CN107400646A - 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 PDFInfo
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Abstract
本发明涉及一株高产丁醇梭菌,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。所述高产丁醇梭菌能够利用葡萄糖或半乳糖发酵转化生物丁醇,而且发酵全程不需要调节pH。本发明的梭菌经发酵培养基富集,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。本发明利用海洋生物质产生物丁醇的潜质,能够高效利用葡萄糖或半乳糖发酵转化生物丁醇;进行发酵时,其主要发酵产物为丁醇和丙酮,而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,发酵全程不需要调节pH,发酵成本大幅降低。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。
Description
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,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,MnCl2·4H2O 0.1g/L,ZnCl2 0.07g/L,H3BO30.006g/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
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。
SEQUENCE LISTING
<110> 汕头大学
<120> 一株高产丁醇梭菌及其筛选与应用
<130> 2017
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1419
<212> DNA
<213> 人工序列
<400> 1
cgaaccggcg tgctttaccc ttgcaagtcg agcgatgaag ctccttcggg agcggattag 60
cggcggacgg gtgagtaaca cgtgggtaac ctgcctcata gaggggaata gcctttcgaa 120
aggaagatta ataccgcata agattgtagt gccgcatggc atagcaatta aaggagtaat 180
ccgctatgag atggacccgc gtcgcattag ctagttggtg aggtaacggc tcaccaaggc 240
gacgatgcgt agccgacctg agagggtgat cggccacatt gggactgaga cacggcccag 300
actcctacgg gaggcagcag tggggaatat tgcacaatgg gggaaaccct gatgcagcaa 360
cgccgcgtga gtgatgacgg tcttcggatt gtaaagctct gtcttcaggg acgataatga 420
cggtacctga ggaggaagcc acggctaact acgtgccagc agccgcggta atacgtaggt 480
ggcaagcgtt gtccggattt actgggcgta aagggagcgt aggtggatat ttaagtggga 540
tgtgaaatac tcgggcttaa cctgggtgct gcattccaaa ctggatatct agagtgcagg 600
agaggaaagt agaattccta gtgtagcggt gaaatgcgta gagattagga agaataccag 660
tggcgaaggc gactttctgg actgtaactg acactgaggc tcgaaagcgt ggggagcaaa 720
caggattaga taccctggta gtccacgccg taaacgatga atactaggtg taggggttgt 780
catgacctct gtgccgccgc taacgcatta agtattccgc ctggggagta cggtcgcaag 840
attaaaactc aaaggaattg acgggggccc gcacaagcag cggagcatgt ggtttaattc 900
gaagcaacgc gaagaacctt acctagactt gacatctcct gaattaccct taatcgggga 960
agcccttcgg ggcaggaaga caggtgktgc atggttgtcg tcagctcgtg tcgtgagatg 1020
ttgggttaag tcccgcaacg agcgcaaccc ttattgttag ttgctaccat ttagttgagc 1080
actctagcga gactgcccgg gttaaccggg aggaaggtgg ggatgacgtc aaatcatcat 1140
gccccttatg tctagggcta cacacgtgct acaatggctg gtacagagag atgctaaacc 1200
gtgaggtgga gccaaacttt aaaaccagtc tcagttcgga ttgtaggctg aaactcgcct 1260
acatgaagct ggagttgcta gtaatcgcga atcagaatgt cgcggtgaat acgttcccgg 1320
gccttgtaca caccgcccgt cacaccatga gagttggcaa tacccaaagt tcgtgagcta 1380
acgcgcaagc gaggcagcga cctaaggtat gtacagccg 1419
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NaCl1.0g/L,MgCl2·6H2O 0.5g/L,KH2PO4 0.2g/L,NH4Cl 0.3g/L,KCl 0.3g/L,CaCl2·2H2O0.015g/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 0.006g/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16SrDNA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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CN112961799A (zh) * | 2021-02-08 | 2021-06-15 | 汕头大学 | 一株梭菌及利用其制备生物丁醇的方法 |
CN115141816A (zh) * | 2022-06-09 | 2022-10-04 | 广州市乾相生物科技有限公司 | 一种提高梭菌利用碳源转化丁醇的方法及应用 |
CN115141816B (zh) * | 2022-06-09 | 2023-08-08 | 广州市乾相生物科技有限公司 | 一种提高梭菌利用碳源转化丁醇的方法及应用 |
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