CN113214999A - Geotrichum TN42 and application thereof in sewage treatment - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to the technical field of microbial sewage treatment. The invention discloses a Geotrichum (Geotrichum sp) TN42 with the preservation number of CGMCC No. 20967. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 29.10.2020 for short, the address is No. 3 of West Lu No.1 of Beijing Korean district, and the preservation number of the strain is CGMCC No. 20967. The Geotrichum (Geotrichum sp.) TN42 disclosed by the invention has the degradation effect on ammonia nitrogen, total nitrogen and COD, and is high in removal rate.

Description

Geotrichum TN42 and application thereof in sewage treatment

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to the technical field of microbial sewage treatment.

Background

Ammonia nitrogen in sewage is a nutrient in a water body, can cause water eutrophication, is a main oxygen-consuming pollutant in the water body, and is toxic to fishes and some aquatic organisms, so that research on efficient denitrification of sewage is always a research hotspot in the field of sewage treatment. .

Biological sewage denitrification, particularly microbial sewage treatment, has been widely studied and applied in recent years as a green, efficient and low-cost treatment method. The method has the core problem of research and development of high-efficiency denitrification strains, and realizes the denitrification of the sewage by efficiently converting and degrading ammonia nitrogen in the sewage through the physiological metabolic process of functional strains. At present, the microbial strain resources applied to the sewage denitrification treatment are mainly bacteria, such as pseudomonas, bacillus, alcaligenes, paracoccus and the like, and fungi which are another important component of the microorganisms, only a few strain resources which can be used for the denitrification treatment, such as hansenula grapevine, rhizobia and the like, are excavated.

Compared with bacteria, the fungal denitrification is usually more excellent in aspects of nitrification and denitrification rate, resistance to inhibitory compounds, improvement of impact load resistance of denitrification process and the like (Guest et al.2002), so that the excavation of more fungal strain resources for efficient biological denitrification has important significance for promoting the improvement of the efficiency of the traditional biological denitrification process and the research and application of novel biological denitrification process.

Disclosure of Invention

The first purpose of the invention is to provide a Geotrichum (Geotrichum sp.) TN 42. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 29.10.2020 for short, the address is No. 3 of West Lu No.1 of Beijing Korean district, and the preservation number of the strain is CGMCC No. 20967.

The Geotrichum (Geotrichum sp.) TN42 provided by the invention is a high-efficiency denitrification fungus, is applied to the field of sewage microbial treatment, has a good effect, has a good degradation effect on ammonia nitrogen, and has an obvious removal effect on COD (chemical oxygen demand) and total nitrogen.

Drawings

FIG. 1 is a Geotrichum sp TN42 morphology.

Wherein A is the strain morphology of the positive side on the PDA culture medium, B is the morphology of the hyphae under a stereomicroscope of 50 times, C is the morphology of the hyphae under an optical microscope of 80 times, and D is the morphology of the spores under an optical microscope of 10 times 40 times.

FIG. 2 is a phylogenetic tree diagram of Geotrichum sp (Geotrichum sp.) TN42 constructed based on LSU sequence homology of 26s rDNA.

FIG. 3 is a graph showing the 24-hour degradation of Geotrichum sp TN42 on ammonia nitrogen.

FIG. 4 is a graph showing the effect of Geotrichum sp TN42 on COD degradation in domestic wastewater.

FIG. 5 is a graph showing the effect of Geotrichum sp TN42 on the degradation of total nitrogen in domestic wastewater.

FIG. 6 is a graph showing the effect of Geotrichum sp TN42 on the degradation of ammonia nitrogen in domestic wastewater.

Detailed Description

The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.

Example 1

Breeding, separating and identifying the high-efficiency ammonia nitrogen degrading strain:

10g of a mixture of pig manure, cow manure, sheep manure and chicken manure which is collected from Beijing Daxing (collection person: YupengGao, collection time: 2019) of a certain farmer is mixed according to a ratio of 1:1:1:1, and then is subjected to shaking table natural fermentation for 12 hours at a temperature of 200rpm in 100mL of sterile water, 5mL of fermentation liquor is inoculated into screening and domesticating culture media A and B, the mixture is subjected to shaking table cultivation for 3 days at a temperature of 30 ℃ and 200rpm, 50mg/L of ammonium sulfate is supplemented every 12 hours, 5mL of culture solution is inoculated into a new screening and domesticating culture medium every 3 days, and the culture is repeatedly carried out for 10 generations.

Screening and domesticating a culture medium A: 30g of sucrose, 2g of ammonium sulfate, 0.5g of sodium chloride, 0.01g of ferrous sulfate heptahydrate, 1g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate and deionized water with constant volume of 1L, subpackaging in 250ml triangular bottles with 100ml of each bottle, and sterilizing at 115 ℃ for 30 min.

Screening and domesticating a culture medium B: 5g of glucose, 2g of ammonium sulfate, 2.0g of sodium chloride, 0.04g of ferrous sulfate heptahydrate, 1g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate and deionized water to a constant volume of 1L, subpackaging in 250ml triangular bottles, 100ml of each bottle, and sterilizing at 115 ℃ for 30 min.

Taking culture solution after 10 generations of screening and domestication according to the proportion of 10^-3-10^-7The different proportion of the dilution, each gradient takes 100 mul of dilution liquid to spread on the purification culture medium, the culture is carried out for 24-48h at 30 ℃, the growth condition of the colony is observed, the colony with fast growth and large number is selected to be purified by adopting a multi-time partition marking method, and the purified single colony is frozen and preserved for later use.

And (3) purifying a culture medium: 10g of glucose, 5g of peptone, 0.05g of ferrous sulfate heptahydrate, 1g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 15.0g of agar and deionized water to a constant volume of 1L, wherein the pH value is natural. Sterilizing at 115 deg.C for 30 min. Temporarily, every 100ml of culture medium is added with 1% aqueous solution of mengladesh and 1% streptomycin solution.

The bacterial strain with high-efficiency ammonia nitrogen degradation effect is obtained through the separation and purification process, the colony morphology of the bacterial strain on a Potato Dextrose Agar (PDA) culture medium at the temperature of 25-35 ℃ is characterized by white powder and villus, the surface of the colony is flat and dry, the bacterial strain is not easy to pick up, and the circular radiation of the colony grows outwards; a small number of separated short hyphae and a large number of clustered round or oval microconidia were observed as shown in FIG. 1.

Potato Dextrose Agar (PDA) medium: 200g of potatoes, 16g of glucose, 20g of agar and deionized water to reach the constant volume of 1L. Sterilizing at 115 deg.C for 30 min.

And carrying out molecular biological identification on the obtained strain, and carrying out sequencing comparison after carrying out PCR amplification on an ITS sequence and an LSU sequence of the fungus.

An amplification primer:

ITS1：5'-TCCGTAGGTGAACCTGCGG-3'

ITS4：5'-TCCTCCGCTTATTGATATGC-3'

NL1：5'-GCATATCAATAAGCGGAGGAAAAG-3'

NL4：5'-GGTCCGTGTTTCAAGACGG-3'

the reaction system is as follows: 10 XBuffer 2 uL, 2.5mM dNTP 1.5 uL, Primer 11 uL, Primer 21 uL, template 1 uL, enzyme 0.3 uL, water 13.2 uL, total volume 20 uL;

the ITS amplification PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30sec at 30 cycles, annealing at 54 ℃ for 30sec, extension at 72 ℃ for 40sec, extension at 72 ℃ for 10min, and incubation at 4 ℃ for forever.

The LSU amplification PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30sec at 30 cycles, annealing at 53 ℃ for 30sec, extension at 72 ℃ for 50sec, extension at 72 ℃ for 10min, and incubation at 4 ℃ for forever.

And (3) detecting the PCR product by agarose gel electrophoresis, and sequencing the Beijing Huada gene.

The ITS sequence (SEQ ID NO.1) and the LSU sequence (SEQ ID NO.2) after sequencing are subjected to Blast alignment in NCBI database, and the ITS sequence alignment result shows that the homology with Geotrichum silvicola NR _077071.1 is the highest and reaches 92.96%. Further, the LSU sequence alignment result shows that the homology with Geotrichum silvicola NG _060622.1 is the highest and reaches 98.90%, the first 15 sequences with the highest homology are selected according to the LSU alignment result to establish a phylogenetic tree (as shown in FIG. 2), and the strain is named Geotrichum sp TN42 and is abbreviated as strain TN42 in the application in combination with morphological identification.

Geotrichum sp (Geotrichum sp.) TN42 has been deposited in China general microbiological culture Collection center (CGMCC) at 29.10.2020, abbreviated as CGMCC, with the accession number of CGMCC No. 3, which is deposited at the national institute of Soy 1, North Chen of the Chaoyang district, Beijing, and the accession number of CGMCC No. 20967.

Example 2

Under the laboratory simulation condition, the 24-hour degradation curve of the strain TN42 on ammonia nitrogen is as follows:

the ammonia nitrogen detection method comprises the following steps: salicylic acid spectrophotometry (see national standard HJ 536-2009).

Selecting single-strain hyphae and spores of a PDA (potato dextrose agar) plate, inoculating the hyphae and the spores into a PDB liquid test tube containing 5mL, carrying out shake culture at 30 ℃ and 200rpm, activating for 24h, inoculating TN42 seed solution which is activated for 24h into a PDB culture medium containing ammonium sulfate according to the volume fraction of 2% under the aseptic condition, setting the culture condition to be 30 ℃ and 200rpm, sampling every 3h to detect the ammonia nitrogen content, and repeating the experiment for 3 times.

Potato Dextrose (PDB) medium: 20g of glucose, 200g of potatoes and deionized water to a constant volume of 1L. Sterilizing at 115 deg.C for 30 min. Ammonium sulfate was added at an initial concentration of 100mg/L for filter sterilization.

The experimental results (table 1) show that the initial ammonia nitrogen concentration is about 180mg/L, the ammonia nitrogen removal rate after the bacterial strain is inoculated for 24 hours is up to about 100%, and the ammonia nitrogen removal rate after the bacterial strain is inoculated for 18 hours can be up to more than 95%.

TABLE 1

The experimental results show that the strain has obvious ammonia nitrogen removal capability and has great application potential in the microbial treatment process of sewage deamination.

Example 3

In practical application, the strain TN42 has the following degradation effect on domestic sewage:

domestic sewage is collected from a certain municipal sewage treatment plant, glucose is added to adjust the carbon-nitrogen ratio to be 20, and the obtained product is used for degradation experiments of the strain after high-temperature and high-pressure sterilization. Selecting flat single-strain hypha and spores, inoculating the flat single-strain hypha and the spores into a 5mL PDB liquid test tube, shaking at 30 ℃, shaking at 200rpm, activating for 24h, inoculating TN42 seed solution for activating for 24h into domestic sewage according to the volume fraction of 1% under the aseptic condition, setting the culture condition to be 30 ℃ and 200rpm, sampling every 12h to detect the content of COD, ammonia nitrogen and total nitrogen in the domestic sewage, and repeating the experiment for 3 times. .

The ammonia nitrogen detection method comprises the following steps: salicylic acid spectrophotometry (see standard HJ 536-2009).

The COD detection method comprises the following steps: potassium dichromate oxidation method (see standard HJ 828-2017 for specific detection method).

The total nitrogen detection method comprises the following steps: alkaline potassium persulfate digestion ultraviolet spectrophotometry (the specific detection method is shown in the standard GB/T11894-1989).

The experimental results (table 2) show that the initial COD, ammonia nitrogen and total nitrogen content mean values of the domestic sewage are 1655mg/L, 70mg/L and 67.3mg/L respectively, and after aerobic treatment for 24 hours by using the strain TN42, the experimental results show that the COD degradation rate in the domestic sewage in the process is 96.7%, the ammonia nitrogen degradation rate is 100% and the total nitrogen removal rate is 58.5%.

TABLE 2

The experimental results show that the strain has good ammonia nitrogen and COD removal capability in the sewage, and has good removal effect on total nitrogen, so the strain has great application potential in the microbial treatment process of the sewage.

Sequence listing

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<120> geotrichum TN42 and application thereof in sewage treatment

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gttctcgtat cgatgaagaa cgcagcgaaa cgcgatattt cttgtgaatt gcagaagtga 180

atcatcagtt tttgaacgca cattgcactt tggggtatcc cccaaagtat acttgtttga 240

gcgttgtttc tctcttggaa ttgctttgct cttctaaaat ttcgaatcaa attcgtttga 300

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tgaaatcggc caccaggtcg agttgtaatt tgtagattgt atcttgagag cggattaaag 120

tctgttggaa cacagcgcct tagagggtga cagccccgta aaatctattc tcattgtaag 180

atactttcga agagtcgagt tgtttgggaa tgcagctcta agtgggaggt aaattccttc 240

taaagctaaa tattgacgag agaccgatag cgaacaagta ctgtgaagga aagatgaaaa 300

gcactttgaa aagagagtga aaaagtacgt gaaattgtta aaagggaagg gtattgaatc 360

agacttggtg ctgttgttca actgtgtttt ggcacagtgt actcagcagt actaggccaa 420

ggtggggtgt ttgggagtga aaaagaagtt ggaacgtaac tcttcggagt gttatagcct 480

actttcatag ctcctcaggc gcctcaggac tgcgcttcgg caaggacctt ggcataatga 540

ttctataccg cccgtct 557

Claims (4)

1. A Geotrichum (Geotrichum sp.) TN42 with the preservation number of CGMCC No. 20967.

2. A microbial preparation, the active ingredient of which is Geotrichum sp TN42 according to claim 1.

3. Use of a Geotrichum (Geotrichum sp.) TN42 for removing ammonia nitrogen, total nitrogen or/and COD from sewage according to claim 1.

4. The use according to claim 3, characterized in that the sewage is sewage with ammonia nitrogen or/and COD exceeding the standard.

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