WO2019041567A1 - 一株高产丁醇梭菌及其筛选与应用 - Google Patents

一株高产丁醇梭菌及其筛选与应用 Download PDF

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WO2019041567A1
WO2019041567A1 PCT/CN2017/110776 CN2017110776W WO2019041567A1 WO 2019041567 A1 WO2019041567 A1 WO 2019041567A1 CN 2017110776 W CN2017110776 W CN 2017110776W WO 2019041567 A1 WO2019041567 A1 WO 2019041567A1
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fermentation
glucose
yield
clostridium
galactose
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吴奕瑞
孙崇然
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汕头大学
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/145—Clostridium
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/16—Butanols
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

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  • the invention belongs to the technical field of microbial biotechnology and bioenergy production, and particularly relates to a high-yield Clostridium butyricum and its screening and application.
  • Bioenergy is mainly obtained by microbial fermentation, with mild production conditions and high safety. Compared with other new energy sources such as hydropower and nuclear power, bioenergy has lower environmental and ecological impacts, so bioenergy is also a substitute for fossil fuels. No choice.
  • lignocellulosic biomass marine biomass is a sustainable biomass that has been gradually paid attention to in recent years. Its distribution is also very extensive, but there are still many problems to be solved in effective use, so how to realize its bio-energy Transformation has become a new and important research topic.
  • Butanol one of the bioenergy products, has low volatility, low hygroscopicity, and is less corrosive to equipment than ethanol, and has high safety and convenience during storage and transportation.
  • butanol has energy density and octane number close to gasoline, and can be miscible with gasoline in any proportion.
  • the promotion and application of butanol fuel can be realized without technical modification of existing power equipment. Therefore, biobutanol has a huge market potential as a new type of bioenergy.
  • Butanol can also be used as an important C4 chemical platform compound. It is a synthetic raw material for various chemical products and organic reagents. Its development value and significance are even more Significantly, it has received wide attention.
  • butanol is generally concentrated in the field of microbial fermentation production.
  • the production of butanol by microbial fermentation shows a good development prospect, but the problems in its production are mainly due to its low substrate conversion rate and high production cost. Fermentation of biobutanol using inexpensive, abundant marine biomass resources (such as red algae) will effectively reduce production costs.
  • most of the strains that are more intensive and widely used in the production of biobutanol belong to the genus Clostridium sp., which are Gram-positive bacteria, strictly anaerobic, and can form endospores. Rod-shaped bacteria.
  • Clostridium is fermented with glucose, galactose and xylose to produce acetone, butanol and ethanol, ie ABE fermentation.
  • the tolerance to butanol is low; and during the fermentation process, a large amount of acetic acid and butyric acid are produced, which inhibits the growth of the cells, and the cells enter irreversible without adjusting the pH value.
  • the spore state leads to a large accumulation of acidic substances. As a result, the fermentation product is not uniform.
  • butanol In addition to the production of butanol, the production of butanol is too low, and the substrate conversion rate is not high, which is accompanied by a large amount of other by-products such as ethanol, acetic acid and butyric acid.
  • the difficulty and cost of separation and purification hinder the industrialization of microbial fermentation to a certain extent.
  • the purpose of the present invention is to provide a Clostridium sp., which is capable of utilizing red algae hydrolysate glucose or galactose to efficiently produce biobutanol, and transforming it into a biofuel research.
  • the rate is high and the by-product species and yield are extremely low, which has the prospect and potential as an excellent industrial butanol fermentation strain.
  • a high-yield Clostridium butyricum is deposited in the General Microbial Culture Collection and Management Center of China, and its deposit number is CGMCC 14506.
  • Clostridium butyricum fermentation does not require pH adjustment throughout the fermentation.
  • Clostridium butyricum can efficiently convert biobutanol by glucose or galactose fermentation.
  • the above screening method for the high-yield Clostridium butyricum mainly includes the following steps:
  • the composition of the fermentation medium is: carbon source substrate (glucose or galactose) 30 g / L, yeast extract 10 g / L, NaHCO 3 2.52 g / L, 100 ⁇ salt solution 10 mL, 1000 ⁇ trace elements 1 mL of solution, 1.952 g/L of 2-(N-morpholinyl)ethanesulfonic acid; wherein the 100 ⁇ salt solution includes NaCl 1.0 g/L, MgCl 2 ⁇ 6H 2 O 0.5 g/L, KH 2 PO 4 0.2 g/L, NH 4 Cl 0.3g/L, KCl 0.3g/L, CaCl 2 ⁇ 2H 2 O 0.015g/L; the 1000 ⁇ trace element solution includes FeCl 2 ⁇ 4H 2 O 1.5g/L, CoCl 2 ⁇ 6H 2 O 0.19g/L, MnCl 2 ⁇ 4H 2 O 0.1g/L, ZnCl 2 0.07g/L, H 3 BO 3
  • the main components of the enhanced Clostridium culture medium are: peptone, 10.0 g/L; beef leaching powder, 10.0 g/L; yeast powder, 3.0 g/L; glucose, 5.0 g/L; soluble starch, 1.0 g / L; sodium chloride, 5.0 g / L; sodium acetate, 3.0 g / L; cysteine hydrochloride, 0.5 g / L; agar, 1.5 g / L.
  • the high temperature treatment in the step (1) is treated at 70 ° C for 0.5 hours.
  • the method further comprises the step (3) gene PCR amplification, which mainly comprises: taking the fermentation liquid obtained in the step (4), collecting the bacteria by centrifugation, extracting the total DNA of the genome, and using the template as a template for PCR amplification of the 16S rDNA;
  • the amplification primers were 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACT-3') respectively;
  • the PCR reaction conditions were: 95 ° C for 5 min; 94 ° C for 30 s, 55 ° C for 30 s, and 72 ° C for 1.5 min. , 30 cycles; 72 ° C for 10 min.
  • Clostridium butyricum application utilizes glucose or galactose (the main hydrolysate of red algae) to efficiently produce biobutanol.
  • the strain can utilize glucose or galactose as a carbon source, and the two carbon sources are the main hydrolyzate of red algae, and it is not directly using the red algae hydrolysis product to produce butanol.
  • the invention separates and obtains a Clostridium strain from the mangrove sediment environment, and is named as WST.
  • the fermentation experiment analysis shows that the strain can utilize the red algae hydrolysate glucose or galactose to efficiently produce biobutanol, and the conversion rate is high and the deputy is high.
  • the product type and yield are extremely low, and it has the prospect and potential as an excellent industrial butanol fermentation strain.
  • the present invention has the following advantages: First, the use of marine biomass (such as red algae, the main components of which are cellulose and agar, whose main hydrolysates are glucose and galactose) produce the potential of butanol, The strain WST can efficiently convert biobutanol by glucose or galactose fermentation. Second, when the strain of the invention is fermented with glucose as a substrate, the main fermentation products are butanol and acetone, and the amount of ethanol and other organic acids is extremely low. It is advantageous to simplify the purification technology of butanol; 3. Compared with other reported wild strains, the strain of the present invention has a relatively high amount of butanol production and product conversion; fourth, the strain of the present invention does not need to adjust pH throughout the fermentation. The fermentation cost is greatly reduced.
  • marine biomass such as red algae, the main components of which are cellulose and agar, whose main hydrolysates are glucose and galactose
  • the strain WST can efficiently convert biobutanol by glucose or
  • Figure 1 is an electron micrograph of the strain WST of the present invention.
  • Figure 2 is a phylogenetic tree diagram of the strain WST of the present invention.
  • Figure 3 is a graph showing the yield of each product, the amount of glucose residual sugar and the growth of the strain of the strain WST of the present invention using 30 g/L glucose as a substrate after anaerobic fermentation for 120 hours;
  • Figure 4 is a graph showing the yield of each product, the amount of galactose residual sugar and the growth of the strain of the strain WST of the present invention using 30 g/L galactose as a substrate after anaerobic fermentation for 120 hours;
  • Figure 5 is a graph showing changes in pH during fermentation of the strain WST of the present invention.
  • the soil samples were obtained from mangrove sediments (below 10 cm), accurately weighed 5.0 g for high temperature (70 ° C) treatment for 0.5 hours, and then added to 45 mL of fermentation medium with 30 g/L of glucose as a carbon source. Anaerobic enrichment culture was carried out at 30 ° C and 150 rpm. After culturing for 24 hours, the enriched cells were serially diluted and applied to a solid culture plate of reinforced Clostridium culture medium (RCM), and cultured at 30 ° C for 24-48 hours, and a single colony was picked. The fermentation was carried out for 96 hours in a fermentation culture concentration, and the yield of butanol in the medium was determined by gas chromatography.
  • RCM Clostridium culture medium
  • the composition of the medium is: glucose, 30g / L; NaHCO 3 , 2.52g / L; yeast extract, 10g / L; 100 ⁇ salt solution (NaCl 1.0g / L; MgCl 2 ⁇ 6H2O, 0.5g / L; KH 2 PO 4 , 0.2 g/L; NH 4 Cl, 0.3 g/L; KCl, 0.3 g/L; CaCl 2 ⁇ 2H 2 O, 0.015 g/L), 10 mL; 1000 ⁇ trace element solution (FeCl 2 ⁇ 4H 2 O, 1.5 g/L; CoCl 2 ⁇ 6H 2 O, 0.19 g/L; MnCl 2 ⁇ 4H 2 O, 0.1 g/L; ZnCl 2 , 0.07 g/L; H 3 BO 3 , 0.006
  • strain WST was observed by scanning electron microscopy.
  • the cells were rod-shaped, often arranged in pairs or short chains, round or tapered ends, showing polymorphism. Gram staining is positive; spores can be produced; strictly anaerobic.
  • the amplification primers were 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACT-3'), respectively.
  • the PCR reaction conditions were: 95 ° C for 5 min; 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 1.5 min, 30 cycles; 72 ° C for 10 min.
  • the sequencing results were subjected to BLAST analysis with the sequences on NCBI.
  • the sequence length was 1419 bp, and the analysis results showed that the strain WST provided in this example had the greatest homology (99%) with Clostridium diolis DMS 5431.
  • the sequences of this example were compared with NCBI-like strains, and the phylogenetic tree was constructed using MEGA6 software. The tree diagram constructed is shown in Fig. 2.
  • the basal medium used in the Clostridium of the present embodiment is prepared, and the composition thereof is mainly: yeast extract, 10 g/L; NaHCO 3 , 2.52 g/L; 100 ⁇ salt solution (NaCl 1.0 g/L; MgCl 2 ⁇ 6H 2 O) , 0.5g / L; KH 2 PO 4 , 0.2g / L; NH 4 Cl, 0.3g / L; KCl, 0.3g / L; CaCl 2 ⁇ 2H 2 O, 0.015g / L), 10mL; 1000 ⁇ trace Element solution (FeCl 2 ⁇ 4H 2 O, 1.5 g/L; CoCl 2 ⁇ 6H 2 O, 0.19 g/L; MnCl 2 ⁇ 4H 2 O, 0.1 g/L; ZnCl 2 , 0.07 g/L; H 3 BO 3 , 0.006g / L; Na 2 MoO 4 ⁇ 2H 2 O, 0.036g
  • Both the glucose mother liquor and the galactose mother liquor were 500 g/L, sterilized at 115 ° C for 15 min, and then added to the basal medium according to the ratio of the concentration of the fermented sugar (30 g/L) to finally obtain a fermentation medium.
  • the WST strain stored at -80 ° C was inoculated into the above fermentation medium for activation.
  • 1 mL of the bacterial solution was inoculated to fresh 49 mL of glucose or galactose containing 30 g/L.
  • anaerobic fermentation was carried out at 30 ° C and 150 rpm for 120 hours, and the fermentation broth was collected every 24 hours, and the content of the fermentation product, the amount of residual sugar, and the biomass of the cells were measured by GC.
  • the yield of biobutanol can also reach 12.11g / L (see Figure 4), the conversion rate of 0.40g butanol / g galactose, the final concentration of ethanol Very low (0.13g/L).
  • the strain can use glucose or galactose (the main hydrolysate of red algae) as a substrate to convert biobutanol, and the amount of butanol produced by the strain of the present example and the product conversion ratio are higher.
  • Table 1 compares the fermentation effect of the present embodiment with the fermentation effect of the prior art.
  • the pH value of the Clostridium in the present embodiment during the fermentation process is as shown in Fig. 5.
  • the pH value gradually decreases in the first 12 hours until about 4.7, and then slowly rises, and after 24 hours, slowly rises back to about 5.0. After that, it has been fluctuating around 5.0. Therefore, the Clostridium of the present embodiment does not need to adjust the pH value throughout the process, and the fermentation cost can be greatly reduced.

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Abstract

一株高产丁醇梭菌，保藏于中国普通微生物菌种保藏管理中心，其保藏编号为CGMCC 14506，经发酵培养基富集，再由强化型梭菌培养基平板在厌氧条件下培养并分离而得。所述梭菌利用葡萄糖或半乳糖发酵转化生物丁醇，且发酵全程不需要调节pH。

Description

一株高产丁醇梭菌及其筛选与应用 技术领域
本发明属于微生物生物技术和生物能源生产技术领域，尤其涉及一株高产丁醇梭菌及其筛选与应用。
背景技术
随着煤、石油等化石燃料的快速消耗，寻找新能源引起了政府及学者的高度关注。生物能源主要通过微生物发酵的方式获取，具有生产条件温和及较高的安全性；与水电、核电等其他新型能源相比，生物能源具有较低的环境生态影响，因此生物能源也是替代化石燃料的不二选择。除木质纤维素生物质外，海洋生物质是近年来被逐步关注的可持续性生物质，其分布也十分广泛，但有效利用一直还存在诸多善待解决的问题，因此如何实现其向生物能源的转化也就成为了新的重要研究课题。
作为生物能源产品之一的丁醇具有低挥发性、低吸湿性，且对于设备的腐蚀性也较乙醇更低，在储存与运输的过程中具有较高的安全性和便利性。此外，丁醇具有接近汽油的能量密度和辛烷值，并且可与汽油以任意比例互溶，在应用过程中不需要对现有的动力设备进行技术改造就可以实现丁醇燃料的推广与应用。因此生物丁醇作为一种新型生物能源，具有巨大的市场潜力，同时丁醇也可作为一个重要的C4化工平台化合物，是多种化工产品与有机试剂的合成原料，其开发价值和意义更为显著，已经受到广泛重视。
目前丁醇的研究普遍集中在其微生物发酵生产的领域。微生物发酵法生产丁醇展示了良好的发展前景，但在其生产中面临的问题主要是其底物转化率较低，生产成本居高不下。若能利用廉价、丰富的海洋生物质资源(如红藻)发酵生产生物丁醇将能有效的降低生产成本。另一方面，作为生产生物丁醇研究较为深入且应用较为广泛的菌株大多数属于梭状芽孢杆菌属(Clostridium sp.)，都是革兰氏阳性菌，严格厌氧的，且能形成内孢子的杆状细菌。绝大多数梭菌以利用葡萄糖、半乳糖和木糖等发酵产生丙酮、丁醇和乙醇，即ABE发酵。但是由于菌株转化机能不高，对丁醇耐受性较低；而且发酵过程中，大量的乙酸和丁酸的产生，抑制菌体生长，在没有调节pH值的情况下，菌体进入不可逆的 孢子状态，导致酸性物质的大量积累。从而导致其发酵产物的不单一性，在产生丁醇的同时，伴随大量其他副产物如乙醇、乙酸及丁酸等的产生，丁醇产量过低，且底物转化率不高，加大了分离纯化的难度和成本，在一定程度上阻碍了微生物发酵的工业化进程。
发明内容
本发明的目的在于针对化石燃料资源日益匮乏，在对生物能源研究的基础上，提供一株梭菌Clostridium sp.，该菌株能利用红藻水解产物葡萄糖或半乳糖高效转化生产生物丁醇，转化率高且副产物种类和产量均极低，具有作为优秀工业丁醇发酵菌株的前景和潜力。
为了实现上述的目的，采用如下的技术方案：
一株高产丁醇梭菌，保藏于中国普通微生物菌种保藏管理中心，其保藏编号为CGMCC 14506。
进一步的，所述高产丁醇梭菌发酵全程不需要调节pH。
进一步的，所述高产丁醇梭菌能够高效利用葡萄糖或半乳糖发酵转化生物丁醇。
上述高产丁醇梭菌的筛选培养方法，主要包括以下步骤：
(1)将红树林底泥进行高温处理后,加入到葡萄糖或半乳糖为碳源的发酵培养基中；
(2)在30℃和150rpm的条件下进行厌氧富集培养，培养24小时；
(3)将富集的菌体采用释稀涂布平板法进行一系列梯度稀释(10-4-10-9)后，将不同稀释度的菌液分别涂布到强化型梭菌培养基(Reinforced Clostridial Medium，RCM)固体培养平板中，于30℃进行恒温培养24-48小时后，挑取单一菌落，转接于发酵培养基中进行发酵培养96小时。
进一步的，所述发酵培养基的组成为：碳源底物(葡萄糖或半乳糖)30g/L，酵母提取物10g/L，NaHCO3 2.52g/L，100×盐溶液10mL，1000×微量元素溶液1mL，2-(N-吗啉基)乙磺酸1.952g/L；其中所述100×盐溶液包括NaCl 1.0g/L，MgCl2·6H2O 0.5g/L，KH2PO4 0.2g/L，NH4Cl 0.3g/L，KCl 0.3g/L，CaCl2·2H2O 0.015g/L；所述1000×微量元素溶液包括FeCl2·4H2O 1.5g/L，CoCl2·6H2O 0.19g/L，MnCl2·4H2O 0.1g/L，ZnCl2 0.07g/L，H3BO3 0.006 g/L，Na2MoO4·2H2O 0.036g/L，NiCl2·6H2O 0.024g/L，CuCl2·2H2O 0.002g/L。
进一步的，所述强化型梭菌培养基的主要成分为：蛋白胨，10.0g/L；牛肉浸出粉，10.0g/L；酵母粉，3.0g/L；葡萄糖，5.0g/L；可溶性淀粉，1.0g/L；氯化钠，5.0g/L；醋酸钠，3.0g/L；盐酸半胱氨酸，0.5g/L；琼脂，1.5g/L。
进一步的，步骤(1)所述高温处理为70℃处理0.5小时。
进一步的，还包括步骤(3)基因PCR扩增，主要包括：取步骤(4)得到的发酵菌液，离心收集菌体，提取基因组总DNA，并以此为模板进行16S rDNA的PCR扩增；其中扩增引物分别为27F(5’-AGAGTTTGATCCTGGCTCAG-3’)和1492R(5’-GGTTACCTTGTTACGACT-3’)；PCR反应条件为：95℃5min；94℃30s，55℃30s，72℃1.5min，30个循环；72℃10min。
上述高产丁醇梭菌的应用，利用葡萄糖或半乳糖(红藻主要水解产物)，高效转化生产生物丁醇。菌株能够利用葡萄糖或半乳糖为碳源，而该两碳源为红藻的主要水解产物，还不是利用红藻水解产物直接产丁醇。
本发明从红树林底泥环境中分离获得一株梭菌，命名为WST，发酵实验分析显示，该菌株能利用红藻水解产物葡萄糖或半乳糖，高效转化生产生物丁醇，转化率高且副产物种类和产量均极低，具有作为优秀工业丁醇发酵菌株的前景和潜力。
与现有技术相比，本发明具有以下优势：一、利用海洋生物质(如红藻，主要成分为纤维素和琼胶，其主要水解产物为葡萄糖和半乳糖)产生物丁醇的潜质，菌株WST能够高效利用葡萄糖或半乳糖发酵转化生物丁醇；二、本发明的菌株以葡萄糖为底物进行发酵时，其主要发酵产物为丁醇和丙酮，乙醇和其他有机酸产生量极低，有利于简化丁醇的纯化技术；三、与其他已报道的野生菌株相比，本发明的菌株的产丁醇量及产物转化率是比较高的；四、本发明的菌株发酵全程不需要调节pH，发酵成本大幅降低。
附图说明
图1为本发明菌株WST的电镜照片；
图2为本发明菌株WST的系统进化树图；
图3为本发明菌株WST以30g/L葡萄糖为底物，经厌氧发酵120小时后，各产物的产量、葡萄糖残糖量及菌株生长情况；
图4为本发明菌株WST以30g/L半乳糖为底物，经厌氧发酵120小时后，各产物的产量、半乳糖残糖量及菌株生长情况；
图5为本发明菌株WST发酵过程中pH变化情况。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述。
实施例
1、菌株WST的分离筛选方法
土壤样品来自于红树林底泥(表层10cm以下)，准确称取5.0g进行高温(70℃)处理0.5小时后,加入到45mL以30g/L的葡萄糖为碳源的发酵培养基中。在30℃和150rpm的条件下进行厌氧富集培养。培养24小时后，将富集的菌体进行梯度稀释并涂布到强化型梭菌培养基(RCM)的固体培养平板中，于30℃进行恒温培养24-48小时后，挑取单一菌落，转接于发酵培养集中进行发酵培养96小时，通过气相色谱测定培养基中丁醇的产量。通过测定，最终确定分离得到一株高产丁醇的菌株，即Clostridium sp.WST。其中培养基的组成为：葡萄糖,30g/L；NaHCO3,2.52g/L；酵母提取物,10g/L；100×盐溶液(NaCl 1.0g/L；MgCl2·6H2O,0.5g/L；KH2PO4,0.2g/L；NH4Cl,0.3g/L；KCl,0.3g/L；CaCl2·2H2O,0.015g/L),10mL；1000×微量元素溶液(FeCl2·4H2O,1.5g/L；CoCl2·6H2O,0.19g/L；MnCl2·4H2O,0.1g/L；ZnCl2,0.07g/L；H3BO3,0.006g/L；Na2MoO4·2H2O,0.036g/L；NiCl2·6H2O,0.024g/L；CuCl2·2H2O,0.002g/L)1mL；2-(N-吗啉基)乙磺酸(MES),1.952g/L。将得到的菌株WST进行扫描电镜观察，如图1所示，细胞呈杆状，常排列成对或短链，圆的或渐尖的末端，显示多态性。革兰氏染色为阳性；可产生芽孢；严格厌氧。
2、菌株WST的16S rDNA基因PCR扩增和序列测定方法。
取5-10mL上述菌株WST发酵菌液，离心收集菌体，通过基因组提取试剂盒提取基因组总DNA，并以此为模板进行16S rDNA的PCR扩增。扩增引物分别为27F(5’-AGAGTTTGATCCTGGCTCAG-3’)和1492R(5’-GGTTACCTTGTTACGACT-3’)。PCR反应条件为：95℃5min；94℃30s，55℃30s，72℃1.5min，30个循环；72℃10min。扩增结束后，将PCR产物 进行纯化后连接pMD-19T载体，然后进行测序。测序结果与NCBI上的序列进行BLAST分析。序列长度为1419bp，分析结果显示，本实施例提供的菌株WST与Clostridium diolis DMS 5431具有最大的同源性(99％)。将本实施例的序列与NCBI相似的菌株进行比对，采用MEGA6软件构建系统进化树，所构建的树状图如图2所示。
3、菌株WST发酵生产生物丁醇的特性分析。
首先，配制本实施例梭菌所用的基础培养基，其组成主要为：酵母提取物,10g/L；NaHCO3,2.52g/L；100×盐溶液(NaCl 1.0g/L；MgCl2·6H2O,0.5g/L；KH2PO4,0.2g/L；NH4Cl,0.3g/L；KCl,0.3g/L；CaCl2·2H2O,0.015g/L),10mL；1000×微量元素溶液(FeCl2·4H2O,1.5g/L；CoCl2·6H2O,0.19g/L；MnCl2·4H2O,0.1g/L；ZnCl2,0.07g/L；H3BO3,0.006g/L；Na2MoO4·2H2O,0.036g/L；NiCl2·6H2O,0.024g/L；CuCl2·2H2O,0.002g/L)1mL；2-(N-吗啉基)乙磺酸(MES),1.952g/L。加入蒸馏水并使其完全溶解，定容至900mL。随后依次加入还原剂Na2S·9H2O,0.048g/L；L-半胱氨酸(Cys)，0.0242g/L和DL-二硫苏糖醇(DTT)0.077g/L，并用4mol/L的HCl调节pH值至6.0，121℃灭菌20min。葡萄糖母液和半乳糖母液均为500g/L,115℃灭菌15min，之后根据发酵糖浓度的比例(30g/L)加入基础培养基中，最终得到发酵培养基。
接下来，取于-80℃保存的WST菌种，接种到上述发酵培养基中进行活化，培养10-12小时后，取1mL菌液接种到新鲜的49mL含有30g/L的葡萄糖或半乳糖的发酵培养基中，置于30℃和150rpm条件下进行厌氧发酵120小时，每隔24小时，收集发酵液，并通过GC测定发酵产物含量、残糖量及菌体生物量。
发酵结果说明，本实施例的菌株可以利用30g/L葡萄糖为底物的发酵培养基中，在30℃和150rpm条件下发酵120小时后，转化生物丁醇的产量可达到16.52g/L(见图3)，转化率为0.55g丁醇/g葡萄糖，且菌株WST发酵产物中乙醇量极低(0.27g/L)，副产物丁酸和乙酸的量在发酵到48小时后，也逐渐减少至接近0g/L，且发酵过程全程无需调节pH值，这些特性均为简化生物丁醇的分离提纯带来了便利。另外，在以30g/L半乳糖为底物的情况下，生物丁醇的产量也可达到12.11g/L(见图4)，转化率为0.40g丁醇/g半乳糖，乙醇终浓度也极低(0.13g/L)。实验证明该菌株可利用葡萄糖或半乳糖(红藻的主要水解产物)为底物转化生物丁醇，且本实施例的菌株的产丁醇量及产物转化率是比 较高的。表1为本实施例的发酵效果跟现有技术的发酵效果的对比。
另外，本实施例的梭菌在发酵过程中pH值的变化如图5所示，在前12小时pH值逐渐降低，直至4.7左右，然后缓慢回升，在24小时后，缓慢回升到5.0左右。之后一直在5.0上下波动。因此本实施例的梭菌在整个过程中无需调节pH值，可大大降低发酵成本。
表1不同丁醇发酵菌株的发酵效果比较
Figure PCTCN2017110776-appb-000001
以上所揭露的仅为本发明的较佳实施例而已，当然不能以此来限定本发明之权利范围，因此依本发明权利要求所作的等同变化，仍属本发明所涵盖的范围。

Claims (10)

  1. 一株高产丁醇梭菌，其特征在于，保藏于中国普通微生物菌种保藏管理中心，其保藏编号为CGMCC 14506。
  2. 根据权利要求1所述高产丁醇梭菌，其特征在于，发酵全程不需要调节pH。
  3. 根据权利要求1所述高产丁醇梭菌，其特征在于，能够利用葡萄糖或半乳糖发酵转化生物丁醇。
  4. 根据权利要求1所述高产丁醇梭菌的筛选培养方法，其特征在于，主要包括以下步骤：
    (1)将红树林底泥进行高温处理后,加入到葡萄糖或半乳糖为碳源的发酵培养基中；
    (2)在30℃和150rpm的条件下进行厌氧富集培养，培养24小时；
    (3)将富集的菌体采用释稀涂布平板法进行10-4-10-9的系列梯度稀释后，将不同稀释度的菌液分别涂布到强化型梭菌培养基固体培养平板中，于30℃进行恒温培养24-48小时后，挑取单一菌落，转接于发酵培养基中进行发酵培养96小时。
  5. 根据权利要求4所述筛选培养方法，其特征在于，所述发酵培养基的组成为：含葡萄糖或半乳糖的碳源底物30g/L，酵母提取物10g/L，NaHCO3 2.52g/L，100×盐溶液10mL，1000×微量元素溶液1mL，2-(N-吗啉基)乙磺酸1.952g/L；其中所述100×盐溶液包括NaCl 1.0g/L，MgCl2·6H2O 0.5g/L，KH2PO40.2g/L，NH4Cl 0.3g/L，KCl 0.3g/L，CaCl2·2H2O 0.015g/L；所述1000×微量元素溶液包括FeCl2·4H2O 1.5g/L，CoCl2·6H2O 0.19g/L，MnCl2·4H2O0.1g/L，ZnCl2 0.07g/L，H3BO3 0.006g/L，Na2MoO4·2H2O 0.036g/L，NiCl2·6H2O 0.024g/L，CuCl2·2H2O 0.002g/L。
  6. 根据权利要求4所述筛选培养方法，其特征在于，所述强化型梭菌培养 基的主要成分为：蛋白胨，10.0g/L；牛肉浸出粉，10.0g/L；酵母粉，3.0g/L；葡萄糖，5.0g/L；可溶性淀粉，1.0g/L；氯化钠，5.0g/L；醋酸钠，3.0g/L；盐酸半胱氨酸，0.5g/L；琼脂，1.5g/L。
  7. 根据权利要求4所述筛选培养方法，其特征在于，步骤(1)所述高温处理为70℃处理0.5小时。
  8. 根据权利要求4所述筛选培养方法，其特征在于，所述红树林底泥为表层10cm以下。
  9. 根据权利要求4所述筛选培养方法，其特征在于，还包括步骤(4)基因PCR扩增，主要包括：取步骤(3)得到的发酵菌液，离心收集菌体，提取基因组总DNA，并以此为模板进行16S rDNA的PCR扩增；其中扩增引物分别为27F(5’-AGAGTTTGATCCTGGCTCAG-3’)和1492R(5’-GGTTACCTTGTTACGACT-3’)；PCR反应条件为：95℃ 5min；94℃ 30s，55℃ 30s，72℃ 1.5min，30个循环；72℃ 10min。
  10. 根据权利要求1-3任一项所述高产丁醇梭菌的应用，其特征在于，利用红藻主要水解产物葡萄糖或半乳糖高效转化生产生物丁醇。
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