Strain for waste treatment and use thereof

Abstract

The application relates to a strain for waste treatment and application thereof, and relates to a strain and application thereof in waste treatment. The strain for waste treatment is Streptomyces sp. ZZ10(5) which is preserved in the China General Microbiological Culture Collection Center and has a preservation number of CGMCC No. 25737. The strain has high urease activity, iron carrier production capacity and high-temperature-resistant streptomycete. The Streptomyces sp. ZZ10(5) provided by the application is separated from solid biogas residue after anaerobic fermentation of cow manure and corn straw. On one hand, the strain has high-temperature resistance and salt and alkali resistance; on the other hand, the strain can quickly convert nitrogen in urea, and application of the strain in fermentation of biogas residue for preparation of organic fertilizer can reduce accumulation of nitrate nitrogen in the environment. The application of the Streptomyces sp. ZZ10(5) can realize waste treatment, in particular, biogas residue treatment.

Description

Technical Field

[0001] This invention relates to a bacterial strain and its application in waste treatment. Background Technology

[0002] Biogas residue and other organic wastes are the residues left after crop straw, grass, and livestock manure are fermented by microorganisms in a biogas digester to produce biogas. It consists of some undecomposed raw materials and newly formed microbial communities. It contains macro-elements such as nitrogen, phosphorus, and potassium required for plant growth, organic matter, and medium and trace elements such as copper, iron, manganese, and zinc. It can be used as high-quality fertilizer, cultivation substrate, and livestock feed.

[0003] Currently, in order to accelerate the reduction, resource utilization, and harmless treatment of organic waste such as biogas residue, fermentation agents can be applied to the resource utilization of biogas residue to optimize the fermentation and composting processes, thereby improving the fertilizer efficiency of biogas residue. However, the continuous application of fermentation agents to biogas residue may increase the risk of soil salinity and nitrate nitrogen in groundwater. Existing technologies address this issue by using a combination of chemical fertilizers and biogas fertilizers to reduce the accumulation of nitrate nitrogen in soil and water. Summary of the Invention

[0004] This invention provides a strain for waste treatment and its application. This Streptomyces strain is a thermotolerant Streptomyces with high urease activity and siderophore production ability.

[0005] The strain used in this invention for waste treatment is Streptomyces sp. ZZ10(5), which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 25737.

[0006] Application of the above-mentioned Streptomyces sp. ZZ10(5) in waste treatment.

[0007] Application of the above-mentioned Streptomyces sp. ZZ10(5) in the treatment of biogas residue.

[0008] The Streptomyces sp. ZZ10(5) strain described in this invention forms red colonies with white hyphae when cultured in LB medium. The medium surrounding the colonies does not change color, and it produces intracellular red pigment.

[0009] The Streptomyces sp. ZZ10(5) described in this invention has a temperature growth range of 15–70°C and can tolerate high temperatures of 50–70°C; Streptomyces sp. ZZ10(5) can tolerate 0–5% NaCl (w / v) and a pH range of 6.5–9.5, exhibiting a certain degree of salt and alkali tolerance.

[0010] The urease activity of Streptomyces sp. ZZ10(5) provided by this invention can reach up to 0.85 g / L NH4+. + -N has high urease activity.

[0011] The *Streptomyces* sp. ZZ10(5) provided in this invention is isolated from solid biogas residue after anaerobic fermentation of cow dung and corn straw. *Streptomyces* sp. ZZ10(5) exhibits high temperature and salt tolerance, as well as high urease activity, enabling rapid conversion of nitrogen in urea. Organic fertilizer prepared from biogas residue fermented with *Streptomyces* sp. ZZ10(5) and applied in combination with chemical fertilizers can reduce the accumulation of nitrate nitrogen in the environment. The *Streptomyces* sp. ZZ10(5) described in this invention can be used for waste treatment, particularly biogas residue treatment.

[0012] The Streptomyces sp. ZZ10(5) described in this invention was inoculated onto CAS medium. After a period of cultivation, an orange-yellow halo formed around the colony of Streptomyces sp. ZZ10(5), indicating that Streptomyces could produce siderophores. Siderophore production was measured, showing that the siderophore yield of Streptomyces sp. ZZ10(5) reached 90.42%, demonstrating that Streptomyces sp. ZZ10(5) has a strong siderophore production capacity. Siderophores are low-molecular-weight compounds that can efficiently chelate ferric ions and are secreted extracellularly to capture Fe. 3+ And it is transported across the cell membrane to the cytoplasm, where the iron is released. The Streptomyces sp. ZZ10(5) of this application can occupy iron ions (F... 3+ Iron ions, as a scarce resource in soil, can effectively inhibit the invasion of pathogens. They can effectively inhibit the invasion of pathogens in the plant rhizosphere and inhibit the survival of harmful bacteria such as Escherichia coli and mold in compost.

[0013] The Streptomyces sp. ZZ10(5) of this invention has growth-promoting effects, as well as anti-infective, antifungal, antitumor, and immunosuppressive effects; it also has antibacterial effects, which can be used as target bacteria, and it is not easy to develop drug resistance during use. The strain of this application can play a role in overcoming bacterial drug resistance by antibiotic coupling with siderophores, which is an important direction for the development of new antibiotic drugs.

[0014] The results of secretory system protein prediction indicate that the Streptomyces sp. ZZ10(5) of the present invention has good plant safety.

[0015] The Streptomyces strain of this invention is Streptomyces sp. ZZ10(5), which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 25737 and deposit date of September 16, 2022. Attached Figure Description

[0016] Figure 1 This refers to the utilization of urea by strain Streptomyces sp. ZZ10(5);

[0017] Figure 2 These are morphological photographs of strain ZZ10(5) and Streptomyces anthocyanicus;

[0018] Figure 3 This is a phylogenetic tree constructed based on the 16S rRNA gene sequence of strain ZZ10(5);

[0019] Figure 4 This is the whole genome phylogenetic tree of strain ZZ10(5);

[0020] Figure 5 This is a photograph of a chromium azurite plate of iron carriers produced by Streptomyces sp. ZZ10(5);

[0021] Figure 6 The siderophore production of Streptomyces sp. ZZ10(5);

[0022] Figure 7 The temperature tolerance of Streptomyces sp. ZZ10(5);

[0023] Figure 8 This refers to the alkali resistance of Streptomyces sp. ZZ10(5);

[0024] Figure 9 This refers to the pH tolerance of Streptomyces sp. ZZ10(5);

[0025] Figure 10 The standard curves for ammonia nitrogen and urease activity and cell dry weight of Streptomyces sp. ZZ10(5) are shown. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 are within the scope of protection of the present invention.

[0027] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0028] Specific implementation method one: The strain used for waste treatment in this implementation method is Streptomyces p. ZZ10(5), which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 25737.

[0029] The Streptomyces sp. ZZ10(5) of this embodiment was isolated from the biogas residue sample after an anaerobic biogas project using straw and cow dung as raw materials in December 2021.

[0030] The culture medium for Streptomyces sp. ZZ10(5) of this invention consists of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride (NaCl), with a pH of 7.0. Culture conditions: colonies appear after 48 hours at 28°C; the logarithmic growth phase is reached after one week of culture.

[0031] I. Method for obtaining Streptomyces sp. ZZ10(5):

[0032] In December 2021, 1g of solid biogas residue from anaerobic fermentation of cow dung and corn straw was weighed and added to 50ml of urea solution (5mol / L). The solution was incubated at 28℃ and 150r / min for 24h with shaking to obtain an enriched solution. Using the dilution plating method, the enriched solution was diluted and plated onto urease screening medium [sodium chloride 5g / L, potassium dihydrogen phosphate 2g / L, glucose 0.1g / L, 0.2% phenol red solution (4ml / L), urea 2%, pH 6.8, agar 18g / L]. (Note: Urea was first dissolved in sterile water, then sterilized using a 0.45μm microporous membrane. After the medium temperature dropped to 50-60℃, the urea was thoroughly mixed with the medium before being poured into plates). The plates were inverted in a 28℃ incubator and incubated statically for at least one week. Colonies that grew fastest and turned the medium red in the shortest time were selected. The utilization of urea by strain Streptomyces sp. ZZ10(5) is as follows: Figure 1 As shown, where Figure 1 As shown in Figure a, the dominant strain ZZ10(5) was selected, which could cause the culture medium to turn red after 24 hours of cultivation. Figure 1 (b) After 48 hours, the culture medium turned completely red. Figure 1 (c)

[0033] II. Identification of strain ZZ10(5)

[0034] Morphological photographs of strain ZZ10(5) and Streptomyces anthocyanicus are shown below. Figure 2 As shown, Figure 2 In the image, a represents the colony morphology of strain ZZ10(5) on LB medium, b represents the morphology of strain ZZ10(5) on Gao's No. 1 medium, c represents the state of strain ZZ10(5) in LB liquid medium, and d represents the colony morphology of *Streptomyces anthocyanicus* on LB medium; from... Figure 2 It can be seen that strain ZZ10(5) forms red colonies with white hyphae in LB medium, and the surrounding medium does not change color, preliminarily indicating that it produces intracellular red pigment. The state of strain ZZ10(5) in LB liquid medium, where the medium does not turn red, indicates that strain ZZ10(5) produces a non-water-soluble pigment. This characteristic is significantly different from its close relative, *Streptomyces anthocyanicus*, which produces a blue pigment.

[0035] The 16S rRNA gene sequence of strain ZZ10(5) was amplified and sequenced using universal bacterial primers 27F and 1492R, yielding a 1407 bp target fragment. BlastN alignment revealed that this sequence showed the highest homology with members of the genus *Streptomyces*, confirming that strain ZZ10(5) is *Streptomyces*, and the genome of this strain was subsequently sequenced. The 16S rRNA gene sequence of strain ZZ10(5) is shown in SEQ ID NO: 1.

[0036] Phylogenetic tree of strain ZZ10(5) based on 16S rRNA gene sequence ( Figure 3 ) and phylogenetic tree of whole genome sequence ( Figure 4 The results showed that the closest relative of strain ZZ10(5) was Streptomyces anthocyanicus, but the morphological characteristics of the patented strain were completely different from those of Streptomyces anthocyanicus (producing different pigments).

[0037] III. The predicted results of the secretory system proteins of Streptomyces sp. ZZ10(5) are shown in Table 1. Pathogens secrete these proteins into extracellular or host cells through the secretory system TNSS (currently identified as 7 types, type I-VII), causing pathological reactions by controlling immune responses and cell death. The predicted results of Streptomyces sp. ZZ10(5) in this invention are shown in the table below, indicating that Streptomyces sp. ZZ10(5) is a non-pathogenic fungus. Among the ten currently reported plant pathogenic Streptomyces species (https: / / encyclopedia.thefreedictionary.com / Streptomyces), the closely related species of strain ZZ10(5) is not Streptomyces anthocyanicus NBRC 14892. T This indicates that Streptomyces sp. ZZ10(5) has good plant safety.

[0038] Table 1 TNSS Prediction Results

[0039]

[0040] Example 1: Detection of siderophores in Streptomyces p. ZZ10(5) according to the Chromeazurol (CAS) assay.

[0041] Among them, the chromium azurite plate photograph of the iron carrier produced by Streptomyces sp. ZZ10(5) is shown in the figure. Figure 5 As shown, Figure 5 An orange-yellow halo formed around the colony in the culture medium, which qualitatively indicated that Streptomyces sp. ZZ10(5) could produce siderophores.

[0042] In the CAS liquid assay, Streptomyces sp. ZZ10(5) was inoculated onto LB plates and incubated at 28°C for 10 days. After the spores matured, they were gently scraped off with a sterile inoculation loop and diluted with sterile water to a concentration of 10. 8 A spore suspension with a spore count of 1 / ml was used as the inoculum. This spore suspension was inoculated at a 1% inoculum volume into iron-free LB liquid medium and cultured at 28℃ and 180 rpm for 8 days until the logarithmic growth phase (the growth curve of the strain was determined in the early stage) to obtain the fermentation broth. The fermentation broth was centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.45 μm microporous membrane to obtain sterile fermentation filtrate. The fermentation filtrate was mixed with an equal volume of CAS detection solution, allowed to stand at room temperature for 1 h, and the OD was measured using a spectrophotometer.680 The siderophore production of Streptomyces sp. ZZ10(5) was calculated. The siderophore production of Streptomyces sp. ZZ10(5) is as follows: Figure 6 As shown, during the logarithmic growth phase, siderophore production can reach 90.42%. The *Streptomyces* p. ZZ10(5) described in this embodiment has excellent siderophore production capabilities. Some beneficial microorganisms in the soil have inhibitory effects on pathogens, while others have indirect effects. The strain described in this application can play this indirect role, because the soil environment itself contains iron ions (F... 3+ The limitation of ) means that if the Streptomyces of this application occupies (F) in advance. 3+ This scarce resource will effectively inhibit the invasion of pathogens. It will effectively inhibit the invasion of pathogens in the plant rhizosphere and also inhibit the survival of harmful bacteria (E. coli, mold) in compost.

[0043] Example 2: High Temperature Resistance and Choline Resistance Test

[0044] After 8 days of shaking culture in LB liquid medium at different constant temperatures, the *Streptomyces* sp. ZZ10(5) of this invention was used to determine the dry weight of the fermentation broth. The temperature tolerance of *Streptomyces* sp. ZZ10(5) was as follows: Figure 7 As shown, the temperature growth range of this strain is 15-70℃, and it can tolerate high temperatures of 50-70℃. Streptomyces sp. ZZ10(5) is a thermotolerant bacterium.

[0045] in Figure 8 The alkali resistance of Streptomyces sp. ZZ10(5) is described. Figure 9 pH tolerance of Streptomyces sp. ZZ10(5) was measured from pH 8 and... Figure 9 It can be seen that Streptomyces sp. ZZ10(5) can tolerate 0-5% NaCl (w / v) and a pH range of 6.5-9.5. The Streptomyces sp. ZZ10(5) described in this invention also has excellent salt and alkali resistance.

[0046] Example 3 Urease Activity Detection

[0047] Streptomyces sp. ZZ10(5) was inoculated into LB fermentation medium and fermented at 28°C and 150 rpm for 7 days to achieve a Streptomyces concentration of 10. 10cfu / ml. The urease activity of Streptomyces was determined using the concentration of ammonia nitrogen released from urea decomposition as the standard. The ammonia nitrogen concentration was calculated by colorimetric determination at a wavelength of 625 nm using the indophenol blue reaction. The standard curves for ammonia nitrogen and the curves for urease activity and cell dry weight of Streptomyces sp. ZZ10(5) are shown below. Figure 10 As shown, where a is NH4 + -N is the standard curve, b is the curve of urease activity and cell dry weight. The urease activity of Streptomyces sp. ZZ10(5) can reach as high as 0.85 g / L NH4. + -N. It can be seen that Streptomyces sp. ZZ10(5) has high urease activity.

[0048] Example 4: Treatment of biogas residue by Streptomyces sp. ZZ10 (5).

[0049] The specific methods for treating biogas residue are as follows:

[0050] I. Raw materials: crushed straw segments (3-5cm), biogas residue with a moisture content of less than 50% after anaerobic fermentation in biogas projects, urea, and Streptomyces sp. ZZ10(5) bacterial liquid with a spore count of more than 200 million / ml;

[0051] 2. Mix urea, straw, biogas residue and urea in a dry weight ratio of 1:20:100 to obtain a mixed material. Inoculate the mixed material with Streptomyces inoculum at 1% dry weight. The total moisture content of the mixed material is controlled at 60%-65%.

[0052] In this embodiment, the biogas residue composting process can rapidly raise the temperature to over 60 degrees Celsius during the initial stage (3-5 days). At this point, most bacteria and fungi die or have low activity due to the high temperature. Because the *Streptomyces* strain of this invention exhibits resistance to high temperatures and salinity, and its logarithmic growth phase occurs late, a secondary temperature rise occurs around 7-10 days of composting. This reactivates enzymes in the compost that function at high temperatures, such as cellulase and urease, thus shortening the overall composting process by 3-5 days. This achieves the treatment of biogas residue.

Claims

1. A strain for waste treatment, characterized in that... The strain used for waste treatment is Streptomyces sp. ZZ10(5), which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.25737.

2. The application of the strain described in claim 1 in the treatment of biogas residue.

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