WO2019033264A1 - 一种新型甲基杆菌mr1及其应用 - Google Patents

一种新型甲基杆菌mr1及其应用 Download PDF

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WO2019033264A1
WO2019033264A1 PCT/CN2017/097497 CN2017097497W WO2019033264A1 WO 2019033264 A1 WO2019033264 A1 WO 2019033264A1 CN 2017097497 W CN2017097497 W CN 2017097497W WO 2019033264 A1 WO2019033264 A1 WO 2019033264A1
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formaldehyde
culture medium
sodium
novel
strain
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陈丽梅
李昆志
王茹
冯永
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云南万魁生物科技有限公司
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    • B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
    • B01D53/34—Chemical or biological purification of waste gases
    • B01D53/74—General processes for purification of waste gases; Apparatus or devices specially adapted therefor
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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    • Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters

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  • the invention belongs to the field of environmental microorganisms, and in particular relates to a novel methyl bacterium having a special metabolic pathway of formaldehyde, which can be applied to the removal of formaldehyde and benzene-based pollution.
  • Formaldehyde (HCHO) is an air pollutant commonly found in indoor environments. Formaldehyde contamination is found in various renovated locations, many factories and drug reagent repositories, and it is also produced as an endogenous metabolite. In living organisms, formaldehyde can be produced spontaneously by the dissociation of 5,10-methylenetetrahydrofolate; in addition, various demethylation reactions also produce low concentrations of formaldehyde. In the long-term evolution process, almost all organisms have a mechanism of formaldehyde detoxification to prevent the lethal and mutated effects of formaldehyde.
  • Benzene is a general term for benzene and derivatives. It is a kind of aromatic organic compound, environmentally polluting benzene source industrial production, automobile exhaust, decoration and decoration materials (such as paint, sheet, decorative materials, etc.), office equipment (such as Copiers, printers, fax machines, computers, etc.), human activities (such as smoking, cooking, burning incense, etc.).
  • the four types of benzene, toluene, ethylbenzene and xylene are representative substances of environmentally contaminated benzene series.
  • These benzene compounds are neurotoxic, and thus can cause direct harm to human health, causing symptoms such as neurasthenia, headache, insomnia, dizziness, and fatigue of the lower limbs. Long-term exposure can cause anemia and leukemia in the human body.
  • benzenes can also destroy DNA and are therefore genotoxic.
  • Methylotrophic bacteria are a group of microorganisms that can grow with a single carbon compound as a carbon source and energy source. Since one carbon compound is organic, methylotrophic bacteria are some heterotrophic bacteria. Studies on the metabolism of mono-carbon compounds by methylotrophic bacteria show that such microorganisms have both formaldehyde degradation pathways for the oxidation of formaldehyde to formic acid and then CO 2 , and assimilation of formaldehyde as a cellular component. Therefore, a large number of studies have investigated these microorganisms. The ability and effect of removing formaldehyde pollution.
  • Pseudomonas putida is a solvent-resistant bacterium with genes encoding formaldehyde dehydrogenase and formate dehydrogenase, so P. putida has the ability to oxidize formaldehyde to produce formic acid and CO 2 .
  • the formaldehyde assimilation pathways found to date in methylotrophic bacteria include the ribulose monophosphate pathway (RuMP), the serine pathway, and the cyclic oxidative pathway of ribulose monophosphate.
  • RuMP ribulose monophosphate pathway
  • Formaldehyde assimilation in facultative methylotrophic bacteria is accomplished by the serine pathway, in which HCHO first combines with non-enzymatic reaction and tetrahydrofolate to form 5,10-methylenetetrahydrofolate, followed by serine methylol
  • the transferase catalyzes the condensation of 5,10-methylenetetrahydrofolate with glycine to form serine (Ser) while releasing tetrahydrofolate.
  • PEP phosphoenolpyruvate
  • Mal malic acid
  • Malic acid decomposes to form two C2 compounds, and the C2 compound is converted to glyoxylic acid (Ox).
  • glycine Gly is produced from glyoxylic acid to complete the entire cycle of the serine pathway.
  • the RuMP pathway is present in many methylotrophic and non-methylotrophic bacteria in which HCHO is first condensed with 5-nucleoside ribulose (Ru5P) to produce 6-hexyl hexose (Hu6P).
  • This step is 6- It is catalyzed by phosphohexulose synthase (HPS); Hu6P is isomerized to form F6P by 6-phosphohexose isomerase (PHI), and F6P is subsequently cleaved to form fructose 1,6-diphosphate (FBP), FBP Re-cracking forms two C3 compounds: glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). DHAP is then used for the synthesis of cellular components, while GAP regenerates the receptor for the HCHO, Ru5P, through a series of reactions.
  • HPS phosphohexulose synthase
  • Hu6P is isomerized to form F6P by 6-phosphohexose isomerase (PHI)
  • F6P is subsequently cleaved to form fructose 1,6-diphosphate (FBP)
  • FBP Re-cracking forms two C3 compounds: glyceral
  • methanol is first oxidized by ethanol oxidase to HCHO, a key intermediate in methanol metabolism, which is at the branching point of the methanol assimilation and dissimulation pathways.
  • HCHO can react with reduced glutathione to form S-methylol glutathione under non-enzymatic conditions, and S-hydroxymethylglutathione is dependent on glutathione-containing HCHO dehydrogenase (FALDH)
  • FALDH glutathione-containing HCHO dehydrogenase
  • the final oxidation of CO 2 is carried out by a series of enzymes such as S-formate hydrolase (FGH) and NADH-dependent formate dehydrogenase (FDH).
  • HCHO xylulosole monophosphate pathway
  • DAS dihydroxyacetone synthase
  • GAP glyceraldehyde triphosphate
  • DHA dihydroxyacetone
  • DAK dihydroxyacetone kinase
  • GAP and DHAP are then catalyzed by aldolase to form 1,6-diphosphate fructose (FBP), FBP is dephosphorylated to form 6-phosphate fructose (F6P), and a portion of F6P is used to regenerate HCHO.
  • FBP 1,6-diphosphate fructose
  • F6P 6-phosphate fructose
  • Xu5P is used for the synthesis of cellular components by other means.
  • Methanol can induce activity of a promoter of a protein gene involved in methanol metabolism in methylotrophic yeast.
  • Pichia pastoris is a common methylotrophic yeast. Its methanol oxidase (AOX) is localized in peroxisomes. When methanol is used as a carbon source, the AOX protein expression is increased to The cell total protein is 35%-40%, so the AOX promoter is often used in genetic engineering to control the expression of the foreign gene-encoded protein in methanol yeast.
  • AOX methanol oxidase
  • the bioreactor is prepared by using domesticated activated sludge.
  • the analysis results show that this kind of bioreactor can remove and degrade the formaldehyde and other organic substances polluted in the air, but the activated sludge contains many kinds of microorganisms, the composition is not clear, and may contain some Pathogenic bacteria.
  • the organic matter contained in high temperature and high humidity conditions may cause mold growth and growth.
  • the object of the present invention is to provide a novel methyl strain having a special metabolic pathway of formaldehyde, which can be applied to the removal of formaldehyde and benzene-based pollution, and provides a new microbial resource for purifying formaldehyde and benzene-based pollution.
  • the present invention provides the following technical solutions:
  • a new type of methyl bacterium MR1 which is deposited in the China Center for Type Culture Collection, is CCTCC NO: M2017322, and the address is: Wuhan University, Wuchang District, Wuhan City, Hubei province.
  • the 16S rRNA gene sequence of this strain is SEQ ID NO. 1 is shown.
  • the invention also discloses two formaldehyde assimilation pathways of the novel methyl bacterium MR1, namely the serine pathway and the glyoxylate pathway.
  • the invention also discloses the application of the novel methyl bacterium MR1 as a biological resource in purifying high concentration formaldehyde pollution.
  • the invention also discloses the application of the novel methyl bacterium MR1 as a biological resource in degrading benzene series.
  • the invention also discloses the application of the novel methyl bacterium MR1 as a biological resource in degrading benzene series when sodium acetate or sodium malate or sodium citrate is used as a carbon source.
  • the novel methyl bacterium MR1 of the present invention is obtained by first embedding activated sludge collected from a river channel with alginate and a sponge, acclimating microorganisms in the activated sludge with air contaminated with high concentration of formaldehyde, and then using methyl groups.
  • the inorganic salt medium of the bacteria separates methyl bacteria which grow fast and are highly resistant to formaldehyde.
  • the novel methyl bacterium MR1 of the present invention is an activated sludge collected in a river channel first embedded with alginate and a sponge, and domesticated the microorganism in the activated sludge by using air contaminated with a high concentration of formaldehyde, and then using the methyl bacterium.
  • the inorganic salt medium separates methyl bacteria which grow fast and is highly resistant to formaldehyde, and has a special formaldehyde assimilation pathway and can purify formaldehyde and remove contaminated benzene series.
  • the MR1 of the present invention assimilates H 13 CHO mainly through the serine pathway and the glyoxylate pathway, and converts formaldehyde metabolism into serine, glycine, malic acid, glyoxylic acid, phosphoenolpyruvate, citric acid, isocitric acid, etc. Organic acids and amino acids that are non-toxic and have no side effects.
  • MR1 can also be grown in an inorganic salt medium with sodium acetate, sodium malate or sodium citrate as the sole carbon source, with sodium malate as the carbon source for best growth. The added benzene series can be removed when grown in a medium containing sodium acetate or sodium malate or sodium citrate, and the removal rate is 100%.
  • Figure 1 Alignment of homology between strain MR1 and Methylobacterium zatmanni strain DSM 5688 16SrRNA gene sequence;
  • Figure 2 Effect of adding different concentrations of formaldehyde on solid medium on the growth of MR1, Candida and Pseudomonas putida;
  • Figure 3 Effect of adding different concentrations of formaldehyde on the growth of MR1, Candida and Pseudomonas putida;
  • Figure 5 Comparison of the removal rates of formaldehyde in different concentrations of MR1, Candida and Pseudomonas putida;
  • Figure 6 Pathway analysis of the metabolism of formaldehyde by Pseudomonas putida and Candida;
  • Figure 7 Pathway analysis of methylation of formaldehyde by Methylbacterium MR1.
  • the present invention firstly uses the alginate and the sponge to embed the activated sludge collected in the river channel, acclimates the microorganisms in the activated sludge with the air contaminated with the high concentration of formaldehyde, and then utilizes the inorganic salts of the methyl bacteria.
  • the medium is isolated and has a strong resistance to formaldehyde, and the specific screening steps are as follows:
  • the sludge collected from the river is mixed with water, filtered with gauze to remove large particles such as gravel, and then mixed with 2% sodium alginate (1:1), soaked in the sponge and removed the excess liquid mixture, and then placed in 4
  • the embedding was carried out in a % CaCl 2 solution.
  • the sponge embedded with sludge is rolled into a column and placed in a plastic storage box for domestication.
  • a small centrifugal fan is installed at the bottom of the storage box, and the artificially prepared formaldehyde gas is blown into the sponge through a centrifugal fan.
  • the formaldehyde concentration is from the initial 1 mg/ m 3 was increased from day to day to a final 15 mg/m 3 for 15 days of continuous fumigation, and the water content in the sponge matrix was maintained at 25-40% throughout the acclimation period.
  • the supernatant was removed by centrifugation of the cells cultured for 7 days, and then suspended in 500 ⁇ l of sterile water and applied to a solid medium supplemented with 2 mM of formaldehyde as the sole carbon source.
  • the cells were cultured in an incubator at 28 ° C to obtain a single colony for subsequent identification.
  • Candida could not grow in the medium supplemented with 6 mM, 8 mM and 10 mM formaldehyde, while MR1 and Pseudomonas putida could grow, indicating that MR1 Formaldehyde resistance is comparable to Pseudomonas putida, but stronger than Candida.
  • Insert MR1, Candida Pseudomonas putida in liquid medium supplemented with methanol, and add 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM formaldehyde, respectively, when the OD600 of each bacterium is 0.4-0.5.
  • the cells were cultured in a °C shaker, and the OD600 of the cells was measured at 12, 24, 36, 48, 60, 72, 84 and 96 hours, and the growth state of each of the cells was observed (Fig. 3).
  • the amount of volatilization and formaldehyde removal rate are calculated according to the formula: 100% (process liquid starting formaldehyde) - residual formaldehyde % of the treatment liquid - volatile formaldehyde %.
  • the results showed that the strain MR1 had a good effect on removing formaldehyde.
  • the removal rate of 4 and 8 mM formaldehyde solution could reach more than 90% (Fig. 4)
  • the removal rate of 10 mM formaldehyde was about 80% (Fig. 4)
  • 15 and 20 mM formaldehyde The removal rate is approximately 50% and 40% (Figure 4).
  • the culture was carried out in a shaker at 28 ° C. After 96 hours of treatment, the residual formaldehyde concentration (OD 410) of the treatment liquid was measured, and the formaldehyde removal efficiency of the three strains was compared. The results showed that the removal efficiency of Candida by 2 mM formaldehyde was better than that of MR1 and Pseudomonas putida (Fig. 5).
  • the carbohydrates in the metabolites include glucose (Gluc), fructose (Fruc) and the phosphorylation products of these two sugars (G6P and F6P), indicating that some of the formaldehyde is assimilated into carbohydrates, presumably the Xu5P pathway.
  • the formaldehyde assimilation plays a role in the subsequent production of carbohydrates into other metabolic pathways that are converted to organic acids such as acetic acid (Ac), pyruvic acid (PA) and amino acids such as alanine (Ala).
  • Ac acetic acid
  • PA pyruvic acid
  • Al amino acids
  • some of the signal peaks in the formaldehyde assimilation product did not identify the assigned metabolite U1-U6.
  • MR1 was cultured in an inorganic salt medium containing 5 mM methanol, 2 g of bacterial cells were collected by centrifugation, and 4 mM H 13 CHO solution (100 ml) was added to the methanol-free inorganic salt medium for 2 h and 24 h, and the untreated sample was used as a control.
  • CK detects background 13 C signal levels of MR1 bacterial cells. After the treatment, the cells were collected by centrifugation, 3 mL of 10 mM potassium phosphate buffer (KPB, pH 7.4) was added, and the soluble metabolites were extracted by sonication.
  • the enzyme was inactivated by heat treatment in a boiling water bath for 3 min, centrifuged at 12000 rpm at 4 ° C for 30 min, and the supernatant was collected. After the solution was freeze-dried in vacuo, it was dissolved in 0.6 mL of sterile water, centrifuged at 12000 rpm for 3 min, 0.5 mL of the supernatant was placed in a nuclear magnetic tube, and an appropriate amount of deionized formamide was added as an internal reference for nuclear magnetic resonance analysis in a Brooke nuclear magnetic resonance apparatus (DRX 500). The 13 C-NMR analysis of the formaldehyde metabolism profile of MR1 methyl bacteria was carried out on -MHz) (Fig. 7).
  • a plate was prepared by adding a concentration of 4 mM, 8 mM, 12 mM, and 20 mM ethanol, isopropyl alcohol, xylene, benzene, and toluene to a solid medium containing 5 mM methanol.
  • 1 ⁇ l, 2 ⁇ l, 3 ⁇ l, 4 ⁇ l, 5 ⁇ l, 6 ⁇ l, 7 ⁇ l, 8 ⁇ l, and 9 ⁇ l of MR1 bacterial solution having an OD600 of 0.5 were placed on a solid plate, and cultured in a 28 ° C incubator for 48 hours, and the growth was observed.

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Abstract

一株甲基菌(Methylobacterium zatmanni strain)MR1，其在中国典型培养物保藏中心的保藏编号为CCTCC M 2017322，该甲基菌是以甲醛驯化海藻酸钠包埋的活性污泥为材料，用甲基菌的无机盐培养基分离而得的一种甲基营养型细菌，能在以甲醛为唯一碳源的培养基中生长，其在固体培养基上对甲醛的抗性高达30mM，在添加5mM甲醇和2-15mM甲醛的液体培养基中正常生长，对培养基中高浓度的甲醛有较高的去除率。MR1还能在以乙酸钠、苹果酸钠或柠檬酸钠为唯一碳源的无机盐培养基中生长，其中以苹果酸钠为碳源时生长最好。在含有乙酸钠或苹果酸钠或柠檬酸钠培养基中生长时可去除添加的苯系物，去除率达100％。

Description

一种新型甲基杆菌MR1及其应用 技术领域
本发明属于环境微生物领域，具体的说，涉及一株具有特殊甲醛代谢途径的新型甲基杆菌，其可应用于甲醛及苯系物污染的去除。
背景技术
甲醛(HCHO)是一种普遍存在于室内环境的空气污染物，经过装修的各种场所、许多工厂和药品试剂储存库中都发现存在甲醛污染，同时它也作为一个内源代谢物产生于所有生物中，在生物体内甲醛可通过5，10-亚甲基四氢叶酸自发解离产生；此外，各种脱甲基化反应也会产生低浓度的甲醛。在长期的进化过程中，几乎所有生物体内都产生甲醛解毒机制，用以阻止甲醛的致命以及突变作用。
苯系物为苯及衍生物的总称，是一类芳香族有机化合物，环境污染的苯系物来源工业生产、汽车尾气、装修装饰材料(如油漆、板材、装饰材料等)、办公设备(如复印机、打印机、传真机、电脑等)、人为活动(如吸烟、烹饪、燃香等)等。苯、甲苯、乙苯、二甲苯四类为环境污染苯系物的代表性物质。这些苯系物具有神经毒性，因此对人体健康能够产生直接危害，引起神经衰弱、头痛、失眠、眩晕、下肢疲惫等症状，长期接触可以导致人体患上贫血症和白血病。此外，苯系物还能破坏DNA，因此也具有遗传毒性。
甲基营养菌是一群能够利用一碳化合物作为碳源和能源生长的微生物。由于一碳化合物是有机物，所以甲基营养菌是一些化能异养菌。对甲基营养菌代谢一碳化合物的研究表明，这类微生物体内同时具有氧化甲醛为甲酸然后产生CO2的甲醛降解途径和同化甲醛为细胞组成成分的同化途径，因此有大量研究考察这类微生物去除甲醛污染的能力和效果。恶臭假单胞菌(Pseudomonas putida)是一种对溶剂有抗性的细菌，有编码甲醛脱氢酶和甲酸脱氢酶的基因，所以P.putida有氧化甲醛产生甲酸和CO2的能力。有些研究用恶臭假单胞菌来制备生物反应器，结果表明生物反应器对室内甲醛污染有一定的净化作用。
至今在甲基营养型细菌中发现的甲醛同化途径包括核酮糖单磷酸途径(RuMP)、丝氨酸途径及核酮糖单磷酸环状氧化途径。在兼性甲基营养型细菌中甲醛同化通过丝氨酸途径完成，在该途径中HCHO首先通过非酶促反应和四氢叶酸结合生成5，10-亚甲基四氢叶酸，然后由丝氨酸羟甲基转移酶催化5，l0-亚甲基四氢叶酸和甘氨酸缩合形成丝氨酸(Ser)，同时释放四氢叶酸。Ser经过一系列的转化反应生成磷酸烯醇式丙酮酸(PEP)，PEP又经过羧化作用形成苹果酸(Mal)。苹果酸分解生成2个C2化合物，C2化合物转化为乙醛酸(Ox)，而 后在丝氨酸-乙醛酸氨基转移酶的作用下，从乙醛酸产生甘氨酸(Gly)，完成丝氨酸途径的整个循环。RuMP途径存在于很多甲基营养型和非甲基营养细菌中，在该途径中HCHO首先与5-磷酸核酮糖(Ru5P)缩合产生6-磷酸己酮糖(Hu6P)，这一步由6-磷酸己酮糖合成酶(HPS)催化完成；Hu6P在6-磷酸己酮糖异构酶(PHI)作用下异构化生成F6P，F6P随后裂解形成1，6-二磷酸果糖(FBP)，FBP再裂解形成二个C3化合物：3-磷酸甘油醛(GAP)和磷酸二羟丙酮(DHAP)。DHAP之后被用于细胞组成成分的合成，而GAP则通过一系列反应再生固定HCHO的受体Ru5P。
在以甲醇为碳源生长的甲基营养型酵母菌中，甲醇首先被乙醇氧化酶氧化为HCHO，HCHO是甲醇代谢的一个关键性中间产物，它处于甲醇同化和异化途径的分支点上，一部分HCHO可以在非酶催化条件下与还原型谷胱甘肽反应生成S-羟甲基谷胱甘肽，S-羟甲基谷胱甘肽在依赖谷胱甘肽的HCHO脱氢酶(FALDH)、S-甲酸水解酶(FGH)和依赖NADH的甲酸脱氢酶(FDH)等一系列酶的作用下最终氧化生成CO2。此外，有研究结果证实由乙醇脱氢酶催化甲酸和甲醇合成甲酸甲酯的途径也参与HCHO的解毒作用。还一部分HCHO通过木酮糖单磷酸途径(XuMP)同化，在XuMP途径中HCHO在二羟丙酮合成酶(DAS)的作用下直接和5-磷酸木酮糖(Xu5P)结合生成三磷酸甘油醛(GAP)和二羟丙酮(DHA)。DHA对酵母细胞来说是有毒的，在酵母菌中二羟丙酮激酶(DAK)参与二羟丙酮的脱毒作用，DAK催化的反应使二羟丙酮磷酸化，形成无毒性的磷酸二羟丙酮(DHAP)。这两个反应的产物GAP和DHAP随后通过醛缩酶催化的反应形成1，6-二磷酸果糖(FBP)，FBP去磷酸化形成6-磷酸果糖(F6P)，F6P的一部分用于再生HCHO的受体Xu5P，另一部分通过其他途径用于细胞组分的合成。
甲醇在甲基营养酵母中可以诱导与甲醇代谢相关酶蛋白基因启动子的活性。巴斯德毕赤酵母(Pichia pastoris)是一种常见的甲基营养型酵母菌，其甲醇氧化酶(AOX)定位于过氧化物酶体中，以甲醇作碳源时AOX蛋白表达量增至细胞总蛋白的35％-40％，因此基因工程操作中常利用AOX启动子控制外源基因编码蛋白在甲醇酵母中的表达。目前已用甲醇诱导基因的强启动子在毕赤酵母(Pichia pastoris)、多形汉森酵母和假丝酵母(Candida boidinii)中建立了高效的异源基因表达系统，这些表达系统在学术研究和工业领域中有广泛的应用。通过突变体和酶学特性分析证实了DAS是甲基营养酵母甲醇同化作用的关键酶，已从假丝酵母中克隆到编码DAS的基因，DAS酶蛋白是个含有焦磷酸硫胺素的同型二聚体，在细胞质中经历二聚体化后被运送到过氧化物酶体中。
活性污泥中有丰富的微生物，有些微生物因为具有甲醛代谢途径，因此很多研究结 果利用驯化的活性污泥制备生物反应器，分析结果说明这类生物反应器能去除并降解空气中污染的甲醛和其他有机物，但是活性污泥中的微生物种类很多，组成不明确，可能含有一些致病菌。此外，由于活性污泥的成分复杂，在高温和高湿条件下其所含的有机物可能会导致霉菌的繁殖和生长。
发明内容
针对以上技术问题，本发明的目的在于提供一株具有特殊甲醛代谢途径的新型甲基菌，可应用于甲醛及苯系物污染的去除，为净化甲醛和苯系物污染提供新的微生物资源。
为达到上述目的，本发明提供如下技术方案：
一株新型甲基菌MR1，该菌株在中国典型培养物保藏中心的保藏编号为CCTCC NO：M2017322，地址为：湖北省武汉市武昌区武汉大学，该菌株的16S rRNA基因序列如SEQ ID NO.1所示。
本发明还公开了新型甲基菌MR1具有的两种甲醛同化途径，即丝氨酸途径和乙醛酸途径。
本发明还公开了新型甲基菌MR1在净化高浓度甲醛污染中作为生物资源的应用。
本发明还公开了新型甲基菌MR1在降解苯系物中作为生物资源的应用。
本发明还公开了新型甲基菌MR1在乙酸钠或苹果酸钠或柠檬酸钠为碳源时，在降解苯系物中作为生物资源的应用。
本发明的新型甲基菌MR1的获得步骤为：首先用海藻酸盐和海绵包埋从河道中收集的活性污泥，利用污染高浓度甲醛的空气驯化活性污泥中的微生物，然后利用甲基菌的无机盐培养基分离生长快且对甲醛抗性强的甲基菌。
本发明的有益效果：
1、本发明的新型甲基菌MR1是以首先用海藻酸盐和海绵包埋河道中收集的活性污泥，利用污染高浓度甲醛的空气驯化活性污泥中的微生物，然后利用甲基菌的无机盐培养基分离生长快且对甲醛抗性强的甲基菌，其具有特殊甲醛同化途径且可以净化甲醛并能去除污染的苯系物。
2、本发明的MR1主要通过丝氨酸途径和乙醛酸途径同化H13CHO，将甲醛代谢转化为丝氨酸、甘氨酸、苹果酸、乙醛酸、磷酸烯醇式丙酮酸、柠檬酸、异柠檬酸等无毒和无副作用的有机酸和氨基酸。MR1还能在以乙酸钠、苹果酸钠或柠檬酸钠为唯一碳源的无机盐培养基中生长，其中以苹果酸钠为碳源时生长最好。在含有乙酸钠或苹果酸钠或柠檬酸钠培养基中生长时可去除添加的苯系物，去除率达100％。
附图说明
图1：菌株MR1与甲基菌(Methylobacterium zatmanni strain DSM 5688)16SrRNA基因序列的同源性比对；
图2：固体培养基上添加不同浓度甲醛对MR1、假丝酵母和恶臭假单胞菌生长的影响；
图3：液体培养基中添加不同浓度甲醛对MR1、假丝酵母和恶臭假单胞菌生长的影响；
图4：MR1对不同浓度甲醛的去除效率；
图5：MR1、假丝酵母和恶臭假单胞菌对不同浓度甲醛去除率的比较；
图6：恶臭假单胞菌和假丝酵母代谢甲醛的途径分析；
图7：甲基菌MR1代谢甲醛的途径分析。
具体实施方式
下面将结合本发明实施例和附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1菌株的筛选、鉴定及检测
为了克服现有技术的缺陷，本发明首先用海藻酸盐和海绵包埋河道中收集的活性污泥，利用污染高浓度甲醛的空气驯化活性污泥中的微生物，然后利用甲基菌的无机盐培养基分离生长快且对甲醛抗性强的甲基菌，其具体筛选步骤为：
1、活性污泥的包埋及微生物的驯化
采集河道的污泥与水混合，用纱布过滤去除碎石等大颗粒杂物，然后与2％的海藻酸钠(1：1)混合，浸泡海绵并移除多余液体混合物，随后放入有4％的CaCl2溶液中进行固化包埋。将包埋污泥的海绵卷成柱状放入塑料收纳箱中进行驯化处理，收纳箱底部安装一个小型离心风扇，人工制备的甲醛气体通过离心风扇吹入海绵中，甲醛浓度从起始的1mg/m3逐天递增至最终的15mg/m3，连续熏蒸处理15天，在整个驯化期间，海绵基质中的含水量保持在25-40％。
2、驯化微生物的分离
驯化处理结束后，取塑料箱中不同高度和位置的海绵，剪5cm左右的一段，剪碎后置于装有200ml液体甲基菌的无机盐培养基(1升含有KH2PO4 5g，K2HPO4 1g，(NH4)2SO4 2.6g，MgSO4·7H2O 0.2g，CaCl2 0.01g，FeSO4·7H2O 0.001g，酵母膏1.6g(0.16％)，固体培养基加1.5％琼脂)中，使用5mM甲醇作为碳源，28℃下摇床振荡培养7天。经过7天培养出 的菌体离心去掉上清，然后用500μl无菌水悬浮，涂在添加2mM甲醛为唯一碳源的固体培养基上。于28℃的培养箱内培养，获得单菌落做后续的鉴定。
3、形态学观察和分子生物学鉴定
对固体培养基形成的单菌落进行形态学观察，用接种环挑取少量菌体，放入载玻片的水珠中，使用结晶紫和番红染色，在显微镜下观察，结果发现分离菌是细菌，能以甲醛为唯一碳源生长，菌株的菌落呈圆形，菌落颜色为红色，菌落表面光滑湿润，液体培养基培养的菌液无异味。16SrRNA测序数据如SEQ ID NO.1所示。分析结果显示该分离菌与甲基菌(Methylobacterium zatmanni strain DSM 5688)同源性达到99％(图1)，因此将其称为甲基菌MR1。
实施例2MR1对甲醛的抗性、去除甲醛的效果及代谢转化甲醛的途径分析
1.MR1对甲醛的抗性分析
在添加和没有添加酵母膏的固体培养基分别加入4mM、8mM、12mM和20mM的甲醛，将MR1的菌液1％(v/v)接种于含有5mM甲醇和酵母膏的液体培养基中，28℃摇床培养，待OD600达到0.5时各取1μlμ、2μl、3μl、4μl、5μl、6μl、7μl、8μl、9μl菌液点于含有不同浓度甲醛的固体培养基上，28℃温箱中培养48小时后观察其生长状况。结果说明菌株MR1在含有酵母膏和没有酵母膏的无机培养基中生长状况无显著差异，说明MR1的生长不依赖酵母膏。在30mM甲醛胁迫下依然可以生长，说明MR1菌株对甲醛的抗性很高，可达到30mM。
在没有添加酵母膏的甲醇固体培养基上分别加入4mM、6mM、8mM和10mM的甲醛，将MR1、甲基营养型酵母菌假丝酵母(Candida boidinii)、恶臭假单胞菌(Pseudomonas putida)菌液(OD600为0.5)各取2μl、4μl、6μl、8μl、10μl点于含有甲醛的固体培养基上，28℃温箱中培养2-3天后观察生长状况(图2所示)。结果说明三种菌株在含有4mM甲醛的培养基中都能生长，在添加6mM、8mM和10mM甲醛的培养基中假丝酵母菌不能生长，而MR1和恶臭假单胞菌能生长，这说明MR1的甲醛抗性与恶臭假单胞菌相当，但强于假丝酵母。
在添加甲醇的液体培养基中接入MR1、假丝酵母菌、恶臭假单胞菌，待每种菌的OD600为0.4-0.5时分别加入2mM、4mM、6mM、8mM、10mM、15mM甲醛，28℃摇床中培养，在12、24、36、48、60、72、84和96小时测定菌体的OD600，观察每种菌体的生长状况(图3)。结果说明在12-24h菌株MR1在所有培养基中的生长速度迅速增加，在随后的36-96h维持相同的生长速率，而假丝酵母菌与恶臭假单胞菌仅有微小的生长速率， 说明这两种菌株的生长速率显著低于MR1，在72-96h假丝酵母菌的生长速率增加的幅度稍大于恶臭假单胞菌，在24-96h内MR1的生长均显著快于假丝酵母与恶臭假单胞菌。这证明在添加甲醛的液体培养基中MR1比假丝酵母菌与恶臭假单胞菌有更好的生长优势。
2.MR1去除甲醛的效果分析
在不含酵母膏的液体培养基中分别添加4mM、8mM、10mM、15mM和20mM的甲醛和5mM甲醇，然后接种OD600为0.5的MR1菌液1％(v/v)，28℃摇床培养，在8h、12h、24h、36h、48h、60h、72h取1毫升菌液，离心后取上清，使用Nash法测剩余甲醛浓度，用不加菌液但含有相同浓度甲醛的溶液检测处理系统甲醛的挥发量，甲醛去除率按照公式：100％(处理液起始甲醛)-处理液剩余甲醛％-挥发甲醛％计算。结果说明菌株MR1去除甲醛效果很好，处理72h后对于4和8mM甲醛溶液除率能达到90％以上(图4)，对10mM甲醛除率约为80％(图4)，对15和20mM甲醛除率约为50％和40％(图4)。
在添加甲醇的培养基中接入MR1、假丝酵母菌、恶臭假单胞菌菌液，待每种菌的OD600为0.4-0.5时分别加入2mM、4mM、6mM、8mM、10mM、15mM的甲醛，28℃摇床中培养，处理96小时后测定处理液残留甲醛的浓度(OD410)，比较三种菌株的甲醛去除效率。结果说明假丝酵母菌对2mM甲醛的去除效率好于MR1和恶臭假单胞菌(图5)，对4-15mM甲醛的去除效率恶臭假单胞菌低于MR1和假丝酵母(图5)，对于2-6mM低浓度甲醛的去除效率假丝酵母高于MR1(图5)，对8-15mM高浓度甲醛MR1的去除效率大于假丝酵母(图5)。
3.MR1代谢转化甲醛的途径分析
挑选恶臭假单胞菌和假丝酵母单菌落在含5mM甲醇的无机盐液体培养液(500ml)中分别扩大培养2-3天，4000rpm 4°离心10min，收集2克鲜重菌体，加入4mM H13CHO(4mM甲醛处理)溶液(100ml)处理24小时，离心收集菌体，加入3毫升无菌水悬浮，超声波处理10min破碎细胞，同时用不加甲醛培养液处理的相同鲜重菌体作为对照(CK)。超声波破碎之后95°加热5分钟使酶失活，12000rpm 4°离心10min，收集上清后离心浓缩干燥，加入500μl无菌水溶解，加甲酰胺做内参，13C-NMR核磁共振检测分析甲醛代谢产物(图6)。结果说明假丝酵母的代谢产物种类较多，其甲醛代谢谱中有很强的甲酸信号峰，说明甲醛氧化途径在甲醛代谢和脱毒过程中发挥作用。代谢产物中的糖类物质包括葡萄糖(Gluc)、果糖(Fruc)及这两种糖的磷酸化产物(G6P和F6P)，这说明有部分甲醛被同化为糖类物质，推测应该是Xu5P途径在甲醛同化过程中发挥作用，产生的糖类物质随后进入其他代谢途径被转化为有机酸如乙酸(Ac)、丙酮酸(PA)和氨基酸如丙氨酸(Ala)。此外，甲醛同化产 物中还有些信号峰未鉴定归属的代谢产物U1-U6。恶臭假单胞菌的甲醛代谢产物种类很少，其代谢谱中甲酸的信号峰最强，说明甲醛氧化是其甲醛代谢和脱毒的主要途径。此外还有乙酸(Ac)、丙酮酸(PA)和丙氨酸(Ala)及未知产物U1和U2，但是这些代谢产物的信号峰很弱，说明甲醛同化在恶臭假单胞菌甲醛代谢和脱毒过程中发挥的作用很有限。
在含有5mM甲醇的无机盐培养基中培养MR1，离心收集菌体细胞2g，在不含甲醇的无机盐培养基中加入4mM H13CHO溶液(100ml)处理2h和24h，以不处理样品作为对照(CK)检测MR1菌体细胞的背景13C信号水平。处理结束后离心收集菌体细胞，加入3mL10mM磷酸钾缓冲液(KPB，pH7.4)，超声破碎抽提可溶性代谢产物，沸水浴加热处理3min使酶失活，12000rpm 4℃离心30min，收集上清液经真空冷冻干燥后，用0.6mL无菌水溶解，12000rpm离心3min，取0.5mL上清装入核磁管，加入适量去离子甲酰胺作为核磁共振分析的内参，在布鲁克核磁共振仪(DRX 500-MHz)上进行13C-NMR分析MR1甲基菌的甲醛代谢谱(图7)。H13CHO标记样品中化学位移参照内参的共振峰(166.85ppm)，13C-NMR谱中的共振峰通过和已知化合物的13C-NMR谱进行比较鉴定其归属。13C-NMR数据分析结果发现MR1处理液中含有甲酸(H13COOH)，说明MR1可将H13CHO氧化为H13COOH。在MR1的H13CHO代谢谱中观察到4mM H13CHO处理2h导致很多内源性的代谢产物信号峰降低，说明短时间的甲醛胁迫会消耗这些内源性的代谢产物，4mM H13CHO处理24h后这些内源性的代谢产物信号峰恢复到接近处理前的水平，说明H13CHO代谢产生这些内源性的代谢产物。此外，H13CHO代谢产物中还有一些与假丝酵母相同的未知产物U1-U6。对代谢产物信号峰进行归属后发现H13CHO代谢谱中有很多丝氨酸途径的代谢产物包括丝氨酸(Ser)、甘氨酸(Gly)、苹果酸(Mal)、乙醛酸(Ox)和磷酸烯醇式丙酮酸(PEP)及乙醛酸途径的代谢产物如柠檬酸(Cit)和异柠檬酸(Icit)，这证实甲基菌MR1主要通过丝氨酸途径和乙醛酸途径同化H13CHO，将甲醛代谢转化为无毒性的有机酸和氨基酸，这两种代谢途径在MR1的甲醛代谢和脱毒中发挥重要作用，甲基菌MR1具有与恶臭假单胞菌和假丝酵母不同的甲醛代谢机制。
实施例3MR1对苯系物和其他有机污染物的耐受性及去除苯系物的效果分析
1.MR1对苯系物和其他有机污染物的耐受性分析
在含有5mM甲醇的固体培养基中分别添加浓度为4mM、8mM、12mM和20mM乙醇、异丙醇、二甲苯、苯、甲苯做成平板。取OD600为0.5的MR1菌液1μl、2μl、3μl、4μl、5μl、6μl、7μl、8μl、9μl点于固体平板上，28℃温箱中培养48小时后观察其生长状况。结果说明乙醛、乙醇、异丙醇的存在对MR1菌株的生长没有明显影响，它对乙醛、乙醇、 异丙醇的耐受性可达20mM，但在含有异丙醇的平板上生长状况较差。在添加二甲苯、苯、甲苯的平板上能生长，对二甲苯、苯、甲苯的抗性达到20mM，但在添加甲苯的平板上生长状况较差。
2.MR1去除苯系物的效果分析
在含有5mM甲醇的液体培养基(50ml)中分别添加0.5mM、1mM、2mM和3mM的苯、甲苯、二甲苯，接种OD600为0.5的MR1菌液100μl。28℃振荡培养，在24、48、72小时测定其OD600值，结果说明MR1能在含有苯系物培养基中生长。12000rp离心15min取上清用高效液相色谱法测定培养基中剩余苯系物含量。结果说明MR1在24h内能完全去除培养基中污染的低浓度苯系物，去除率达到100％。此外，在液体培养基中添加5mM乙酸钠或苹果酸钠或柠檬酸钠为碳源时生长速度比甲醇为碳源的快，其中以苹果酸钠为碳源生长最快，能在含有乙酸钠或苹果酸钠或柠檬酸钠为碳源的培养基中生长时去除添加的苯系物。
最终，以上实施例和附图仅用以说明本发明的技术方案而非限制，尽管通过上述实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。

Claims (5)

  1. 一种新型甲基菌MR1，其特征在于：该菌株在中国典型培养物保藏中心的保藏编号为CCTCC NO：M 2017322，该菌株的16S rRNA基因序列如SEQ ID NO.1所示。
  2. 如权利要求1所述的新型甲基菌MR1，其特征在于：该菌株具有丝氨酸途径和乙醛酸途径两种甲醛同化途径。
  3. 如权利要求1所述的新型甲基菌MR1，其特征在于：所述的新型甲基菌MR1在净化高浓度甲醛污染中作为生物资源的应用。
  4. 如权利要求1所述的新型甲基菌MR1，其特征在于：所述的新型甲基菌MR1在降解苯系物中作为生物资源的应用。
  5. 如权利要求4所述的新型甲基菌MR1，其特征在于：所述的新型甲基菌MR1在乙酸钠或苹果酸钠或柠檬酸钠为碳源时，在降解苯系物中作为生物资源的应用。
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