CN106480109A - 一种利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法 - Google Patents
一种利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法 Download PDFInfo
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- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 23
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title abstract description 13
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- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 6
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- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 claims description 6
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 6
- ZBIBSMMVRNDSNI-UHFFFAOYSA-N diazanium;selenate Chemical compound [NH4+].[NH4+].[O-][Se]([O-])(=O)=O ZBIBSMMVRNDSNI-UHFFFAOYSA-N 0.000 claims description 6
- IMBKASBLAKCLEM-UHFFFAOYSA-L ferrous ammonium sulfate (anhydrous) Chemical compound [NH4+].[NH4+].[Fe+2].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O IMBKASBLAKCLEM-UHFFFAOYSA-L 0.000 claims description 6
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- AGBQKNBQESQNJD-UHFFFAOYSA-M lipoate Chemical compound [O-]C(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-M 0.000 claims description 6
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
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- 239000000203 mixture Substances 0.000 claims description 6
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 claims description 6
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- 235000019319 peptone Nutrition 0.000 claims description 6
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 claims description 6
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- 239000011684 sodium molybdate Substances 0.000 claims description 6
- 235000015393 sodium molybdate Nutrition 0.000 claims description 6
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 6
- XMVONEAAOPAGAO-UHFFFAOYSA-N sodium tungstate Chemical compound [Na+].[Na+].[O-][W]([O-])(=O)=O XMVONEAAOPAGAO-UHFFFAOYSA-N 0.000 claims description 6
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
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- 238000009835 boiling Methods 0.000 description 1
- DNZWLJIKNWYXJP-UHFFFAOYSA-N butan-1-ol;propan-2-one Chemical compound CC(C)=O.CCCCO DNZWLJIKNWYXJP-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- 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
-
- 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
- C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
-
- 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- 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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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Life Sciences & Earth Sciences (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法,其以稀硫酸预处理的玉米秸秆为原料,通过添加纤维素酶和木聚糖酶对玉米秸秆进行酶解糖化获得玉米秸秆水解液,然后再以玉米秸秆水解液为原料,首先利用酪丁酸梭菌Clostridium tyrobutyricum ATCC25755进行丁酸发酵,然后接种能够转化有机酸为对应醇的菌株Clostridium ljungdahlii ATCC 55380并通入合成气实现发酵生产丁醇。本发明方法中所用材料来源广泛、副产物少,不仅实现了农业废弃物资源的合理利用,而且减少了环境污染,同时也为丁醇的生产提供了一条新途径。
Description
技术领域
本发明属于农业生物质能源技术领域,具体涉及一种利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法。
背景技术
我国的生物质资源十分丰富,玉米秸秆作为我国第一大秸秆资源,年产量超过2亿吨,约占总秸秆资源的35%,主要成分为木质纤维素,经酸或酶水解后可以产生低聚糖、戊糖和己糖等,是一种优良的生物质资源。露天焚烧或者作为生活燃料燃烧是目前秸秆的主要处理方式,既浪费了资源又对环境造成了污染。随着能源危机和环境污染等问题的日益严重,生物质能源以其清洁和可再生的特性受到广泛的关注,以木质纤维素为原料生产乙醇已成为当前领域的热点。
与乙醇相比,丁醇无论是在能量密度还是与汽油的配伍性上都具有明显的优势,是一种优良的生物质燃料。目前的生产方法主要有化工合成法和生物发酵法。然而随着化石能源的枯竭,化工合成法在可持续性上受到很大的限制。而传统的丙酮丁醇发酵法副产物较多,底物转化率低,并且原料来源主要是玉米和糖蜜,成本较高。探索一条新的丁醇生产途径具有十分重要的意义。
合成气来源于煤、石油、生物质和有机废物的气化,其主要成份为 CO、H2 和 CO2,还含有少量甲烷和硫氮化合物,可通过厌氧发酵转化为甲烷、乙醇、乙酸、丁醇和丁酸等燃料和化学品,来源广泛且价格低廉。生物质的气化过程可以消除不同原料间的化学差异性,一些有毒物质和难以降解的有机物也可以被利用,可以直接越过传统生物转化过程中木质素难以充分利用的障碍,因此合成气厌氧发酵技术被认为是一项极具潜力和竞争力的技术。目前合成气的研究主要集中在制乙醇和丁醇上,将有机酸转化为相应醇的研究也越来越多。本发明利用混合菌发酵的技术,研究了以玉米秸秆水解液为原料耦合合成气发酵生产丁醇的方法。
发明内容
本发明目的在于克服现有技术缺陷,提供一种利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法,其材料来源广泛,副产物少,不仅实现了资源的合理利用,而且减少了环境污染,同时也为丁醇的生产提供了一条新途径。
为实现上述目的,本发明采用如下技术方案:
一种利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法,其以玉米秸秆水解液为原料,首先利用酪丁酸梭菌(Clostridium tyrobutyricum ATCC25755)进行丁酸发酵,然后接种能够转化有机酸为对应醇的菌株Clostridium ljungdahlii ATCC 55380并通入合成气进行丁醇发酵;具体包括如下步骤:
(1)玉米秸秆水解液制备:将玉米秸秆与1wt%的稀硫酸按固液比1g:7-9ml的比例混合均匀,121℃灭菌处理1-2h,调节pH至4.7-4.9后加入纤维素酶和木聚糖酶进行酶解糖化,获得玉米秸秆水解液;
(2)玉米秸秆水解液脱毒:将D301阴离子交换树脂置于三角瓶中,然后加入步骤(1)制备所得的玉米秸秆水解液进行摇床震荡处理,处理结束后过滤,取滤液备用;
(3)菌种活化:将酪丁酸梭菌Clostridium tyrobutyricum ATCC 25755、菌株Clostridium ljungdahlii ATCC 55380分别接种到液体活化培养基中,37℃厌氧静置培养1.5-2天,获得酪丁酸梭菌种子液、菌株Clostridium ljungdahlii种子液;
(4)丁酸发酵:向步骤(2)所得滤液中添加发酵培养基,然后接种酪丁酸梭菌种子液于37℃厌氧培养60-72h,获得丁酸发酵后的水解液;
(5)合成气丁醇发酵:以步骤(4)所得丁酸发酵后的水解液为基质,加入储备液和维生素溶液,然后接种菌株Clostridium ljungdahlii种子液,最后通入合成气于37℃厌氧培养5-6天即得。
具体的,步骤(1)中,纤维素酶的用量为15-20U/g秸秆,木聚糖酶的用量为200-220U/g秸秆;酶解糖化的条件为48℃、120rpm-150 rpm摇床振荡48-60h。
步骤(2)具体为:将100g D301阴离子交换树脂置于500ml三角瓶中,然后加入300ml步骤(1)制备所得的玉米秸秆水解液,在摇床中于28-32℃、120rpm震荡处理1-1.5h后过滤。
步骤(3)中,酪丁酸梭菌Clostridium tyrobutyricum ATCC 25755使用的液体活化培养基组成为:30g/ L葡萄糖,5g/ L酵母膏,5g/L蛋白胨,6g/L NaC1,3g/L ( NH4)2·SO4,1.5 g/L K2HPO4,0.6g/L MgSO4·7H2O,0.03 g/L FeSO4·7H2O,0.3 g/L L-盐酸半胱氨酸,0.5mg/L刃天青(用作无氧指示剂),pH6.0。
步骤(3)中,菌株Clostridium ljungdahlii ATCC 55380使用的活化培养基组成为:0.80g/L NaC1,1.0g/L NH4C1,0.10g/L KCl,0.2g/L MgSO4·7H2O,0.04g CaCl2,0.2g/LKH2PO4,1.0g/L NaHCO3,1.0g/L酵母膏,5.0g/L MES(2-吗啉乙磺酸),5.0g/L木糖,0.4g/LL-盐酸半胱氨酸,10 ml/L微量金属元素溶液,10 ml/L维生素溶液,0.5mg/L刃天青(用作无氧指示剂),pH5.80;其中,
微量金属元素溶液的配方如下:2.0g/L氨三乙酸,1g/L硫酸锰,0.8g/L硫酸亚铁铵,0.2g/L氯化钴,0.2g/L硫酸锌,0.02g/L氯化铜,0.02g/L氯化镍,0.02g/L钼酸钠,0.02g/L硒酸铵,0.02g/L钨酸钠;
维生素溶液的配方如下:10mg/L维生素B6,5 mg/L硫胺素,5 mg/L维生素B2,5 mg/L泛酸钙,5mg/L硫辛酸,5mg/L对氨基苯甲酸,5 mg/L烟碱酸,5mg/L维生素B12,2mg/L生物素,2mg/L叶酸。
上述的活化培养基均经过沸水浴煮沸除氧,添加刃天青脱氧至瓶内由粉色转为无色,121℃灭菌20min。接种前添加维生素溶液(经0.22μm微孔滤膜过滤)。酪丁酸梭菌Clostridium tyrobutyricum的接种量为5%,菌株Clostridium ljungdahlii的接种量为10%,接种操作均在厌氧培养箱中完成,培养条件均为37℃厌氧静置培养。
步骤(4)中,所述发酵培养基组成为:5g/ L 酵母膏,5g/ L蛋白胨,6g/L NaC1,3g/L ( NH4)2·SO4,1.5 g/L K2HPO4,0.6g/L MgSO4·7H2O,0.03 g/L FeSO4·7H2O,0.5mg/L刃天青,pH用10mol/L NaOH溶液调至6.0。步骤(4)制备所得丁酸发酵后的水解液分装至250ml厌氧瓶,装液量20ml。酪丁酸梭菌Clostridium tyrobutyricum的接种量为5%。
步骤(5)具体为:向步骤(4)所得丁酸发酵后的水解液中添加60ml 储备液和0.8ml维生素溶液,然后接种10%菌株Clostridium ljungdahlii种子液,通入200ml合成气,于37℃、150rpm厌氧培养5-6天,每天补加100ml合成气;
所述合成气中各原料气的体积百分比为:CO 60%、H2 35%和CO2 5%;
所述储备液配方如下:1L储备液中含有20g MES,10ml微量金属元素溶液,0.2g L-盐酸半胱氨酸,pH用10mol/L NaOH溶液调至7.0,经0.22μm微孔滤膜过滤;其中,
微量金属元素溶液的配方如下:2.0g/L氨三乙酸,1g/L硫酸锰,0.8g/L硫酸亚铁铵,0.2g/L氯化钴,0.2g/L硫酸锌,0.02g/L氯化铜,0.02g/L氯化镍,0.02g/L钼酸钠,0.02g/L硒酸铵,0.02g/L钨酸钠;
所述维生素溶液的配方如下:10mg/L维生素B6,5 mg/L硫胺素,5 mg/L维生素B2,5 mg/L泛酸钙,5mg/L硫辛酸,5mg/L对氨基苯甲酸,5 mg/L烟碱酸,5mg/L维生素B12,2mg/L生物素,2mg/L叶酸。
和现有技术相比,本发明的有益效果:
本发明使用的菌种酪丁酸梭菌(Clostridium tyrobutyricum ATCC25755)和菌株Clostridium ljungdahlii ATCC 55380 均为普通市售产品。酪丁酸梭菌可以同时利用木糖和葡糖糖在厌氧条件下发酵生产丁酸,Clostridium ljungdahlii 是一种目前研究的较多的合成气乙醇发酵菌株,并且在合成气发酵过程中能够将丁酸转化为丁醇。本发明方法以稀硫酸预处理的玉米秸秆为原料,通过添加纤维素酶和木聚糖酶对玉米秸秆进行酶解糖化获得玉米秸秆水解液,然后再以玉米秸秆水解液为原料,首先利用酪丁酸梭菌Clostridium tyrobutyricum ATCC25755进行丁酸发酵,然后接种能够转化有机酸为对应醇的菌株Clostridium ljungdahlii ATCC 55380并通入合成气实现了发酵生产丁醇。本发明方法中所用材料来源广泛、副产物少,不仅实现了农业废弃物资源的合理利用,而且减少了环境污染,同时也为丁醇的生产提供了一条新途径。
具体实施方式
以下结合实施例对本发明的技术方案作进一步地详细介绍,但本发明的保护范围并不局限于此。
下述实施例中,所使用菌种酪丁酸梭菌(Clostridium tyrobutyricumATCC25755)和菌株Clostridium ljungdahlii ATCC 55380均购自美国菌种保藏中心(ATCC)。
实施例1
一种利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法,具体步骤如下:
1)玉米秸秆水解液的制备
称取玉米秸秆15g置于300mL三角瓶中,添加120 mL 质量浓度1%的稀硫酸(w/v=1:8),混合搅拌均匀并于121℃灭菌处理60 min。冷却至室温后用10mol/L的氢氧化钠溶液调节pH值为4.8,按15U/g玉米秸秆、200U/g玉米秸秆的用量添加纤维素酶和木聚糖酶,于48℃、120rpm摇床振荡48h用以进行酶解糖化。结束后用布氏漏斗过滤,除去未糖化的秸秆残渣,获得玉米秸秆水解液。
2)玉米秸秆水解液脱毒
在500ml三角瓶中加入100g D301阴离子交换树脂,然后加入300ml步骤(1)制备所得的玉米秸秆水解液,在摇床中于30℃、120rpm震荡处理1h,处理结束后过滤,取滤液备用。
3)菌种活化
将酪丁酸梭菌Clostridium tyrobutyricum ATCC 25755、菌株Clostridium ljungdahlii ATCC 55380分别接种到液体活化培养基中,37℃厌氧静置培养2天,获得酪丁酸梭菌种子液、菌株Clostridium ljungdahlii种子液;其中,
酪丁酸梭菌Clostridium tyrobutyricum ATCC 25755使用的液体活化培养基组成为:30g/ L葡萄糖,5g/ L酵母膏,5g/L蛋白胨,6g/L NaC1,3g/L ( NH4)2·SO4,1.5 g/L K2HPO4,0.6g/L MgSO4·7H2O,0.03 g/L FeSO4·7H2O,0.3 g/L L-盐酸半胱氨酸,0.5mg/L刃天青(用作无氧指示剂),pH6.0;
菌株Clostridium ljungdahlii ATCC 55380使用的活化培养基组成为:0.80g/LNaC1,1.0g/L NH4C1,0.10g/L KCl,0.2g/L MgSO4·7H2O,0.04g CaCl2,0.2g/L KH2PO4,1.0g/L NaHCO3,1.0g/L酵母膏,5.0g/L MES(2-吗啉乙磺酸),5.0g/L木糖,0.4g/L L-盐酸半胱氨酸,10 ml/L微量金属元素溶液,10 ml/L维生素溶液,0.5mg/L刃天青(用作无氧指示剂),pH5.80;其中,
微量金属元素溶液的配方如下:2.0g/L氨三乙酸,1g/L硫酸锰,0.8g/L硫酸亚铁铵,0.2g/L氯化钴,0.2g/L硫酸锌,0.02g/L氯化铜,0.02g/L氯化镍,0.02g/L钼酸钠,0.02g/L硒酸铵,0.02g/L钨酸钠;
维生素溶液的配方如下:10mg/L维生素B6,5 mg/L硫胺素,5 mg/L维生素B2,5 mg/L泛酸钙,5mg/L硫辛酸,5mg/L对氨基苯甲酸,5 mg/L烟碱酸,5mg/L维生素B12,2mg/L生物素,2mg/L叶酸。
上述的活化培养基均经过沸水浴煮沸除氧,添加0.5mg/L刃天青脱氧至瓶内由粉色转为无色,121℃灭菌20min。接种前添加维生素溶液(经0.22μm微孔滤膜过滤)。酪丁酸梭菌Clostridium tyrobutyricum的接种量为5%,菌株Clostridium ljungdahlii的接种量为10%,接种操作均在厌氧培养箱中完成,培养条件均为37℃厌氧静置培养。
(4)丁酸发酵:
向步骤(2)所得滤液中添加发酵培养基,然后接种酪丁酸梭菌种子液于37℃厌氧培养72h,获得丁酸发酵后的水解液并测定丁酸含量;制备所得丁酸发酵后的水解液分装至250ml厌氧瓶,装液量20ml;
所述发酵培养基组成为:5g/ L 酵母膏,5g/ L蛋白胨,6g/L NaC1,3g/L ( NH4)2·SO4,1.5 g/L K2HPO4,0.6g/L MgSO4·7H2O,0.03 g/L FeSO4·7H2O,0.5mg/L刃天青,pH用10mol/L NaOH溶液调至6.0。酪丁酸梭菌Clostridium tyrobutyricum的接种量为5%。
(5)合成气丁醇发酵:
步骤(5)具体为:向步骤(4)所得丁酸发酵后的水解液中添加60ml 储备液和0.8ml维生素溶液,然后接种10%菌株Clostridium ljungdahlii种子液,通入200ml合成气,于37℃、150rpm厌氧培养6天,每天补加100ml合成气,测定丁醇含量。
所述合成气中各原料气的体积百分比为:CO 60%、H2 35%和CO2 5%;
所述储备液(stock solution)配方如下:1L储备液中含有20g MES,10ml微量金属元素溶液,0.2g L-盐酸半胱氨酸,pH用10mol/L NaOH溶液调至7.0,经0.22μm微孔滤膜过滤;其中,
微量金属元素溶液的配方如下:2.0g/L氨三乙酸,1g/L硫酸锰,0.8g/L硫酸亚铁铵,0.2g/L氯化钴,0.2g/L硫酸锌,0.02g/L氯化铜,0.02g/L氯化镍,0.02g/L钼酸钠,0.02g/L硒酸铵,0.02g/L钨酸钠;
所述维生素溶液的配方如下:10mg/L维生素B6,5 mg/L硫胺素,5 mg/L维生素B2,5 mg/L泛酸钙,5mg/L硫辛酸,5mg/L对氨基苯甲酸,5 mg/L烟碱酸,5mg/L维生素B12,2mg/L生物素,2mg/L叶酸。
试验结果:
步骤(4)丁酸发酵后的水解液中丁酸浓度为66.6 mmol/L。添加储备液和维生素溶液后稀释为16.7 mmol/L,经合成气丁醇发酵后丁醇产量为8.2 mmol/L,转化率为49.1%。由此可见:本发明以玉米秸秆水解液为原料耦合合成气发酵生产丁醇在技术上是完全可行的,是一种生产丁醇的新途径。
Claims (7)
1.一种利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法,其特征在于,包括如下步骤:
(1)玉米秸秆水解液制备:将玉米秸秆与1wt%的稀硫酸按固液比1g:7-9ml的比例混合均匀,121℃灭菌处理1-2h,调节pH至4.7-4.9后加入纤维素酶和木聚糖酶进行酶解糖化,获得玉米秸秆水解液;
(2)玉米秸秆水解液脱毒:将D301阴离子交换树脂置于三角瓶中,然后加入步骤(1)制备所得的玉米秸秆水解液进行摇床震荡处理,处理结束后过滤,取滤液备用;
(3)菌种活化:将酪丁酸梭菌Clostridium tyrobutyricum ATCC 25755、菌株Clostridium ljungdahlii ATCC 55380分别接种到液体活化培养基中,37℃厌氧静置培养1.5-2天,获得酪丁酸梭菌种子液、菌株Clostridium ljungdahlii种子液;
(4)丁酸发酵:向步骤(2)所得滤液中添加发酵培养基,然后接种酪丁酸梭菌种子液于37℃厌氧培养60-72h,获得丁酸发酵后的水解液;
(5)合成气丁醇发酵:以步骤(4)所得丁酸发酵后的水解液为基质,加入储备液和维生素溶液,然后接种菌株Clostridium ljungdahlii种子液,最后通入合成气于37℃厌氧培养5-6天即得。
2.根据权利要求1 所述利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法,其特征在于,步骤(1)中,纤维素酶的用量为15-20U/g秸秆,木聚糖酶的用量为200-220U/g秸秆;酶解糖化的条件为48℃、120-150rpm摇床振荡48-60h。
3. 根据权利要求1 所述利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法,其特征在于,步骤(2)具体为:将100g D301阴离子交换树脂置于500ml三角瓶中,然后加入300ml步骤(1)制备所得的玉米秸秆水解液,在摇床中于28-32℃、120rpm震荡处理1-1.5h后过滤。
4.根据权利要求1所述利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法,其特征在于,步骤(3)中,酪丁酸梭菌Clostridium tyrobutyricum ATCC 25755使用的液体活化培养基组成为:30g/ L葡萄糖,5g/ L酵母膏,5g/L蛋白胨,6g/L NaC1,3g/L ( NH4)2·SO4,1.5g/L K2HPO4,0.6g/L MgSO4·7H2O,0.03 g/L FeSO4·7H2O,0.3 g/L L-盐酸半胱氨酸,0.5mg/L刃天青,pH6.0。
5.根据权利要求1所述利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法,其特征在于,步骤(3)中,菌株Clostridium ljungdahlii ATCC 55380使用的活化培养基组成为:0.80g/L NaC1,1.0g/L NH4C1,0.10g/L KCl,0.2g/L MgSO4·7H2O,0.04g CaCl2,0.2g/LKH2PO4,1.0g/L NaHCO3,1.0g/L酵母膏,5.0g/L MES,5.0g/L木糖,0.4g/L L-盐酸半胱氨酸,10 ml/L微量金属元素溶液,10 ml/L维生素溶液,0.5mg/L刃天青,pH5.80;其中,
微量金属元素溶液的配方如下:2.0g/L氨三乙酸,1g/L硫酸锰,0.8g/L硫酸亚铁铵,0.2g/L氯化钴,0.2g/L硫酸锌,0.02g/L氯化铜,0.02g/L氯化镍,0.02g/L钼酸钠,0.02g/L硒酸铵,0.02g/L钨酸钠;
维生素溶液的配方如下:10mg/L维生素B6,5 mg/L硫胺素,5 mg/L维生素B2,5 mg/L泛酸钙,5mg/L硫辛酸,5mg/L对氨基苯甲酸,5 mg/L烟碱酸,5mg/L维生素B12,2mg/L生物素,2mg/L叶酸。
6. 根据权利要求1所述利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法,其特征在于,步骤(4)中,所述发酵培养基组成为:5g/ L 酵母膏,5g/ L蛋白胨,6g/L NaC1,3g/L( NH4)2·SO4,1.5 g/L K2HPO4,0.6g/L MgSO4·7H2O,0.03 g/L FeSO4·7H2O,0.5mg/L刃天青,pH用10mol/L NaOH溶液调至6.0。
7. 根据权利要求1所述利用玉米秸秆水解液耦合合成气发酵生产丁醇的方法,其特征在于,步骤(5)具体为:向步骤(4)所得丁酸发酵后的水解液中添加60ml 储备液和0.8ml维生素溶液,然后接种10%菌株Clostridium ljungdahlii种子液,通入200ml合成气,于37℃、150rpm厌氧培养5-6天,每天补加100ml合成气;
所述合成气中各原料气的体积百分比为:CO 60%、H2 35%和CO2 5%;
所述储备液配方如下:1L储备液中含有20g MES,10ml微量金属元素溶液,0.2g L-盐酸半胱氨酸,pH用10mol/L NaOH溶液调至7.0,经0.22μm微孔滤膜过滤;其中,
微量金属元素溶液的配方如下:2.0g/L氨三乙酸,1g/L硫酸锰,0.8g/L硫酸亚铁铵,0.2g/L氯化钴,0.2g/L硫酸锌,0.02g/L氯化铜,0.02g/L氯化镍,0.02g/L钼酸钠,0.02g/L硒酸铵,0.02g/L钨酸钠;
所述维生素溶液的配方如下:10mg/L维生素B6,5 mg/L硫胺素,5 mg/L维生素B2,5 mg/L泛酸钙,5mg/L硫辛酸,5mg/L对氨基苯甲酸,5 mg/L烟碱酸,5mg/L维生素B12,2mg/L生物素,2mg/L叶酸。
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