii*, *Geobacillus stearothermophilus*, *Thermoactinomyces vulgaris*, and *Pallidibacillus thermolactis* is 12%:30%:25%:18%:15%.

[0032] The second aspect of the present invention discloses a composite microbial agent for the treatment of chemical pulp wastewater, comprising a composite microbial community for the treatment of chemical pulp wastewater.

[0033] The third aspect of this invention discloses a composite microbial community for treating chemical pulp wastewater or a composite microbial agent for treating chemical pulp wastewater.

[0034] The fourth aspect of this invention discloses a method for using a composite microbial community or a composite microbial agent for treating chemimechanical pulp wastewater in chemimechanical pulp wastewater, comprising:

[0035] Add a compound microbial community or compound microbial agent to the biochemical system and mix it with the chemical slurry wastewater.

[0036] Furthermore, the methods of application include:

[0037] The biochemical system temperature should be between 30℃ and 60℃, and the DO value should be greater than 0.5 mg / L.

[0038] The dosing point is at the inlet of the aerobic tank of the biological system, where the dissolved oxygen level is not lower than 0.5 mg / L;

[0039] For every 1 kg of chemical oxygen demand (COD) removed from chemimechanical slurry wastewater, the dosage of microbial flora or compound microbial agents is 0.01 kg to 0.03 kg.

[0040] Furthermore, the optimal temperature for the biochemical system is 40℃.

[0041] The beneficial effects of this invention are:

[0042] Extensive experiments have verified the following ability of each microbial strain in the composite microbial community or agent for treating chemical pulp wastewater disclosed in this invention to secrete different enzymes under specific conditions:

[0043] Geobacillus thermodenitrificans secretes nitrate reductase, nitrite reductase, and cellulase in environments with temperatures above 40 degrees Celsius and high salinity; it has the ability to remove nitrogenous pollutants, lignin, and phenolic pollutants from chemical pulp wastewater.

[0044] Calidifermentibacillus hisashii secretes α-amylase and xylanase, which have the ability to open rings and degrade pollutants with complex carbon chain structures in chemical pulp wastewater;

[0045] Geobacillus stearothermophilus maintains good activity under high temperature and high salt conditions and secretes large amounts of carbohydrate-metabolizing enzymes, α-amylase and pullulanase, which have good degradation capabilities for carbon chain pollutants in wastewater.

[0046] Common thermophilic actinomycetes (Thermoactinomyces vulgaris) are adapted to living environments above 42 degrees Celsius and to highly alkaline and saline environments. They secrete lipase, xylanase, and cellulase. They have a good degradation effect on lignin, cellulose, and hemilignin in chemical pulp wastewater, and also have a good degradation effect on cell tissue fluid and lipids in wood.

[0047] Pallidibacillus thermolactis, adapted to environments above 45 degrees Celsius, secretes pyruvate kinase and cellulase, a carbohydrate metabolism enzyme. It exhibits excellent degradation capabilities for ketones, lignin, cellulose, and other complex pollutants in chemi-mechanical pulp wastewater.

[0048] The aforementioned strains secrete various enzymes to decompose lignin, cellulose, and hemicellulose in chemi-mechanical pulp wastewater. Simultaneously, these enzymes also act on aging or dead microbial cells in the biochemical system, decomposing their different components and providing energy for the complex microbial community / inoculant. The decomposed organic matrix (including sludge cells) is ultimately converted into carbon dioxide and water, thus achieving COD removal. Furthermore, the strains can also decompose some of the adsorbed organic matrix in the wastewater, converting it into carbon dioxide and water. At the same time, this microbial community effectively inhibits the growth of some miscellaneous bacteria through competitive inhibition among microorganisms.

[0049] By adding the composite microbial flora or bacterial agent disclosed in this invention for the treatment of chemimechanical pulp wastewater to the biochemical system, the biochemical treatment efficiency of chemimechanical pulp wastewater is significantly improved, and it has the following characteristics:

[0050] 1. Without additional investment in structures or equipment, the operating cost increases by 0.3 to 0.8 yuan per ton of water, which reduces the operating cost of the chemical pulp wastewater treatment process and is expected to reduce the cost of chemical treatment after biochemical treatment by more than 1.0 yuan per ton.

[0051] 2. Compared with traditional biochemical treatment technologies, the COD removal rate is increased by 20-50%;

[0052] 3. The temperature tolerance of the biochemical system can be increased from about 38 degrees Celsius to 45 degrees Celsius compared to the traditional biological treatment, effectively solving the problem of high temperature and difficulty in cooling chemical wastewater.

[0053] 4. This technology is a feasible solution for achieving standard discharge of chemical pulp wastewater through biological treatment process, and the treatment process produces less sludge and is more environmentally friendly than traditional technologies.

[0054] This invention is applicable to situations where the biochemical treatment efficiency of chemimechanical pulp wastewater is low and the COD of the biochemical effluent is high, resulting in significant pressure on downstream chemical treatment. This technology can significantly improve the COD removal efficiency of the biochemical system, reduce treatment costs and equipment investment, and has the advantages of high activity, low investment, low energy consumption, convenient operation, and no secondary pollution.

[0055] Biological Preservation Instructions:

[0056] (1) Biological material: ST-A003, classified name: Geobacillus thermonitrificans, accession number: CGMCC No.31848; accession date: September 3, 2024, accession institution: China General Microbiological Culture Collection Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0057] (2) Biological material: SW-B104, classified name: Calidifermentibacillus hisashii, accession number: CGMCC No.31843; accession date: September 3, 2024, depositary institution: China General Microbiological Culture Collection Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0058] (3) Biological material: ST-A005, classified name: Geobacillus stearothermophilus, accession number: CGMCC No.31849; accession date: September 3, 2024, accession institution: China General Microbiological Culture Collection Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0059] (4) Biological material: SW-B108, classified name: Thermoactinomyces vulgaris, accession number: CGMCC No.31846; accession date: September 3, 2024, accession institution: China General Microbiological Culture Collection Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0060] (5) Biological material: SW-B109, classified name: Pallidibacillus thermomolactis, accession number: CGMCC No.31847; accession date: September 3, 2024, depositary institution: China General Microbiological Culture Collection Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0061] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0062] Figure 1 This is a microscopic photograph of Geobacillus thermonitrificans provided by the present invention.

[0063] Figure 2 This is a microscope image of Calidifermentibacillus hisashii provided by the present invention.

[0064] Figure 3 This is a microscopic photograph of Geobacillus stearothermophilus provided by the present invention.

[0065] Figure 4 This is a microscope image of the common thermoactinomycetes vulgaris provided by the present invention.

[0066] Figure 5 This is a microscope image of Pallidibacillus thermolactis provided by the present invention.

[0067] Figure 6 These are photos of the inside of the bacterial strain preparation equipment provided in Case Study 1 of the verification experiment of this invention.

[0068] Figure 7 This is a photo of the bacterial culture preparation and addition point provided in Case Study 1 of the verification experiment of this invention.

[0069] Figure 8 These are on-site photos of the bacterial culture preparation and addition point provided in Case 2 of the verification experiment of this invention. Detailed Implementation

[0070] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0071] This invention, through analysis of water pollutants and environmental conditions in chemical pulp wastewater, screens microbial strains capable of degrading target pollutants under specific environmental conditions. This wastewater contains organic matter such as lignin, cellulose, resin acids, terpenes, and phenols, as well as large amounts of inert salts such as NaCl and Na₂SO₄. Lignin and its derivatives are the main chromogenic substances, resulting in high wastewater color. Due to the high concentration of macromolecular pollutants, poor biodegradability, and the presence of certain antibacterial chemicals, coupled with high salt content and high color, conventional microorganisms have low removal efficiency for these pollutants.

[0072] Therefore, the primary condition for strain screening is that the strains must be adaptable to a relatively high temperature environment of 38-45℃ and possess good high salt tolerance. Through extensive preliminary screening, isolation, purification, and testing experiments, five effective strains for treating chemimechanical slurry wastewater with high efficiency in removing pollutants were finally identified and numbered #HT1, #HT2, #HT3, #HT4, and #HT5. 16S rDNA sequencing was performed, and based on the sequencing results, comparisons were made with the NCBI geneBank database and the 16S rDNA bacterial database (Maidak et al., 2001). A phylogenetic tree was constructed using Mega software to determine the species and classification of the bacteria. Currently, these strains are deposited at the China General Microbiological Culture Collection Center (CGMCC).

[0073] Microscopic images of the morphological characteristics of strain #HT1 are shown below. Figure 1 As shown in the figure. 16S rDNA sequencing and comparison with NCBI GeneBank data showed that the strain was *Geobacillus thermonitrificans*, and the strain was subsequently deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 31848.

[0074] The >16S rDNA sequencing results of this strain are shown in SEQ ID No:1:

[0075] tccacgccgt aaacgatgag tgctaagtgt tagaggggtc acacccttta gtgctgtagc 840

[0076] taacgcgata agcactccgc ctggggagta cggccgcaag gctgaaactc aaaggaattg 900

[0077] acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt 960

[0078] accaggtctt gacatcccct gacaacccaa gagattgggc gttccccctt cggggggaca 1020

[0079] gggtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1080

[0080] gcaacgagcg caacccttgc ctctagttgc cagcattcag ttgggcactc tagagggact 1140

[0081] gccggctaaa agtcggagga aggtggggat gacgtcaaat catcatgccc cttatgacct 1200

[0082] gggctacaca cgtgctacaa tgggcggtac aaagggctgc gaacccgcga gggggagcga 1260

[0083] atcccaaaaa gccgctctca gttcggattg caggctgcaa ctcgcctgca tgaagccgga 1320

[0084] atcgctagta atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac 1380

[0085] cgcccgtcac accacgagag cttgcaacac ccgaagtcgg tgaggtaacc cttacgggag 1440

[0086] ccagccgccg aagtgataac tacgac 1466

[0087] Microscopic images of the morphological characteristics of strain #HT2 are shown below. Figure 2 As shown in the figure. 16S rDNA sequencing and comparison with NCBI GeneBank data showed that the strain was Calidifermentibacillus hisashii, and the strain was subsequently deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 31843.

[0088] The >16S rDNA sequencing results of this strain are shown in SEQ ID No:2:

[0089] cgccgtaaac gatgagtgct aagtgttgga gggtttccgc ccttcagtgc tgcagctaac 840

[0090] gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg 900

[0091] gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960

[0092] ggtcttgaca tctcctgacc gccctggaga cagggtcttc ccttcgggga caggatgaca 1020

[0093] ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080

[0094] cgcaaccctt ggttctagtt gccagcattc agttgggcac tctagagcga ctgccggcga 1140

[0095] caagtcggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200

[0096] cacgtgctac aatggatggt acaaagggca gcgaagcggc gacgcatgag cgaatcccag 1260

[0097] aaaaccattc tcagttcgga ttgcaggctg caactcgcct gcatgaagcc ggaatcgcta 1320

[0098] gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380

[0099] cacaccacga gagtttgtaa cacccgaagt cggtgaggta accgtaagga gccagccgcc 1440

[0100] gaagtggatc agaaccgccc a 1461

[0101] Microscopic images of the morphological characteristics of strain #HT3 are shown below. Figure 3 As shown in the figure. 16S rDNA sequencing and comparison with NCBI GeneBank showed that the strain was *Geobacillus stearothermophilus*, and the strain was subsequently deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 31849.

[0102] The >16S rDNA sequencing results of this strain are shown in SEQ ID No:3:

[0103] ggcggccttt gggctgtcac ttgcggatgg gcccgcggcg cattagctag ttggtgaggt 240

[0104] aacggctcac caaggcgacg atgcgtagcc ggcctgagag ggtgaccggc cacactggga 300

[0105] ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg caatgggcga 360

[0106] aagcctgacg gagcgacgcc gcgtgagcga agaaggcctt cgggtcgtaa agctctgttg 420

[0107] tgagggacga aggagcgccg ttcgaagagg gcggcgcggt gacggtacct cacgagaaag 480

[0108] ccccggctaa ctacgtgcca gcagccgcgg taatacgtag ggggcgagcg ttgtccggaa 540

[0109] ttattgggcg taaagcgcgc gcaggcggtc tcttaagtct gatgtgaaag cccacggctc 600

[0110] aaccgtggag ggtcattgga aactggggga cttgagggca ggagaggaga gcggaattcc 660

[0111] acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcggctctct 720

[0112] ggcctgtacc tgacgctgag gcgcgaaagc gtggggagca aacaggatta gataccctgg 780

[0113] tagtccacgc cgtaaacgat gagtgctaag tgttagaggg gtcacaccct ttagtgctgc 840

[0114] agctaacgg 849

[0115] Microscopic images of the morphological characteristics of strain #HT4 are shown below. Figure 4 As shown in the figure. GeneBank comparison showed that the strain is *Thermoactinomyces vulgaris*, and the strain was subsequently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31846.

[0116] The >16S rDNA sequencing results of this strain are shown in SEQ ID No:4:

[0117] tcggctgtca cttgcggatg ggcccgcggc gcattagctg gttggtgagg taacggctca 240

[0118] ccaaggcgac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300

[0119] ggcccagact cctacggggag gcagcagtag ggaattttcc gcaatgggcg aaagcctgac 360

[0120] ggagcaacgc cgcgtgagtg aggacggcct tcgggttgta aaactctgtt cttgaggaag 420

[0121] aattccttcc aggcgaacag cctgggaggt tgacggtact caaggagaaa gccccggcta 480

[0122] actacgtgcc agcagccgcg gtaatacgta gggggcgagc gttatccgga attattgggc 540

[0123] gtaaagcgcg cgcaggcggc cgattaagtc aggtgtgaaa ggctgcggct caaccgcaga 600

[0124] gcggcacctg aaactggtcg gcttgagtgc aggagagggg agcggaattc ccggtgtagc 660

[0125] ggtggaatgc gtagagatcg ggaggaacac cagtggcgaa ggcggctctc tggcctgtaa 720

[0126] ctgacgctga ggcgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780

[0127] ccgtaaacga tgagtgctag ggggttgggg gggctacgcc cctcagtgcc gaaggtaacc 840

[0128] cattaagcac tccgcctgg 859

[0129] Microscopic images of the morphological characteristics of strain #HT5 are shown below. Figure 5 As shown in the figure. 16S rDNA sequencing and comparison with NCBI GeneBank showed that the strain was *Pallidibacillus thermolactis*, and the strain was subsequently deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 31847.

[0130] The 16S rDNA sequencing result of this strain is shown as SEQ ID No:5:

[0131] ggcttttagc tatcacttac agatgggccc gcggcgcatt agctagttgg tggggtaaag 240

[0132] gcctaccaag gcgacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga 300

[0133] gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat ggacgaaagt 360

[0134] ctgacggagc aacgccgcgt gagcgaagaa ggtcttcgga tcgtaaagct ctgttgttag 420

[0135] ggaagaacaa gtaccggagt aactgacggt accttgacgg tacctaacca gaaagccacg 480

[0136] gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaatcatt 540

[0137] gggcgtaaag cgcgcgcagg cggtccttta agtctgatgt gaaatcttgc ggctcaaccg 600

[0138] taagcggtca ttggaaactg ggggacttga gtgcaggaga ggaaagcgga attccatgtg 660

[0139] tagcggtgaa atgcgtagag atatggagga acaccagtgg cgaaggcggc tttctggcct 720

[0140] gtaactgacg ctgaggcgcg aaagcgtggg gagcaaacag gattagatac cctggtagtc 780

[0141] cacgccgtaa acgatgagtg ctaagtgttg gagggtttcc gcccttcagt gctgcagcta 840

[0142] acgcattaag cactccgcc 859

[0143] Using the above five strains, with the COD removal rate of chemimechanical pulp wastewater as the main evaluation index, the five strains were combined and verified, and finally a composite microbial community for the treatment of chemimechanical pulp wastewater was invented:

[0144] The effective viable count ratio of *Geobacillus thermodenitrificans*, *Calidifermentibacillusshisashii*, *Geobacillus stearothermophilus*, *Thermoactinomyces vulgaris*, and *Pallidibacillus thermolactis* is (10~15%): (20~30%): (20~30%): (10~30%): (10~25%).

[0145] Based on the influent water quality and operating parameters of the biochemical system, the compounding ratio and dosage of different bacterial strains were determined and added to the system, which significantly increased the number and biological activity of effective bacteria within the system. These strains can decompose various organic pollutants in the mechanical pulp wastewater and convert macromolecular pollutants into smaller organic molecules. Simultaneously, through synergistic metabolism among microorganisms, they can degrade recalcitrant or toxic organic compounds in the wastewater, thus creating favorable conditions for subsequent degradation by the microbial strains in the system.

[0146] Adding the above-mentioned composite microbial community to the biochemical system according to the proportion of effective live bacteria can effectively remove COD from chemimechanical slurry wastewater. The microbial community decomposes aged or dead sludge into carbon dioxide and water by secreting a variety of biological enzymes and by means of synergistic metabolism among strains. At the same time, it inhibits the growth of miscellaneous bacteria through competition, ultimately achieving the purpose of removing COD.

[0147] This invention also provides an experimental method for determining the optimal strain ratio of a composite microbial community for treating chemical pulp wastewater, comprising:

[0148] 1. Preparation and fabrication of materials:

[0149] Step S1: Prepare some sterile 10ml centrifuge tubes;

[0150] Step S2: Sterilize a 500ml bottle of sterile water and dispense it into 10ml sterile centrifuge tubes, adding 5ml of sterile water to each centrifuge tube.

[0151] Step S3: Sterilized spreader or yellow sterile pipette tip, 3 sterilized 100ml Erlenmeyer flasks (for dilution).

[0152] Step S4: Incubator and shaker. According to research, the operating temperature range of both the shaker and the incubator can reach above 42℃.

[0153] 2. Experimental steps:

[0154] The above-mentioned bacterial strains were inoculated into their respective culture media and incubated at a constant temperature. The control group was placed in a 37°C incubator. After 2-3 days, when single-clonal colonies had grown, the bacterial isolation on the dilution gradient plates was observed. Endophytic bacteria with good isolation performance were selected by visual inspection. The endophytic bacteria were purified using the streak plating method.

[0155] The corresponding culture medium formulation includes:

[0156] (1) The culture medium formula for Geobacillus thermodenitrificans:

[0157] LB solid medium: peptone 5g / L, yeast extract 2g / L, sodium chloride 5g / L, agar powder 15g / L.

[0158] LB liquid medium: peptone 5g / L, yeast extract 2g / L, sodium chloride 5g / L.

[0159] After preparation, autoclave at 121℃ for 20 minutes; the morphology of the strain after incubation, i.e., electron micrograph, is shown below. Figure 1 As shown.

[0160] (2) The culture medium formula for Calidifermentibacillus hisashii:

[0161] NB solid medium: peptone 15g / L, beef extract 3g / L, agar powder 10g / L.

[0162] NB liquid culture medium: peptone 15g / L, beef extract 3g / L.

[0163] After preparation, autoclave at 121℃ for 20 minutes. The morphology of the cultured strain, i.e., electron micrograph, is shown below. Figure 2 As shown.

[0164] (3) The culture medium formula for Geobacillus stearothermophilus:

[0165] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, agar powder 20 g / L.

[0166] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L.

[0167] After preparation, autoclave at 121℃ for 20 minutes. The morphology of the cultured strain, i.e., electron micrograph, is shown below. Figure 3 As shown.

[0168] (4) The culture medium formula for common thermophilic actinomycetes (Thermoactinomyces vulgaris):

[0169] LB solid medium: 20 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar powder.

[0170] LB liquid medium: peptone 20 g / L, yeast extract 5 g / L, sodium chloride 5 g / L.

[0171] After preparation, autoclave at 121℃ for 20 minutes. The morphology of the cultured strain, i.e., electron micrograph, is shown below. Figure 4 As shown.

[0172] (5) The formulation of the culture medium for Pallidibacillus thermolactis:

[0173] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, agar powder 20 g / L.

[0174] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L.

[0175] After preparation, autoclave at 121℃ for 20 minutes. The morphology of the cultured strain, i.e., electron micrograph, is shown below. Figure 5 As shown.

[0176] This invention also provides a composite microbial agent for treating chemimechanical pulp wastewater, comprising a composite microbial community for treating chemimechanical pulp wastewater with an effective viable count ratio of (10~15%): (20~30%): (20~30%): (10~30%): (10~25%) of *Geobacillus thermodenitrificans*: *Calidifermentibacillus hisashii*: *Geobacillus stearothermophilus*: *Thermoactinomyces vulgaris*: *Pallidibacillus thermolactis*.

[0177] After multiple experiments, two optimal strain ratios were finally determined: the optimal strain ratio of *Geobacillus thermodenitrificans*: *Calidifermentibacillus hisashii*: *Geobacillus stearothermophilus*: *Thermoactinomyces vulgaris*: *Pallidibacillus thermolactis* was 15%: 25%: 20%: 15%: 15%.

[0178] The optimal strain ratio for the following bacteria is: *Geobacillus thermodenitrificans*, *Calidifermentibacillusshisashii*, *Geobacillus stearothermophilus*, *Thermoactinomyces vulgaris*, and *Pallidibacillus thermolactis*: 12%:30%:25%:18%:15%.

[0179] This invention also provides an application of a composite microbial community or a composite microbial agent for treating chemimechanical pulp wastewater. Specifically, it aims to remove COD from the chemimechanical pulp wastewater.

[0180] This invention also provides a method for using a composite microbial community or a composite microbial agent for treating chemimechanical pulp wastewater in chemimechanical pulp wastewater, comprising:

[0181] Add a compound microbial community or compound microbial agent to the biochemical system and mix it with the chemical slurry wastewater.

[0182] The methods of application include:

[0183] The biochemical system temperature should be between 30℃ and 60℃, and the DO value should be greater than 0.5 mg / L.

[0184] The dosing point is at the inlet of the aerobic tank of the biological system, where the dissolved oxygen level is not lower than 0.5 mg / L;

[0185] For every 1 kg of chemical oxygen demand (COD) removed from chemimechanical slurry wastewater, the dosage of microbial flora or compound microbial agents is 0.01 kg to 0.03 kg.

[0186] The optimal temperature for the biochemical system is 40℃, at which the microbial activity is strongest.

[0187] Verification Experiment

[0188] To verify the optimal strain ratio in the technical solution of this invention in the field, two specific verification examples are provided:

[0189] Case 1:

[0190] A pulp and paper manufacturing company in Zhejiang Province uses a chemimechanical pulping process. Its water volume is approximately 20,000 tons per day. The treatment process employs a combined approach: inclined wire mesh + hydrolysis acidification + anaerobic digestion + anaerobic sedimentation tank + A / O + secondary sedimentation tank + coagulation sedimentation + Fenton process. The influent COD from the hydrolysis acidification system is approximately 8000 mg / L, T-N 90 mg / L, and salinity approximately 4000 mg / L. The effluent COD from the anaerobic system is approximately 4000-5000 mg / L, and TN is between 80-90 mg / L. The A / O system has a 48-hour retention time, resulting in a sludge concentration of approximately 7000 mg / L. The biological system temperature is consistently maintained between 39 and 43 degrees Celsius. The effluent from the secondary sedimentation tank has a COD of approximately 3000 mg / L, ammonia nitrogen of approximately 60 mg / L, TN of approximately 80 mg / L, and a color intensity as high as 800 times the standard. The downstream coagulation sedimentation and Fenton processes utilize large quantities of chemical agents, resulting in extremely high costs and a large sludge production.

[0191] On-site preparation: The optimal strain ratio of *Geobacillus thermodenitrificans*, *Calidifermentibacillus hisashii*, *Geobacillus stearothermophilus*, *Thermoactinomyces vulgaris*, and *Pallidibacillus thermomolactis* is 15%:25%:20%:15%:15%. This mixture is added to the biochemical system for chemimechanical slurry wastewater at a dosage of 120 kg of the compound microbial community per day. Photos of the on-site addition are shown below. Figures 6 to 7 As shown in Table 1, the test results are as follows:

[0192] Table 1

[0193]

[0194] Cost: The COD removal rate of the A / O system is increased by 43 percentage points through the technology of this invention, and the cost of downstream deep treatment agents is reduced by 11 yuan / ton of water.

[0195] Case 2:

[0196] A pulp and paper manufacturing company in Guangxi produces approximately 15,000 tons of mixed chemimechanical pulp and some papermaking wastewater per day. The biological treatment system uses a primary sedimentation tank + A / O + secondary sedimentation tank + Fenton process. The COD of the effluent from the primary sedimentation tank is about 2500 mg / L. The A / O system has a retention time of 36 hours, and the system temperature is generally around 40 degrees Celsius. The sludge concentration is 6000 mg / L, and the normal COD of the secondary sedimentation effluent is around 400 mg / L.

[0197] On-site preparation: The optimal strain ratio of *Geobacillus thermodenitrificans*, *Calidifermentibacillus hisashii*, *Geobacillus stearothermophilus*, *Thermoactinomyces vulgaris*, and *Pallidibacillus thermomolactis* is 12%:30%:25%:18%:15%. This mixture is added to the biochemical system for chemimechanical slurry wastewater at a rate of 70 kg of the compound microbial community per day. Photos of the on-site addition are shown below. Figure 8 As shown in Table 2, the operating results are as follows:

[0198] Table 2

[0199]

[0200] In summary, this invention, based on current biochemical treatment methods, employs an optimal ratio of strains from a composite microbial community to significantly increase the number and biological activity of effective bacteria in the biochemical system, thereby greatly improving its COD treatment efficiency and enhancing the system's stability and resilience. Compared to traditional biological treatment technologies, this technology can increase COD removal rates by 20%–50% (traditional treatment achieves approximately 40%, while this invention can increase it to 60%–80%); simultaneously, it significantly reduces operating costs, solving the problem of high investment costs, thus truly achieving a technological revolution in the treatment of chemical pulp wastewater.

[0201] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A composite microbial community for treating chemical pulp wastewater, characterized in that, include: The following bacteria were deposited on September 3, 2024: *Geobacillus thermodenitrificans* (CGMCC No. 31848), *Calidifermentibacillus hisashii* (CGMCC No. 31843), *Geobacillus stearothermophilus* (CGMCC No. 31849), *Thermoactinomyces vulgaris* (CGMCC No. 31846), and *Pallidibacillus thermolactis* (CGMCC No. 31847). All deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The composite microbial community for treating chemical pulp wastewater as described in claim 1, characterized in that, The effective viable count ratio of *Geobacillus thermodenitrificans*, *Calidifermentibacillusshisashii*, *Geobacillus stearothermophilus*, *Thermoactinomyces vulgaris*, and *Pallidibacillus thermolactis* is (10~15%): (20~30%): (20~30%): (10~30%): (10~25%).

3. A composite microbial community for treating chemimechanical pulp wastewater as described in claim 1 or 2, characterized in that, The optimal strain ratio for the following bacteria is: *Geobacillus thermodenitrificans*, *Calidifermentibacillusshisashii*, *Geobacillus stearothermophilus*, *Thermoactinomyces vulgaris*, and *Pallidibacillus thermolactis*: 15%: 25%: 20%: 15%: 15%.

4. A composite microbial community for treating chemical pulp wastewater as described in claim 1 or 2, characterized in that, The optimal strain ratio for the following bacteria is: *Geobacillus thermodenitrificans*, *Calidifermentibacillusshisashii*, *Geobacillus stearothermophilus*, *Thermoactinomyces vulgaris*, and *Pallidibacillus thermolactis*: 12%:30%:25%:18%:15%.

5. A compound microbial agent for treating chemical pulp wastewater, characterized in that, It includes a composite microbial community for treating chemical pulp wastewater as described in any one of claims 1-4.

6. The application of a composite microbial community for treating chemical pulp wastewater as described in any one of claims 1-4, or a composite microbial agent for treating chemical pulp wastewater as described in claim 5, in chemical pulp wastewater.

7. The method of using a composite microbial community for treating chemimechanical pulp wastewater as described in any one of claims 1-4, or a composite microbial agent for treating chemimechanical pulp wastewater as described in claim 5, in chemimechanical pulp wastewater, characterized in that... include: Add a compound microbial community or compound microbial agent to the biochemical system and mix it with the chemical slurry wastewater.

8. The method of use as described in claim 7, characterized in that, The methods of application include: The biochemical system temperature should be between 30℃ and 60℃, and the DO value should be greater than 0.5 mg / L. The dosing point is at the inlet of the aerobic tank of the biological system, where the dissolved oxygen level is not lower than 0.5 mg / L; For every 1 kg of chemical oxygen demand (COD) removed from chemimechanical slurry wastewater, the dosage of microbial flora or compound microbial agents is 0.01 kg to 0.03 kg.

9. The method of use as described in claim 8, characterized in that, The optimal temperature for the biochemical system is 40℃.

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