WO2020134728A1 - 嗜盐反硝化菌yl5-2及其应用 - Google Patents

嗜盐反硝化菌yl5-2及其应用 Download PDF

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WO2020134728A1
WO2020134728A1 PCT/CN2019/119640 CN2019119640W WO2020134728A1 WO 2020134728 A1 WO2020134728 A1 WO 2020134728A1 CN 2019119640 W CN2019119640 W CN 2019119640W WO 2020134728 A1 WO2020134728 A1 WO 2020134728A1
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denitrifying bacteria
halophilic
salt
salinity
halovibrio
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徐军
孙文妮
张璐璐
王开春
田凤蓉
李坤
王强
洪磊
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中蓝连海设计研究院有限公司
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    • C02F2103/08—Seawater, e.g. for desalination

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  • the invention relates to the field of environmental protection microorganisms, in particular to halophilic denitrifying bacteria YL5-2 and its application.
  • Nitrogen pollution is one of the important causes of eutrophication of water.
  • Denitrification treatment of wastewater plays an important role in maintaining the quality of water environment and preventing eutrophication of water bodies.
  • the nitrogen-containing wastewater discharged from petroleum, chemical, food processing, chemical fertilizer and other industries has the characteristics of high salt.
  • the salt content of some wastewater generated by chemical and food processing even exceeds that of seawater.
  • Traditional biological methods have advantages in the treatment of low-salinity nitrogen-containing wastewater, but when the wastewater concentration is too high, it will inhibit the metabolism of denitrifying microorganisms.
  • the existence of salt-tolerant and halophilic denitrification bacteria provides theoretical possibilities for biological denitrification of high-salt wastewater.
  • Biological denitrifying bacteria include nitrifying bacteria and denitrifying bacteria.
  • Biological denitrification bacteria under high-salt wastewater conditions can also be divided into nitrifying bacteria and denitrifying bacteria.
  • Denitrifying bacteria are a group of bacteria that can reduce nitrate or compressed nitrate to gaseous nitrogen compounds. Therefore, screening, isolating and cultivating salt-tolerant and halophilic nitrifying bacteria and denitrifying bacteria from the environment has become the key to solving the problem of biological nitrogen removal in high-salt wastewater.
  • a moderately halophilic denitrifying bacteria was isolated from the wastewater purification unit, belonging to the genus Halomonas. Its optimal metabolic growth conditions are temperature 30°C, salinity 100g/L, pH 7.5 ⁇ 8.5, and C/N ratio 4: 1.
  • halophilic denitrifying bacteria there are few reports on halophilic denitrifying bacteria in the prior art, especially halophilic denitrifying bacteria under the condition of salinity> 10%. Obtaining halophilic denitrifying bacteria with a salt tolerance capacity greater than 10% from the environment is a technical problem that needs to be solved in the biological nitrogen removal of such wastewater.
  • the purpose of the present invention is to provide a halophilic denitrifying bacteria for solving the problem of biological denitrification under high-salt conditions, especially for high-salt wastewater under conditions of salinity greater than 10%, which can provide salinity of 10% Under the above conditions, denitrification is performed using NO 3 -N as an electron acceptor.
  • Another object of the present invention is to provide the application of the halophilic denitrifying bacteria.
  • the halophilic denitrifying bacteria YL5-2 disclosed in the present invention is deposited in the Deposit Management Center of China General Microorganisms, and its deposit number is CGMCC NO.16315, and the deposit date is August 20, 2018. Based on 16SrRNA phylogenetic analysis, genome sequencing, DNA hybridization test, fatty acid composition, respiratory quinone category, physiological and biochemical and phenotypic characteristics, it can be determined that strain YL5-2 is a new species of the genus Halovibrio, named Halovibrio salipaludis sp.nov.
  • GenBank/EMBL/DDBJ accession number of the 16S rRNA sequence of the halophilic denitrifying bacteria YL5-2 disclosed by the present invention is MF782425, and the nucleotide sequence is shown in SEQ ID NO.1; GenBank/EMBL/DDBJ of the whole genome sequence The login number is NSKD00000000.1.
  • the halophilic denitrifying bacteria YL5-2 disclosed in the present invention are Gram-negative, facultative aerobic, straight rod-shaped or 0.5-0.8 ⁇ m ⁇ 1.0-3.5 ⁇ m small vibrio traits, and through unipolar flagella movement, The colonies on its solid medium are smooth and light yellow.
  • the Halobacterium halobrio sp.YL5-2 of the present invention can grow in a salt concentration of 3% to 32%, a pH of 6.5 to 11.0, and a temperature of 15 to 45°C; the optimal growth salt concentration is 10% to 25%.
  • the suitable growth pH is 7.5 ⁇ 8.0, and the optimum growth temperature is 30 ⁇ 35°C.
  • the main respiratory quinone of Halovibrio sp.YL5-2 of the present invention is Q-9
  • the main fatty acids are C18:1 ⁇ 9c, C16:0,C19:0 cyclo ⁇ 8c and SummedFeature 8
  • the main polar lipid is Diphosphatidylglycerol (DPG), Phosphatidylethanolamine (PE), Phosphatidylglycerol (PG), Phosphatidylcholine (PC).
  • DPG Diphosphatidylglycerol
  • PE Phosphatidylethanolamine
  • PG Phosphatidylglycerol
  • PC Phosphatidylcholine
  • the Halobrio sp. YL5-2 of the present invention can utilize bromo-succinic acid, propionic acid and acetic acid as the sole carbon source; however, it cannot utilize D-maltose, D-fructose, D-galactose, D-cellobiose , Stachyose, D-melose, N-acetyl-D-galactosamine, D-fucose, L-rhamnose, D-mannitol, D-galacturonic acid, D-aspartame Acid, D-serine, D-glucuronic acid, L-lactic acid, quinic acid, mucic acid, D-malic acid, ⁇ -amino-butyric acid, formic acid, acetoacetic acid as the sole carbon source.
  • the present invention has discovered a new species of the genus Halovibrio, named Halovibrio salipaludis sp.nov.
  • the strain is a facultative or aerobic denitrifying bacteria, its salt tolerance range is 3% ⁇ 32%, and it can degrade many pollutants in wastewater within the salt concentration of 5% ⁇ 25%.
  • the halophilic denitrifying bacteria YL5-2 disclosed in the present invention can be used in the process of degradation, transformation and biological denitrification of pollutants under high-salt conditions, including treatment of high-salt wastewater, treatment of polluted seawater, repair of saline-alkali land, consumption of nitrogen nutrients, and suppression Algae overproliferate, purify water, improve bottom quality, etc.
  • Fig. 2 is a polar lipid map of Halovibrio sp.YL5-2(a) and Halovibrio denitrificans DSM15503(b), Halovibrio variabilis DSM 3050(c).
  • DPG diphosphatidylglycerol
  • PG phosphatidylglycerol
  • AL Aminolipid
  • PL Phospholipid
  • PE Phosphatidylethanolamine
  • PGNL Phosphoaminoglycolipid
  • f first dimension
  • s second dimension
  • Fig. 3 is a 16S rRNA-based phylogenetic tree constructed by Halovibrio sp.YL5-2 and Halovibrio denitrificans DSM15503.
  • Figure 4 is a phylogenetic tree constructed by the Halobrio sp. YL5-2 and Halovibrio variabilis DSM 3050T and other strains based on the 16S rRNA maximum parsimony method (MP).
  • Fig. 5 is a phylogenetic tree diagram of Halovibrio sp.YL5-2 constructed based on GGD matrix.
  • Example 1 Isolation and preservation of the halophilic denitrifying bacteria
  • the halophilic denitrifying bacterium YL5-2 was isolated from the sedimentary soil of Qarhan Chaerhan Salt Lake (36°51′N, 94°95′E) in Qinghai province, China.
  • the water of the Qarhan Salt Lake is saturated or close to saturated.
  • LB liquid medium with NaCl concentration of 20% was added, and 250mg/L of glycerol, 250mg/L of glucose, and 50mg/L of methanol were added, and the sedimentary soil of Chaerhan Salt Lake was enriched and cultivated at 30°C for 48h.
  • the strains in the enriched culture liquid were separated using YL solid medium.
  • the 1L medium contains the following components: glucose: 0.6g, trisodium citrate 0.5g, glycerin 2mL, yeast extract 0.8g, peptone 1.6g, dipotassium hydrogen phosphate 0.35g, potassium dihydrogen phosphate 0.1g, ammonium sulfate 0.25g, ammonium chloride 0.25g, MgSO 4 0.5g, CaCl 2 0.1g, NaCl 180g; Microelements SL-4 10mL, pH 7.0-7.2; 2.5% agar.
  • strain YL5-2 is deposited in the China General Microbial Culture Collection Management Center, with the deposit number CGMCC NO.16315, and the deposit date is August 20, 2018.
  • the genomic DNA of strain YL5-2 was extracted using TaKaRa kit (TaKaRa MiniBEST Bacteria Genomic DNA Extraction 68 Kit Ver. 3.0).
  • 16S rRNA amplification uses universal bacterial primers 27F (5'-AGAGTTTGATCMTGGCTCA G-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). PCR sequencing was commissioned by Shanghai Shengong Biotechnology Co., Ltd. The complete 16S rRNA sequence of strain YL5-2 is 1518bp, as shown in SEQ ID NO.1, and the GenBank accession number is MF782425.
  • the whole-genome sequencing of the strain YL5-2 adopts the Illumina MiSeq 2000 high-throughput sequencing platform of Shanghai Shanghai Sensen Biotechnology Co., Ltd.
  • the original sequencing data was filtered and corrected using PRINSEQ (version number v0.20.4) software, and then using SOAP with denovo software (version number v1.05) software with default parameters for base pairing of the genome, and then using CheckM software (version 1.03) Assess the integrity of the genome.
  • the protein coding open reading frame was predicted using Glimmer software (version 1.2).
  • RNA prediction uses RNAmmer software (version 1.2).
  • the whole genome sequence of strain YL5-2 is 3,495,096bp, and the GenBank accession number is NSKD00000000.1.
  • the DNA-DNA hybridization test was carried out by the vibrio haloibrio model strain with the closest genetic development to the strain YL5-2. This method was proposed by De Ley equal to 1970, and the DNA hybridization value (dDDH) was obtained by using the second mode (version number 2.0) of GGDC software to perform one-to-one comparison of gene sequences.
  • DNA test and analysis results show that the DNA-DNA hybridization values of strains YL5-2 and Halovibrio variabilis DSM 3050 T and Halovibrio denitrificans DSM15503 T are 43.5% and 38.2%, respectively, far below the threshold of 70% (species classification is generally accepted Threshold).
  • the average nucleotide identity value was obtained by repeating the topology test 1000 times using the base group of the whole genome sequence. This method was proposed by Goris equal to 2007, and the software used was MUMmer (version number 3.23) and Jspecies (version number 1.2.1). Based on the ANI threshold range (95-96%) of the species classification proposed by Kim et al. and Richter et al., ANI analysis was performed on the genome of strain YL5-2 and the genome closely related to it in GenBank (Table 1).
  • strain YL5-2 had the highest average nucleotide identity (ANI) value with Tamilnaduibacter alinus Mi 7 at 88.5% (Supplementary Table S1), which is a new species of strain YL5-2 T belonging to the genus Halovibrio Provided an argument, named Halovibrio salipaludis sp.nov.
  • ANI nucleotide identity
  • Table 1 Average nucleotide identity (ANI) and DDH values between the YL5-2 T strain and closely related genomes in GenBank.
  • TEM transmission electron microscope
  • Oxidase activity Using an oxidase kit (bioMerieux), catalase activity was determined by pouring a 3.0% H 2 O 2 solution into bacterial colonies and observing the generation of bubbles.
  • the temperature growth conditions were carried out on YL liquid agar medium, the temperatures were 4, 10, 15, 20, 25, 30, 33, 37, 40, 45 and 50°C, and the pH was constant at 7.5, comparing strain YL5- at different temperatures
  • the growth rate of 2 T determines its optimal growth temperature.
  • Salt tolerance was performed on YL agar and YL liquid medium with 0.0-30.0% NaCl (w/v). Adjust the pH to 5.0, 5.5, 6.0, 7.0, 8.0, 9.0 with buffer (Na 2 HPO 4 /NaH 2 PO 4 (pH 5.0-7.0), Na 2 CO 3 /NaHCO 3 (pH 8.0-12.0)) 10.0 and 11.0 (15.0% NaCl, 35°C) to determine the pH range suitable for growth.
  • buffer Na 2 HPO 4 /NaH 2 PO 4 (pH 5.0-7.0), Na 2 CO 3 /NaHCO 3 (pH 8.0-12.0)
  • Carbon source utilization ability and enzyme activity test adopt API 20NE, API ZYM (bioMérieux) and Biolog GENIII microplate. All tests were seeded with pre-grown cells on YL medium and diluted with the relevant seeding medium.
  • the halophilic denitrifying bacteria YL5-2 is Gram-negative, alkaline aerobic, straight rod-shaped or 0.5-0.8x1.0-3.5 ⁇ m in shape of small vibrio, and moves through unipolar flagella ( Figure 1) .
  • Strain YL5-2 grows with acetic acid under aerobic conditions and nitrate (API 20NE) under anoxic conditions.
  • Cellular fatty acid identification uses YL5-2, Halovibrio denitrificans DSM 15503 and Halovibrio variabilis DSM 3050 cells cultured at 30°C for 3 days on YL medium.
  • the main steps are: scraping 100 mg of cells from YL medium and saponifying with 50% methanol containing sodium hydroxide; freeze-drying the cells after saponification, and then using a ratio of 1:2:0.8 (v/v/v) The chloroform/methanol/0.3% (w/v) NaCl aqueous solution was used to extract cell fatty acids.
  • the total amount of fatty acids in the cells was measured using phosphomolybdic acid method.
  • the only breath quinone of YL5-2 is the same as Halovibrio variabilis DSM 3050, which is ubiquinone Q-9.
  • the main cellular fatty acids of YL5-2 include C 18:1 ⁇ 9c, C 16:0 , C 19:0 cyclo ⁇ 8c and Summed Feature 8 (Table 3); the main polar lipids are Diphosphatidylglycerol (DPG), Phosphatidylethanolamine (PE), Phosphatidylglycerol (PG), Phosphatidylcholine (PC) and two unidentified lipids (L). These are similar to Halivobrio variabilis and Halovibrio denitrificans, relatives of YL5-2.
  • DPG Diphosphatidylglycerol
  • PE Phosphatidylethanolamine
  • PG Phosphatidylglycerol
  • PC Phosphatidylcholine
  • L two unidentified lipids
  • Summed features indicate a mixture of two or three fatty acids that cannot be separated from the MIDI system by GLC.
  • Summed feature 3 includes C 16:1 ⁇ 7c and/or C 16:1 ⁇ 6c
  • Summed feature 8 includes C 18:1 ⁇ 6c and/or C 18:1 ⁇ 7c.
  • the medium components are glycerin 500mg/L, glucose 250mg/L, methanol 500mg/L, methylamine 200mg/L, sodium chloride 100 ⁇ 250g/L, sodium acetate 250mg/L, trisodium citrate 250mg/L , Yeast powder 100mg/L, peptone 200mg/L, beef extract 200mg/L, a small amount of trace elements, pH 7.5 ⁇ 8.0. Sterilize the liquid culture medium while controlling the evaporation of water.
  • the medium components are glycerin 50mg/L, glucose 25mg/L, methanol 50mg/L, methylamine 20mg/L, sodium chloride 250g/L, sodium acetate 25mg/L, trisodium citrate 25mg/L, yeast Powder 10mg/L, peptone 20mg/L, beef extract 20mg/L, agar 20g/L.
  • Salinity gradient setting According to the enrichment and screening conditions of YL5-2 strains, set the salinity gradient of the medium to 0%, 0.5%, 1%, 2%, 3%, 5%, 8%, 10%, 12%, 15%, 18%, 21%, 24%, 26%, 28%, 30%, 32%, 34%.
  • medium is prepared according to the set medium formula.
  • the medium with higher salinity is sterilized after hot water is melted.
  • the volatile medium components are to be sterilized
  • Inoculation and culture pick a ring of fresh moss under sterile conditions to connect to each salinity medium, and transfer from a low salinity to a high salinity gradient. After 34% salinity medium is crossed, follow the streak A large amount of salt crystals were deposited on the track. After the inoculation, the petri dish was sealed with parafilm, and cultured at 35-37°C for 3-7 days to observe the growth of the bacteria.
  • Test result The salt tolerance range of YL5-2 strain is 3-32%.
  • the strain YL5-2 can obviously observe newly grown moss in three days in the 3-30% salinity medium, but it can grow on the 32% saturated salinity medium for more than 7 days to grow the visible moss. It shows that YL5-2 grows faster in the salt concentration range of 3% to 30%.
  • Example 7 Salt-tolerant denitrification ability test of halophilic denitrifying bacteria YL5-2
  • Test design The denitrification test was carried out in 10 500mL Erlenmeyer flasks, 300mL of medium was added to each, 8 salinity gradients were set, and the salt content was 3%, 6%, 10%, 12%, 15%, 20%, 25%, 30%, the blank group without salt also set 2 parallel.
  • Example 8 Salt-tolerant denitrification ability test of halophilic denitrifying bacteria YL5-2
  • Culture medium acetic acid 2000mg/L, peptone 20mg/L, beef extract 20mg/L, NO 2 -N 100mg/L, NaCl concentration 3%-30%, adding buffer to make the pH 7.5-8.0.
  • Test design The denitrification test was carried out in 10 500mL Erlenmeyer flasks, 300mL of medium was added to each, 8 salinity gradients were set, and the salt content was 3%, 6%, 10%, 12%, 15%, 20%, 25%, 30%, the blank group without salt also set 2 parallel.

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Abstract

提供了一种嗜盐反硝化菌（Halovibrio sp.）YL5-2及其应用，所述菌株保藏于中国普通微生物菌种保藏管理中心，保藏号为CGMCC NO.16315，保藏日期为2018年8月20日。YL5-2菌株是一种兼性或好氧反硝化菌，其耐盐范围是3％～32％，可以在盐浓度5％～25％范围内以NO 3 -和NO 2 -作为电子受体进行反硝化，可用于盐含量大于10％的高盐废水的生物脱氮处理。

Description

嗜盐反硝化菌YL5-2及其应用 技术领域
本发明涉及环保微生物领域，特别是涉及嗜盐反硝化菌YL5-2及其应用。
背景技术
氮污染是引起水体富营养化的重要原因之一。废水的脱氮处理对于维持水环境质量和防止水体富营养化具有重要作用。石油、化工、食品加工、化肥等行业排放的含氮废水具有高盐的特点。化工、食品加工产生的某些废水，其盐含量甚至超过海水。传统生物法处理低盐度含氮废水具有优势，但当废水浓度过高时，会抑制脱氮微生物的代谢作用。耐盐和嗜盐的脱氮菌的存在，为高盐废水生物脱氮提供里理论上的可能。
生物脱氮菌包括硝化菌和反硝化菌。高盐废水条件下的生物脱氮菌也可以分为硝化菌和反硝化菌。反硝化菌是一类能够将硝酸盐或压硝酸盐还原为气态氮化合物的细菌。因此，从环境中筛选分离并培育耐盐和嗜盐的硝化菌和反硝化菌，就成为解决高盐废水生物脱氮问题的关键。
大连海洋大学焦点等人(焦点、张蕾等.一株中度嗜盐反硝化菌的分离鉴定及其代谢特性研究.《水产科技情报》，2018，45(3)：149～154)从海产养殖废水净化单位中分离得到一株中度嗜盐反硝化菌，属于Halomonas属，其最佳代谢生长条件为温度30℃、盐度100g/L、pH7.5～8.5，C/N比为4:1。
青岛大学郭艳丽等(郭艳丽、张培玉等.一株轻度嗜盐反硝化菌的分离鉴定及特性.《应用与环境生物学报》2010,16(3):394～398)从处理高盐废水的成熟活性污泥中分离得到一株轻度嗜盐好氧反硝化菌YL-1，属于迪茨氏菌(Dietzia sp.)，该菌能够在0％～10％、pH 7.5～8.5、利用乙酸、蔗糖、葡萄糖、柠檬酸钠、丁二酸钠进行反硝化。
南京工业大学訾小利(訾小利.嗜盐反硝化细菌的分离鉴定及其反硝化特性研究.南京工业大学2013年硕士毕业论文)以硝酸钠为唯一氮源且盐浓度为8％的异养反硝化培养基，从盐城盐场的土壤样品中富集、分离和筛选得到了6株嗜盐反硝化菌：NY-1、NY-11与NY-13均为支芽孢杆菌属，(Virgibacillus sp.)，NY-8和NY-10为盐单胞菌属(Hal-monas sp.)，NY-4为海杆菌属(Marinobacter sp.)，其盐度生长范围均为0％～12％，最适生长盐浓度为3％～8％。其中NY-4的反硝化能力最强，在以柠檬酸三钠为碳源、盐度8％、C/N为5、pH为8时，NO 3-N的去除 率为95％。
现有技术关于嗜盐反硝化菌的报道较少，尤其是盐度＞10％的条件下的嗜盐反硝化菌。从环境中获取耐盐能力大于10％的嗜盐反硝化菌是此类废水生物脱氮需要解决的技术问题。
发明内容
本发明的目的是为解决高盐条件下的生物脱氮问题，尤其是盐度大于10％条件下的高盐废水生物脱氮问题，提供一种嗜盐反硝化菌，可以在盐度10％以上的的条件下以NO 3-N作为电子受体进行反硝化。
本发明的另一目的是提供该嗜盐反硝化菌的应用。
本发明的目的可通过以下技术方案实现的：
本发明公开的嗜盐反硝化菌YL5-2保藏于中国普通微生物菌种保藏管理中心，其保藏号为CGMCC NO.16315，保藏日期为2018年8月20日。基于16SrRNA系统发育分析、基因组测序、DNA杂交试验、脂肪酸构成、呼吸醌类别、生理生化和表型特征等差异，可以确定菌株YL5-2为Halovibrio属的新物种，命名为Halovibrio salipaludis sp.nov。
本发明公开的嗜盐反硝化菌YL5-2的16S rRNA序列的GenBank/EMBL/DDBJ的登录号为MF782425，核苷酸序列如SEQ ID NO.1所示；全基因组序列的GenBank/EMBL/DDBJ的登录号为NSKD00000000.1。
本发明公开的嗜盐反硝化菌YL5-2为革兰氏阴性、兼性好氧、直杆状或0.5～0.8μm×1.0～3.5μm的小弧菌性状，并且通过单极性鞭毛运动，其固体培养基上的菌落为光滑和浅黄色。
本发明嗜盐弧菌Halovibrio sp.YL5-2能够在盐浓度3％～32％、pH6.5～11.0、温度15～45℃范围内生长；最适生长盐浓度为10％～25％，最适生长pH为7.5～8.0、最适生长温度为30～35℃。
本发明嗜盐弧菌Halovibrio sp.YL5-2主要的呼吸醌为Q-9，主要的脂肪酸为C18:1ω9c,C16:0,C19:0 cycloω8c和Summed Feature 8，主要的极性脂质是Diphosphatidylglycerol(DPG)，Phosphatidylethanolamine(PE)，Phosphatidylglycerol(PG)，Phosphatidylcholine(PC)。
本发明嗜盐弧菌Halovibrio sp.YL5-2可以利用溴-琥珀酸，丙酸和乙酸作为唯一碳源利用；但不能利用D-麦芽糖，D-果糖，D-半乳糖，D-纤维二糖，水苏糖，D-蜜二糖，N-乙酰基-D半乳糖胺，D-岩藻糖，L-鼠李糖，D-甘露醇，D-半乳糖醛酸，D-天冬氨酸，D-丝氨酸，D-葡萄糖醛酸，L-乳酸，奎尼酸，粘酸，D-苹果酸，γ-氨基-丁酸，甲酸，乙酰乙酸作为唯一碳源。
有益效果：
本发明发现了一个Halovibrio属的新物种，命名为Halovibrio salipaludis sp.nov。该菌株是一种兼性或好氧反硝化菌，其耐盐范围是3％～32％，可以在盐浓度5％～25％范围内降解废水中的多种污染物。本发明公开的嗜盐反硝化菌YL5-2可以用于高盐条件下污染物的降解、转化和生物脱氮过程，包括高盐废水处理，污染海水治理，盐碱地修复，消耗氮素营养，抑制藻类过度繁殖，净化水体，改良底质等。
附图说明：
图1嗜盐弧菌Halovibrio sp.YL5-2细胞的透射电子显微镜(TEM)照片(比例尺为2μm)；
图2是嗜盐弧菌Halovibrio sp.YL5-2(a)与Halovibrio denitrificans DSM15503(b)，Halovibrio variabilis DSM 3050(c)的极性脂质图谱。DPG：diphosphatidylglycerol；PG：phosphatidylglycerol；AL：Aminolipid；PL：Phospholipid；PE：Phosphatidylethanolamine；PGNL：Phosphoaminoglycolipid；f：第一维度；s：第二维
图3是嗜盐弧菌Halovibrio sp.YL5-2与Halovibrio denitrificans DSM15503构建的基于16S rRNA的的系统发育树图。
图4是嗜盐弧菌Halovibrio sp.YL5-2与Halovibrio variabilis DSM 3050T及其它菌种基于16S rRNA的最大简约法(MP)构建的系统发育树图。
图5是嗜盐弧菌Halovibrio sp.YL5-2基于GGD矩阵构建的系统发育树图。
生物材料保藏信息
YL5-2，分类命名为Halovibrio salipaludis，保藏日期为2018年8月20日，保藏单位为中国普通微生物菌种保藏管理中心，保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号：CGMCC NO.16315。
具体实施方式
实施例1 该嗜盐反硝化菌的分离及保藏
嗜盐反硝化菌YL5-2是从中国青海省格尔木察尔汗盐湖(36°51′N,94°95′E)沉积土壤中分离得到。察尔汗盐湖湖水常年为饱和盐浓度或接近饱和盐浓度。
配置NaCl浓度为20％的LB液体培养基，并加入甘油250mg/L、葡萄糖250mg/L、甲醇 50mg/L，在30℃条件下对察尔汗盐湖沉积土壤富集培养48h。利用YL固体培养基对富集培养液中的菌株进行分离。1L培养基中含有以下组分：葡萄糖：0.6g，柠檬酸三钠0.5g，甘油2mL，酵母提取物0.8g，蛋白胨1.6g，磷酸氢二钾0.35g，磷酸二氢钾0.1g，硫酸铵0.25g，氯化铵0.25g，MgSO 40.5g，CaCl 20.1g，NaCl 180g；微量元素SL-4 10mL，pH 7.0-7.2；琼脂2.5％。
菌株YL5-2保藏在中国普通微生物菌种保藏管理中心，保藏号为CGMCC NO.16315，保藏日期为2018年8月20日。
实施例2 嗜盐反硝化菌YL5-2 16S rRNA序列分析与全基因序列分析
菌株YL5-2基因组DNA提取采用TaKaRa试剂盒(TaKaRa MiniBEST Bacteria Genomic DNA Extraction 68 Kit Ver. 3.0)。
16S rRNA扩增采用通用细菌引物27F(5’-AGAGTTTGATCMTGGCTCA G-3’)和1492R(5’-TACGGYTACCTTGTTACGACTT-3’)。PCR测序委托上海生工生物科技有限公司进行。菌株YL5-2完整的16S rRNA序列为1518bp，如SEQ ID NO.1所示，GenBank登录号为MF782425。
菌株YL5-2的全基因组测序采用上海上海派森诺生物科技有限公司Illumina MiSeq 2000高通量测序平台。原始测序数据采用PRINSEQ(版本号v 0.20.4)软件进行过滤和修正，然后采用带有默认参数的SOAP denovo软件(版本号v1.05)软件进行基因组的碱基配对，然后使用CheckM软件(版本1.03)评估基因组的完整性。蛋白质编码开放阅读框架采用Glimmer软件(版本号1.2)进行预测。RNA预测采用RNAmmer软件(版本1.2)。菌株YL5-2全基因组序列共3,495,096 bp，GenBank登录号为NSKD00000000.1。
DNA-DNA杂交试验通过菌株YL5-2与其遗传发育最接近的弧菌属Halovibrio模式菌株进行。该方法由De Ley等于1970年提出，DNA杂交值(dDDH)采用GGDC软件第2种模式(版本号2.0)进行基因序列的一一比对得到。DNA试验和分析结果表明，菌株YL5-2和Halovibrio variabilis DSM 3050 T、Halovibrio denitrificans DSM15503 T的DNA-DNA杂交值分别为43.5％和38.2％，远低于70％的阈值(物种划分通常被接受的阈值)。
核苷酸平均一致性值采用全基因组序列的碱基组进行1000次重复拓扑检验得到。该方法由Goris等于2007提出，使用的软件为MUMmer(版本号3.23)和Jspecies(版本号1.2.1)。基于Kim等人和Richter等人提出的物种划分的ANI阈值范围(95-96％)，对菌株YL5-2的基因组和GenBank中与之密切相关的基因组进行ANI分析(表1)。结果表明，菌株YL5-2其与 Tamilnaduibacter alinus Mi 7的平均核苷酸一致性(ANI)值最高，为88.5％(Supplementary Table S1)，这为菌株YL5-2 T属于Halovibrio属的一种新物种提供了论据，命名为Halovibrio salipaludis sp.nov。
表1菌株YL5-2 T与GenBank中密切相关的基因组之间的平均核苷酸一致性(ANI)和DDH值。
Figure PCTCN2019119640-appb-000001
实施例3 嗜盐反硝化菌YL5-2的表型特征和生理生化特征鉴定
革兰氏染色特性采用BD革兰氏染色试剂盒进行测试。
细胞运动性使用半MA培养基(0.5％琼脂，w/v)测定。
细胞形态采用透射电子显微镜(TEM)分析检测。即从指数生长的培养液中挑取细胞，用0.5％乙酸铀酰染色细胞，并在显微镜(Tecnai Spirit,FEI,Hillsboro,OR,USA)下对细胞进行拍照。
氧化酶活性使用氧化酶试剂盒(bioMérieux)，通过将3.0％H 2O 2溶液倒入细菌菌落并观察气泡产生来测定过氧化氢酶活性。
温度生长条件在YL液体琼脂培养基上进行，温度分别为4、10、15、20、25、30、33、37、40、45和50℃，pH恒定为7.5，比较不同温度下菌株YL5-2 T的生长速率确定其最佳生长温度。
耐盐能力在0.0-30.0％NaCl(w/v)的YL琼脂和YL液体培养基进行。用缓冲液(Na 2HPO 4/NaH 2PO 4(pH 5.0-7.0)，Na 2CO 3/NaHCO 3(pH 8.0-12.0))将pH调至5.0、5.5、6.0、7.0、8.0、9.0、10.0和11.0(15.0％NaCl，35℃)以测定适合生长的pH范围。
碳源利用能力和酶活性测试采用API 20NE，API ZYM(bioMérieux)和Biolog GENIII微孔板。所有测试接种YL培养基上预生长的细胞，并用相关的接种培养基稀释。
菌株YL5-2的表型特征和生理生化特征鉴定结果如表2所示：
表2嗜盐弧菌Halovibrio sp.YL5-2(a)与Halovibrio denitrificans DSM15503 T(b)，Halovibrio variabilis DSM 3050 T(c)在表型与生理生化等方面的区别特征比较
Figure PCTCN2019119640-appb-000002
说明：+，阳性；-，阴性。
嗜盐反硝化菌YL5-2是革兰氏染色阴性、碱性好氧，直杆状或0.5-0.8x1.0-3.5μm的小弧菌形状，并且通过单极性鞭毛运动(图1)。
菌株YL5-2在好氧条件下利用乙酸生长，在缺氧条件下则利用硝酸盐生长(API 20NE)。
实施例4 嗜盐弧菌Halovibrio sp.YL5-2细胞脂肪酸鉴定
细胞脂肪酸鉴定采用YL培养基上30℃培养3天的YL5-2、Halovibrio denitrificans DSM 15503和Halovibrio variabilis DSM 3050细胞。主要步骤为：从YL培养基上刮下100mg细胞的用含氢氧化钠的50％甲醇进行皂化；皂化后的细胞进行冷冻干燥，然后使用比例为1:2:0.8(v/v/v)的氯仿/甲醇/0.3％(w/v)NaCl水溶液提取细胞脂肪酸。
细胞脂肪酸总量使用磷钼酸方法检测。
脂肪酸定性和定量检测采用6890N气相色谱仪(Agilent)和Sherlock微生物鉴定系统中的标准MIS文库生成软件(VERSION 6.0and Date 4,Microbial ID Inc.,Newark,DE,USA)(Sasser，1990)。
基于细胞脂肪酸的菌种鉴定则采用德国DSMZ相关分析工具。
细胞脂肪酸鉴定结果如图2所示。
YL5-2的唯一呼吸醌与Halovibrio variabilis DSM 3050相同，都是泛醌Q-9。
YL5-2主要的细胞脂肪酸包括C 18:1ω9c、C 16:0、C 19:0cycloω8c和Summed Feature 8(表3)；主要的极性脂质是Diphosphatidylglycerol(DPG)，Phosphatidylethanolamine(PE)，Phosphatidylglycerol(PG)，Phosphatidylcholine(PC)和两种未鉴定的脂质(L)。这些均与YL5-2的亲缘种Halovibrio variabilis与Halovibrio denitrificans相似。
表3嗜盐弧菌Halovibrio sp.YL5-2(a)与Halovibrio denitrificans DSM 15503 T(b)，Halovibrio variabilis DSM 3050 T的细胞脂肪酸组成(％)比较。
Figure PCTCN2019119640-appb-000003
Figure PCTCN2019119640-appb-000004
说明： *Summed features表示不能通过GLC与MIDI系统分离的两种或三种脂肪酸的混合。Summed feature 3包括C 16:1ω7c和/或C 16:1ω6c，Summed feature 8包括C 18:1ω6c和/或C 18:1ω7c。
实施例5 嗜盐弧菌Halovibrio sp.YL5-2的发酵培养试验
(1)培养基成分为甘油500mg/L、葡萄糖250mg/L、甲醇500mg/L、甲胺200mg/L、氯化钠100～250g/L，乙酸钠250mg/L，柠檬酸三钠250mg/L、酵母粉100mg/L、蛋白胨200mg/L、牛肉膏200mg/L，微量元素少量，pH为7.5～8.0。液体培养基进行灭菌，同时控制好水分蒸发。
(2)1L的三角瓶中接种后在35℃条件下培养48h，培养过程中补充水分的散失影响盐浓度的变化。48h后测得10％、15％、20％、25％盐浓度条件下OD600分别为1.72、1.65、1.62、1.60、1.63。进行两次转接培养，每次转接后均培养48h，驯化培养完成后OD600分别为2.56、2.68、2.72、2.68、2.52。
(3)1L培养液接种到20L的好氧发酵罐中，反复进行发酵培养，培养基成分保持不变。仍然培养48h，培养温度为35℃、搅拌速度为100rpm，溶解氧为2.0～4.0mg/L。48h后发酵罐中菌液的OD600可达到2.6～3.0。
(4)检验：培养过程中使用显微镜每日取样观察，检查是否有杂菌混入生长；同时观察YL5-2的生长及形态变化情况。
(5)结果：试验结果表明，菌株YL5-2可以快速的进行发酵培养和放大，这表明YL5-2具有大规模工程应用的潜力。
实施例6 嗜盐弧菌Halovibrio sp.YL5-2的耐盐能力试验
(1)培养基成分为甘油50mg/L、葡萄糖25mg/L、甲醇50mg/L、甲胺20mg/L、氯化钠250g/L，乙酸钠25mg/L，柠檬酸三钠25mg/L、酵母粉10mg/L、蛋白胨20mg/L、牛肉膏20mg/L，琼脂20g/L。
(2)使用新鲜的上述培养基活化菌种，培养3天至菌苔生长丰富备用
(3)盐度梯度设置：根据YL5-2菌种的富集筛选条件，设置培养基的盐度梯度分别为0％、0.5％、1％、2％、3％、5％、8％、10％、12％、15％、18％、21％、24％、26％、28％、30％、 32％、34％。
(4)制备固体培养基：根据设置的培养基配方制备培养基，盐度较高的培养基热水融化后灭菌，每瓶多加5ml的蒸发水，易挥发的培养基成分待灭菌后加入摇匀，待冷却至60℃左右，倒制平皿，盐度高极易凝固，因此，倒制平皿快速要快(32％盐度培养基倒制平皿时，冷却凝固后有少量盐析出，34％盐度有大量盐晶体析出)。
(5)接种培养：无菌条件下挑取一环新鲜菌苔接入各盐度培养基，由低盐度往高盐度梯度转接，34％盐度培养基划线后随着划线轨迹有大量盐晶体析出，接种完毕培养皿用封口膜封口，35-37℃培养3-7天，观察菌种生长情况。
(6)试验结果：YL5-2菌种耐盐范围为3-32％。YL5-2菌株在3-30％盐度培养基中三天内可以明显观察到新长出的菌苔，但是在32％饱和盐度培养基中生长7天以上才能长出肉眼可见菌苔。表明YL5-2在3％～30％的盐浓度范围内生长速度均较快。
表4 YL5-2在不同盐浓度培养基上的生长情况
Figure PCTCN2019119640-appb-000005
实施例7 嗜盐反硝化菌YL5-2的耐盐反硝化能力试验
(1)培养基：乙酸2000mg/L、蛋白胨20mg/L、牛肉膏20mg/L，NO 3-N为100mg/L，NaCl浓度3％～30％，加入缓冲液使pH为7.5～8.0。
(2)试验设计：反硝化试验10个500mL的三角瓶中进行，各加入培养基300mL，设置8个盐度梯度，盐含量分别为3％、6％、10％、12％、15％、20％、25％、30％，其中不含盐的空白组也设置2个平行。
(3)反硝化试验：所有样品灭菌后接种YL-5培养液约10mL，在恒温摇床上进行培养，温度为30～35℃；震荡速度分别为10ppm；分别于24h、48h和72h后取样测定三角瓶中NO 3-N的浓度。试验结果如下表数据：
表5 YL5-2在不同盐浓度下反硝化去除NO 3-N的试验结果(单位：mg/L)
  0 0 3％ 6％ 10％ 12％ 15％ 20％ 25％ 30％
24h 99.6 99.5 91.3 76.2 68.8 67.5 66.4 67.6 72.6 89.6
48h 99.1 99.2 55.4 18.6 14.4 11.7 9.9 14.7 19.1 40.6
72h 98.5 98.8 13.5 6.7 4.5 3.6 3.8 4.2 8.2 12.5
实施例8 嗜盐反硝化菌YL5-2的耐盐反硝化能力试验
(1)培养基：乙酸2000mg/L、蛋白胨20mg/L、牛肉膏20mg/L，NO 2-N为100mg/L，NaCl浓度3％～30％，加入缓冲液使pH为7.5～8.0。
(2)试验设计：反硝化试验10个500mL的三角瓶中进行，各加入培养基300mL，设置8个盐度梯度，盐含量分别为3％、6％、10％、12％、15％、20％、25％、30％，其中不含盐的空白组也设置2个平行。
(3)反硝化试验：所有样品灭菌后接种YL-5培养液约10mL，在恒温摇床上进行培养，温度为30～35℃；震荡速度分别为10ppm；分别于24h、48h和72h后取样测定三角瓶中NO 2-N的浓度。试验结果如下表数据：
表6 YL5-2在不同盐浓度下反硝化去除NO 2-N的试验结果(单位：mg/L)
  0 0 3％ 6％ 10％ 12％ 15％ 20％ 25％ 30％
24h 99.7 99.7 95.3 86.2 78.8 77.5 76.4 77.6 81.6 91.6
48h 99.3 99.1 62.4 20.3 16.5 14.8 11.3 15.7 19.1 30.6
72h 99.0 98.6 15.8 9.7 6.6 4.2 3.8 7.3 8.5 15.3

Claims (8)

  1. 一株嗜盐反硝化菌YL5-2，为Halovibrio属的新物种，保藏于中国普通微生物菌种保藏管理中心，保藏号为CGMCC NO.16315,保藏日期为2018年8月20日。
  2. 权利要求1所述的嗜盐反硝化菌YL5-2在盐度3％～32％的高盐废水处理中的应用。
  3. 根据权利要求2所述的应用，其特征在于权利要求1所述的嗜盐反硝化菌YL5-2在盐度3％～32％的高盐废水的生物脱氮处理中的应用。
  4. 根据权利要求3所述的应用，其特征在于所述的嗜盐反硝化菌YL5-2在缺氧条件下或好氧条件下对高盐废水进行反硝化。
  5. 根据权利要求4所述的应用，其特征在于所述的嗜盐反硝化菌YL5-2在缺氧条件下或好氧条件下对盐度大于10％的高盐废水进行反硝化。
  6. 权利要求1所述的嗜盐反硝化菌YL5-2在污染海水治理、盐碱地修复中的应用。
  7. 权利要求1所述的嗜盐反硝化菌YL5-2在消耗氮素营养，抑制藻类过度繁殖，净化水体中的应用。
  8. 权利要求1所述的嗜盐反硝化菌YL5-2在改良水体底质中的应用。
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113699057A (zh) * 2021-07-06 2021-11-26 广州大学 一株具有异养硝化-好氧反硝化功能的椿象红球菌及其应用
CN113832042A (zh) * 2021-10-19 2021-12-24 泸州老窖股份有限公司 一株耐盐扣囊复膜孢酵母及其在高盐高cod污水处理中的应用
CN114292793A (zh) * 2022-01-13 2022-04-08 青岛蔚蓝赛德生物科技有限公司 一株耐盐的盐单胞菌菌株及其在水净化领域的应用
CN114644993A (zh) * 2020-12-21 2022-06-21 有研资源环境技术研究院(北京)有限公司 高耐盐的盐单胞菌及其促进盐碱地植物生长的微生物原位生态修复方法
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Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109385388B (zh) * 2018-12-29 2021-09-14 中蓝连海设计研究院有限公司 嗜盐反硝化菌yl5-2及其应用
CN109928505A (zh) * 2019-03-15 2019-06-25 江苏大学 一种利用盐藻、嗜盐菌处理腌制废水的方法
CN115181694B (zh) * 2022-06-23 2024-03-22 北京工业大学 一株具有高盐废水同化脱氮功能的中度嗜盐菌及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102747014A (zh) * 2012-05-29 2012-10-24 北京盖雅技术中心有限公司 一株高盐生物脱氮的盐弧菌菌株及其在废水处理中的应用
CN103074286A (zh) * 2013-01-28 2013-05-01 北京大学 一株高盐异养硝化-好氧反硝化除磷的盐弧菌及其在废水处理中的应用
CN103805529A (zh) * 2012-11-14 2014-05-21 中国科学院过程工程研究所 一株具有异养硝化好氧反硝化功能的盐田盐单胞菌及其应用
CN109385388A (zh) * 2018-12-29 2019-02-26 中蓝连海设计研究院有限公司 嗜盐反硝化菌yl5-2及其应用
CN109456926A (zh) * 2018-12-29 2019-03-12 中蓝连海设计研究院有限公司 一种含嗜盐反硝化菌yl5-2的微生物菌剂及其应用
CN109534518A (zh) * 2018-12-29 2019-03-29 中蓝连海设计研究院有限公司 一种利用嗜盐菌yl5-2的高盐废水生物膜处理工艺

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5602020A (en) * 1992-02-27 1997-02-11 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Thermostable, salt tolerant, wide PH range novel chitobiase
EP2354098A1 (en) * 2010-02-09 2011-08-10 Biomim-Greenloop Valorisation of divalent cations in brine and associated co2 sequestration by microorganisms
CN102268386B (zh) * 2010-12-01 2013-01-23 中国环境科学研究院 氨氧化细菌及其分离方法与应用
US20120325740A1 (en) * 2011-02-14 2012-12-27 Empire Technology Development Llc Organism metabolites for removal of pollutants from brine
EP2970859A4 (en) * 2013-03-14 2017-07-19 The University Of Wyoming Research Corporation Conversion of carbon dioxide utilizing chemoautotrophic microorganisms systems and methods
NO338103B1 (no) * 2014-05-14 2016-08-01 Yara Int Asa Anvendelse av en gruppe av halofile og/eller halotolerante bakterier i en biokjemisk prosess for denitrifikasjon av en hypersalt avfallsblanding.
CN104611279B (zh) * 2015-03-02 2017-06-09 中蓝连海设计研究院 一种红城红球菌lh‑n13及其微生物菌剂与用途
CN105861359A (zh) * 2016-05-17 2016-08-17 中国石油大学(华东) 一株产絮的异养硝化-好氧反硝化耐高温菌株及其应用
ES2677443B2 (es) * 2017-02-01 2019-02-27 Univ Vigo Procedimiento para el incremento de producción de lipasa en cultivos de Halomonas mediante inducción química y biológica
CN109486699B (zh) * 2018-08-21 2021-04-09 四川大学 一株耐盐好氧反硝化菌及应用
CN109439602B (zh) * 2018-12-29 2021-09-14 中蓝连海设计研究院有限公司 嗜盐弧菌yl5-2及其菌剂在高盐条件下降解转化污染物中的应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102747014A (zh) * 2012-05-29 2012-10-24 北京盖雅技术中心有限公司 一株高盐生物脱氮的盐弧菌菌株及其在废水处理中的应用
CN103805529A (zh) * 2012-11-14 2014-05-21 中国科学院过程工程研究所 一株具有异养硝化好氧反硝化功能的盐田盐单胞菌及其应用
CN103074286A (zh) * 2013-01-28 2013-05-01 北京大学 一株高盐异养硝化-好氧反硝化除磷的盐弧菌及其在废水处理中的应用
CN109385388A (zh) * 2018-12-29 2019-02-26 中蓝连海设计研究院有限公司 嗜盐反硝化菌yl5-2及其应用
CN109456926A (zh) * 2018-12-29 2019-03-12 中蓝连海设计研究院有限公司 一种含嗜盐反硝化菌yl5-2的微生物菌剂及其应用
CN109534518A (zh) * 2018-12-29 2019-03-29 中蓝连海设计研究院有限公司 一种利用嗜盐菌yl5-2的高盐废水生物膜处理工艺

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
D. YU. SOROKIN ET AL.: "Extremely halophilic denitrifying bacteria from hypersaline inland lakes, Halovibrio denitrificans sp. Nov. and Halospina denitrificans gen. nov., sp. Nov., and evidence that the genus name Halovibrio Fendrich 1989 with the type species Halovibrio variabilis should be associated with DSM 3050", INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, vol. 56, no. 2, 1 February 2006 (2006-02-01), XP055721895, ISSN: 1466-5034 *
DATABASE GenBank 4 September 2017 (2017-09-04), ZHANG, X. ET AL., Database accession no. X77833 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114644993A (zh) * 2020-12-21 2022-06-21 有研资源环境技术研究院(北京)有限公司 高耐盐的盐单胞菌及其促进盐碱地植物生长的微生物原位生态修复方法
CN114644993B (zh) * 2020-12-21 2023-06-02 有研资源环境技术研究院(北京)有限公司 高耐盐的盐单胞菌及其促进盐碱地植物生长的微生物原位生态修复方法
CN113699057A (zh) * 2021-07-06 2021-11-26 广州大学 一株具有异养硝化-好氧反硝化功能的椿象红球菌及其应用
CN113699057B (zh) * 2021-07-06 2023-01-20 广州大学 一株具有异养硝化-好氧反硝化功能的椿象红球菌及其应用
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