CN109161566A - 一种利用玉米芯全组分生产丁酸的方法 - Google Patents
一种利用玉米芯全组分生产丁酸的方法 Download PDFInfo
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- CN109161566A CN109161566A CN201811144501.1A CN201811144501A CN109161566A CN 109161566 A CN109161566 A CN 109161566A CN 201811144501 A CN201811144501 A CN 201811144501A CN 109161566 A CN109161566 A CN 109161566A
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- corncob
- lignin
- corncobs
- butyric acid
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- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 8
- GAEKPEKOJKCEMS-UHFFFAOYSA-N gamma-valerolactone Chemical compound CC1CCC(=O)O1 GAEKPEKOJKCEMS-UHFFFAOYSA-N 0.000 claims abstract description 38
- 229920005610 lignin Polymers 0.000 claims abstract description 28
- 241000193452 Clostridium tyrobutyricum Species 0.000 claims abstract description 22
- 238000000855 fermentation Methods 0.000 claims abstract description 22
- 230000004151 fermentation Effects 0.000 claims abstract description 22
- 235000000346 sugar Nutrition 0.000 claims abstract description 18
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
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- 238000000926 separation method Methods 0.000 claims abstract description 4
- 239000000758 substrate Substances 0.000 claims abstract description 3
- IWRMHJYRXMAJNR-UHFFFAOYSA-N dimethyl hydrogen phosphate 1,3-dimethyl-2H-imidazole Chemical compound COP(=O)(OC)O.CN1CN(C=C1)C IWRMHJYRXMAJNR-UHFFFAOYSA-N 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 28
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 8
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- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 3
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 2
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- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 abstract description 4
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 9
- 235000005822 corn Nutrition 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
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- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 3
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- IOCJWNPYGRVHLN-MMALYQPHSA-N (2r)-2-amino-3-[[(2r)-2-amino-2-carboxyethyl]disulfanyl]propanoic acid;hydrochloride Chemical compound Cl.OC(=O)[C@@H](N)CSSC[C@H](N)C(O)=O IOCJWNPYGRVHLN-MMALYQPHSA-N 0.000 description 1
- 108010084185 Cellulases Proteins 0.000 description 1
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- -1 Methyl esters salt Chemical class 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
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- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
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- CDQSJQSWAWPGKG-UHFFFAOYSA-N butane-1,1-diol Chemical compound CCCC(O)O CDQSJQSWAWPGKG-UHFFFAOYSA-N 0.000 description 1
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- FYHXNYLLNIKZMR-UHFFFAOYSA-N calcium;carbonic acid Chemical compound [Ca].OC(O)=O FYHXNYLLNIKZMR-UHFFFAOYSA-N 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
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- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
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- C12P7/00—Preparation of oxygen-containing organic compounds
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- C12P7/52—Propionic acid; Butyric acids
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Abstract
本发明公开了一种利用玉米芯全组分生产丁酸的方法。以γ‑戊内酯和1,3‑二甲基咪唑磷酸二甲酯盐([Mmim]DMP)为溶剂,对玉米芯进行预处理,以水为反溶剂沉淀玉米芯并抽滤分离得到残渣,以干燥后的玉米芯残渣为底物,纤维素酶进行酶水解,得到可发酵糖;预处理溶剂提取出的木质素用酸沉淀出来,用于制备包埋酪丁酸梭菌的大孔海藻酸钙‑木质素微球,从而使其利用可发酵糖发酵产丁酸,实现玉米芯全组分的综合利用。本发明有效利用了玉米芯的全组分发酵生产丁酸,且[Mmim]DMP与γ‑戊内酯的联合使用大大提高了预处理效果,增强了玉米芯的酶解效率,获得了高产率的可发酵糖,酪丁酸梭菌经过大孔海藻酸钙‑木质素微球的包埋,与游离细胞相比丁酸产量也得到了提升。
Description
技术领域
本发明涉及生物质资源玉米芯综合利用技术领域,具体地涉及一种利用玉米芯全组分生产丁酸的方法。
背景技术
玉米是国际粮食作物之一,也是一种可交易的商品。在中国,玉米种植广泛,2016年产量约为2.2亿吨。每年根据玉米粒和玉米芯之间的比例,甜玉米加工业预计生产约4000万吨玉米芯副产品。在南非,玉米是主要的出口商品,主要用来生产二代玉米产品,如玉米片,玉米粉和葡萄糖等。由于每年消耗大量的玉米,玉米芯已成为南非最大的农业废弃物之一,年产量约为900万吨,占玉米残留量的近20%。在过去,人们将大多数玉米芯当做燃料燃烧,作为废弃物丢弃或用作肥料,这不仅造成了巨大的资源浪费,而且造成了严重的环境污染,影响了生态平衡。为了减少人类对不可再生资源的依赖,许多研究人员越来越关注农业生物质的利用,以生产生物燃料及高附加值化学品。木质纤维素生物质是一种理想的原料,因为其可再生且在世界各地大量供应。玉米芯是一种实用的木质纤维素生物质原料,具有低木质素和高碳水化合物含量的优点。因此,已被中国的生物炼制工业开发和利用,用于制备木糖,木寡糖和生物发酵产物,如木糖醇,乙醇,丁二醇和糠醛等。
在综合利用玉米芯之前,必须对原料进行预处理,因为纤维素的高结晶度和木质素的存在将使酶难以进入生物质内部并导致低产率的可发酵糖,因此需要通过预处理破坏木质纤维素的抗性结构,实现三大组分的分离。生物质预处理通常有以下几种方法,包括物理法,如蒸汽***,氨纤维***,微波处理或热解;化学法,如浓酸水解,稀酸水解,碱处理或有机溶剂处理;还有生物学方法。离子液体逐渐被认为是用于木质纤维素预处理的最有希望的新溶剂。离子液体是熔点低于100 ℃的盐,并且具有广泛的物理化学性质,例如低挥发性,高热稳定性,不可燃性,独特的溶解特性,且对环境和人类健康无害。通过选择合适的阴阳离子组分,组合出来的离子液体的物理、化学性质会有很大差异。1,3-二甲基咪唑磷酸二甲酯盐([Mmim]DMP)是一种环保型溶剂,可用于玉米芯的预处理,溶解木质纤维素,且和纤维素酶具有生物相容性。然而,仅使用单个组分预处理生物质是比较浪费的,因为对于许多商业应用来说离子液体仍然太昂贵。生物质衍生的溶剂 γ-戊内酯被证明可用于促进木质纤维素生物质的温和预处理,γ-戊内酯是一种理想的可持续发展的溶剂,可用于生产能源和碳基消费品,在[Mmim]DMP中加入γ-戊内酯用作共溶剂,可以克服传质阻力,辅助提高[Mmim]DMP溶解木质纤维素,从而对木质纤维素生物质进行温和而有效的预处理。
玉米芯经过预处理之后,酶解糖化可以获得可发酵糖,主要成分为葡萄糖、木糖,在预处理过程中提取出的木质素也是一种资源,为了将其利用,考虑到酪丁酸梭菌是一种可以同时利用葡萄糖、木糖为碳源生产丁酸的优势菌株,将其利用木质素、海藻酸钠、碳酸钙成分进行包埋形成微球,以发酵生产丁酸。从而实现玉米芯全组分的综合利用。
发明内容
本发明的目的在于开发一种利用玉米芯全组分生产丁酸的工艺,通过酶水解改善生物质转化,提高可发酵糖生产率,以提取出的木质素制备微球包埋酪丁酸梭菌,使其利用可发酵糖产丁酸。
本发明的目的是通过以下技术方案实现的:
所述的一种利用玉米芯全组分生产丁酸的工艺,具体步骤如下:
(1)玉米芯洗涤干燥后,粉碎;
(2)在氮气氛围保护下,将玉米芯粉末与1,3-二甲基咪唑磷酸二甲酯盐([Mmim]DMP)、γ-戊内酯混合进行预处理,之后冷却至室温,加入去离子水作为反溶剂,搅拌反应以沉淀玉米芯粉末,抽滤分离获得玉米芯残渣和滤液;
(3)将玉米芯残渣用去离子水洗涤并干燥后,与乙酸盐缓冲液混合,加入纤维素酶酶解,反应结束后将溶液煮沸以淬灭酶促反应,将煮沸溶液离心,获得以葡萄糖、木糖为主要成分的可发酵糖液;
(4)向步骤(2)获取的滤液中加酸调节pH,低温沉淀滤液中木质素,将木质素沉淀物用去离子水洗涤并干燥;
(5)将酪丁酸梭菌重悬于含有海藻酸钠、木质素和碳酸钙的混合溶液中,用注射器滴加进磁力搅拌的氯化钙溶液中,挤压法包埋酪丁酸梭菌;
(6)以可发酵糖液为碳源发酵包埋酪丁酸梭菌的微球,生产丁酸。
本发明的方法,所述步骤(1)中,玉米芯粉碎粒径为80-120目。
所述步骤(2)中,将玉米芯粉末与1,3-二甲基咪唑磷酸二甲酯盐按照质量比1:6.5~1:19.5混合,之后添加10~16 wt%的γ-戊内酯,于90~110 ℃下加热搅拌3~4 h。
所述步骤(2)中,向经预处理的玉米芯粉末、1,3-二甲基咪唑磷酸二甲酯盐、γ-戊内酯的共混溶液中加入等体积的等离子水反溶剂。
所述步骤(3)中,将玉米芯残渣按照50 g/L的底物浓度与乙酸盐缓冲液混合;乙酸盐缓冲液浓度为50 mM,pH 4.8,之后在50℃、150 rpm条件下酶解。纤维素酶添加量为45FPU/g 干燥玉米芯残渣。
所述步骤(4)中,加入盐酸缓慢酸化至pH调节至2.0,之后在4℃下冷藏沉淀木质素。
所述步骤(5)中,混合溶液中含质量分数2%海藻酸钠,0.5%木质素和5%碳酸钙;氯化钙溶液浓度为11 mg/mL。
所述步骤(6)中,发酵培养基的组成如下:可发酵糖液50 g/L,酵母粉5 g/L,蛋白胨5 g/L,NaCl 6 g/L,(NH4)2SO4 3 g/L,K2HPO4 1.5 g/L, MgSO4·7H2O 0.6 g/L,FeSO4·7H2O 0.03 g/L,L-半胱氨酸盐酸盐0.3 g/L,0.1%刃天青,pH 6.0。
本发明的方法可实现利用玉米芯全组分生产丁酸,γ-戊内酯与[Mmim]DMP联用进行预处理木质纤维素玉米芯,[Mmim]DMP作为环保型离子液体,绿色可再生,γ-戊内酯作为助溶剂,同样是可持续生产资源,两者结合预处理工艺环境友好,符合绿色可持续发展原则。γ-戊内酯与[Mmim]DMP联用预处理玉米芯粉末,高效破坏木质纤维素的结构,降解提取了木质素,为后续的酶促反应提供了优势,预处理后分离的玉米芯残渣加入纤维素酶酶解获得高产率的可发酵糖液,分离的滤液利用酸沉淀出木质素成分,用于包埋酪丁酸梭菌,相比于游离细胞,包埋后的细胞发酵出更纯、产量更高的丁酸,实现了玉米芯的全组分利用。
附图说明
图1为本发明方法流程图。
具体实施方式
下面结合附图说明和具体实施方式对本发明的技术方案作进一步阐述。
实施例中酪丁酸梭菌培养基组成为:葡萄糖30 g/L,酵母粉5 g/L,蛋白胨5 g/L,NaCl,6 g/L,(NH4)2SO43 g/L,K2HPO4 1.5 g/L,MgSO4·7H2O 0.6 g/L,FeSO4·7H2O 0.03 g/L, L-半胱氨酸盐酸盐0.3 g/L,0.1%刃天青,pH 6.0。
发酵培养基的组成如下:可发酵糖液50 g/L,酵母粉5 g/L,蛋白胨5 g/L,NaCl 6g/L,(NH4)2SO4 3 g/L,K2HPO4 1.5 g/L, MgSO4·7H2O 0.6 g/L,FeSO4·7H2O 0.03 g/L,L-半胱氨酸盐酸盐0.3 g/L,0.1%刃天青,pH 6.0。
实施例1
如图1所示,本发明的方法包括如下步骤:
(1)将玉米芯洗涤干燥后粉碎,粒径为80-120目;
(2)预处理:
取0.3 g 玉米芯粉末,加入到5 mL [Mmim]DMP和 16 wt% γ-戊内酯混合物(16 wt%指γ-戊内酯质量/([Mmim]DMP+γ-戊内酯二者总质量))中,于50 mL三颈圆底烧瓶中氮气氛围下90℃加热,1000 rpm磁力搅拌下反应4 h。反应结束后,将反应混合物用相同体积的去离子水稀释,该去离子水用作反溶剂以沉淀预处理的玉米芯粉末。通过抽滤分离所得玉米芯残渣和滤液。
(3)用去离子水反复洗涤玉米芯残渣以除去可能的[Mmim]DMP和 γ-戊内酯残余物,并在65℃下干燥10小时,即为预处理后的玉米芯粉末。
将0.25g预处理后的玉米芯粉末置于25 mL锥形烧瓶中,加入5 mL 50 mM乙酸盐缓冲液(pH4.8),45 FPU/g纤维素酶(购于宁夏和氏璧公司),于恒温水浴振荡器50℃,150 rpm下进行酶解反应72小时。反应结束后,将酶解液煮沸5分钟以淬灭酶促反应,并以3000×g离心5 min,利用HPLC测定糖浓度。根据预处理前的玉米芯中的纤维素和半纤维素含量,计算得葡萄糖收率94.9%,木糖收率53.3%。
(4)向滤液中加入4M HCl缓慢酸化至pH调节至2.0,在4℃冰箱中储存48小时沉淀木质素。将木质素沉淀物用去离子水洗涤并干燥以备进一步使用。
(5)微球制备
酪丁酸梭菌培养基煮沸以除去氧气,在115 ℃下高压灭菌30 min,碳源和氮源分开灭菌。将5%菌液接种到培养基后,37℃培养48小时。经过三代活化,将第三代种子培养基用作下一步培养的母液。使用相同的培养过程将菌株培养至对数生长期,之后3000×g离心10min以收集菌体,并用0.1M磷酸盐缓冲液(pH 7.4)洗涤,控制细胞浓度为10 g/L,重悬于20mL含有质量分数2%海藻酸钠,0.5%木质素和5%碳酸钙的混合溶液,用10 mL注射器滴加到100 mL磁力搅拌的11 mg/mL CaCl2水溶液中以制备包埋酪丁酸梭菌的微球,搅拌反应30min,微球置4 ℃下孵育4 h。然后用0.5 M HCl洗涤直至不再产生气泡,再用磷酸盐缓冲液洗涤,4 ℃保存于无菌蛋白胨溶液中待用。
(6)发酵产丁酸
发酵在5 L发酵罐中进行,内含2 L发酵培养基。在接种之前,发酵装置115 ℃下灭菌30min。将10 wt%微球接种于发酵罐中开始发酵,通N2维持厌氧条件,以5%接种量的游离细胞为对照,37 ℃下培养48小时,通过添加6 M NaOH和3 M H2SO4在线控制pH 6.0。以单批次和重复批次模式进行发酵。在重复批次模式中,固定化细胞一批一批地连续生长,当可发酵糖浓度降至0时,加入新鲜无菌碳源到培养基中以开始新批次的发酵。经过10次重复批次发酵,包埋后的酪丁酸梭菌发酵产生的丁酸产量为22.5 g/L,产率为0.45 g/g,游离细胞仅为22.2 g/L,产率为0.44 g/g,且包埋的酪丁酸梭菌产的丁酸更纯一些。
实施例2
(1)将玉米芯洗涤干燥后粉碎,粒径为80-120目;
(2)预处理:
取0.3 g 玉米芯粉末,加入到10 mL [Mmim]DMP和 14 wt % γ-戊内酯混合物中,于50mL三颈圆底烧瓶中氮气氛围下90 ℃加热,1000 rpm磁力搅拌下反应3 h。反应结束后,将反应混合物用相同体积的去离子水稀释,该去离子水用作反溶剂以沉淀预处理的玉米芯粉末。通过抽滤分离所得玉米芯残渣和滤液。
(3)用去离子水反复洗涤玉米芯残渣以除去可能的[Mmim]DMP和 γ-戊内酯残余物,并在65℃下干燥10小时,即为预处理后的玉米芯粉末。
将0.25g预处理后的玉米芯粉末置于25 mL锥形烧瓶中,加入5 mL 50 mM乙酸盐缓冲液(pH4.8),45 FPU / g纤维素酶(购于宁夏和氏璧公司),于恒温水浴振荡器50℃,150rpm下进行酶解反应72小时。反应结束后,将酶解液煮沸5分钟以淬灭酶促反应,并以3000×g离心5 min,利用HPLC测定糖浓度。根据预处理前的玉米芯中的纤维素和半纤维素含量,计算得葡萄糖收率89.0%,木糖收率44.7%。
(4)向滤液中加入4M HCl缓慢酸化至pH调节至2.0,在4℃冰箱中储存48小时沉淀木质素。将木质素沉淀物用去离子水洗涤并干燥以备进一步使用。
(5)微球制备
酪丁酸梭菌培养基煮沸以除去氧气,在115 ℃下高压灭菌30 min,碳源和氮源分开灭菌。将5%菌液接种到培养基后,37℃培养48小时。经过三代活化,将第三代种子培养基用作下一步培养的母液。使用相同的培养过程将菌株培养至对数生长期,之后3000×g离心10min以收集菌体,并用0.1M磷酸盐缓冲液(pH 7.4)洗涤,控制细胞浓度为10 g/L,重悬于20mL含有质量分数2%海藻酸钠,0.5%木质素和5%碳酸钙的混合溶液,用10 mL注射器滴加到100 mL磁力搅拌的11 mg/mL CaCl2水溶液中以制备包埋酪丁酸梭菌的微球,搅拌反应30min,微球置4 ℃下孵育4 h。然后用0.5 M HCl洗涤直至不再产生气泡,再用磷酸盐缓冲液洗涤,4 ℃保存于无菌蛋白胨溶液中待用。
(6)发酵产丁酸
发酵在5 L发酵罐中进行,内含2 L发酵培养基。在接种之前,发酵装置115 ℃下灭菌30min。将10 wt%微球接种于发酵罐中开始发酵,通N2维持厌氧条件,以5%接种量的游离细胞为对照,37 ℃下培养48小时,通过添加6 M NaOH和3 M H2SO4在线控制pH 6.0。以单批次和重复批次模式进行发酵。在重复批次模式中,固定化细胞一批一批地连续生长,当可发酵糖浓度降至0时,加入新鲜无菌碳源到培养基中以开始新批次的发酵。经过10次重复批次发酵,包埋后的酪丁酸梭菌发酵产生的丁酸产量为15.3 g/L,产率为0.43 g/g,游离细胞仅为14.7 g/L,产率为0.42 g/g。
实施例3
(1)将玉米芯洗涤干燥后粉碎,粒径为80-120目;
(2)预处理:
取0.3 g 玉米芯粉末,加入到15 mL [Mmim]DMP和 10 wt % γ-戊内酯混合物中,于50mL三颈圆底烧瓶中氮气氛围下110 ℃加热,1000 rpm磁力搅拌下反应3 h。反应结束后,将反应混合物用相同体积的去离子水稀释,该去离子水用作反溶剂以沉淀预处理的玉米芯粉末。通过抽滤分离所得玉米芯残渣和滤液。
(3)用去离子水反复洗涤玉米芯残渣以除去可能的[Mmim]DMP和 γ-戊内酯残余物,并在65℃下干燥10小时,即为预处理后的玉米芯粉末。
将0.25g预处理后的玉米芯粉末置于25 mL锥形烧瓶中,加入5 mL 50 mM乙酸盐缓冲液(pH4.8),45 FPU / g纤维素酶(购于宁夏和氏璧公司),于恒温水浴振荡器50℃,150rpm下进行酶解反应72小时。反应结束后,将酶解液煮沸5分钟以淬灭酶促反应,并以3000×g离心5 min,利用HPLC测定糖浓度。根据预处理前的玉米芯中的纤维素和半纤维素含量,计算得葡萄糖收率82.2%,木糖收率42.7%。
(4)向滤液中加入4M HCl缓慢酸化至pH调节至2.0,在4℃冰箱中储存48小时沉淀木质素。将木质素沉淀物用去离子水洗涤并干燥以备进一步使用。
(5)微球制备
酪丁酸梭菌培养基煮沸以除去氧气,在115 ℃下高压灭菌30 min,碳源和氮源分开灭菌。将5%菌液接种到培养基后,37℃培养48小时。经过三代活化,将第三代种子培养基用作下一步培养的母液。使用相同的培养过程将菌株培养至对数生长期,之后3000×g离心10min以收集菌体,并用0.1M磷酸盐缓冲液(pH 7.4)洗涤,控制细胞浓度为10 g/L,重悬于20mL含有质量分数2%海藻酸钠,0.5%木质素和5%碳酸钙的混合溶液,用10 mL注射器滴加到100 mL磁力搅拌的11 mg/mL CaCl2水溶液中以制备包埋酪丁酸梭菌的微球,搅拌反应30min,微球置4 ℃下孵育4 h。然后用0.5 M HCl洗涤直至不再产生气泡,再用磷酸盐缓冲液洗涤,4 ℃保存于无菌蛋白胨溶液中待用。
(6)发酵产丁酸
发酵在5 L发酵罐中进行,内含2 L发酵培养基。在接种之前,发酵装置115 ℃下灭菌30min。将10 wt%微球接种于发酵罐中开始发酵,通N2维持厌氧条件,以5%接种量的游离细胞为对照,37 ℃下培养48小时,通过添加6 M NaOH和3 M H2SO4在线控制pH 6.0。以单批次和重复批次模式进行发酵。在重复批次模式中,固定化细胞一批一批地连续生长,当可发酵糖浓度降至0时,加入新鲜无菌碳源到培养基中以开始新批次的发酵。经过10次重复批次发酵,包埋后的酪丁酸梭菌发酵产生的丁酸产量为12.8 g/L,产率为0.40 g/g,游离细胞仅为12.2 g/L,产率为0.38 g/g。
Claims (10)
1.一种利用玉米芯全组分生产丁酸的方法,其特征在于,包括如下步骤:
(1)玉米芯洗涤干燥后,粉碎;
(2)在氮气氛围保护下,将玉米芯粉末与1,3-二甲基咪唑磷酸二甲酯盐、γ-戊内酯混合进行预处理,之后冷却至室温,加入去离子水作为反溶剂,搅拌反应以沉淀玉米芯粉末,抽滤分离获得玉米芯残渣和滤液;
(3)将玉米芯残渣用去离子水洗涤并干燥后,与乙酸盐缓冲液混合,加入纤维素酶酶解,反应结束后将溶液煮沸以淬灭酶促反应,将煮沸溶液离心,获得以葡萄糖、木糖为主要成分的可发酵糖液;
(4)向步骤(2)获取的滤液中加酸调节pH,低温沉淀滤液中木质素,将木质素沉淀物用去离子水洗涤并干燥;
(5)将酪丁酸梭菌重悬于含有海藻酸钠、木质素和碳酸钙的混合溶液中,用注射器滴加进磁力搅拌的氯化钙溶液中,挤压法包埋酪丁酸梭菌;
(6)以可发酵糖液为碳源发酵包埋酪丁酸梭菌的微球,生产丁酸。
2.根据权利要求1所述的方法,其特征在于,所述步骤(1)中,玉米芯粉碎粒径为80-120目。
3. 根据权利要求1所述的方法,其特征在于,所述步骤(2)中,将玉米芯粉末与1,3-二甲基咪唑磷酸二甲酯盐按照质量比1:6.5~1:19.5混合,之后添加10~16 wt%的γ-戊内酯,于90~110 ℃下加热搅拌3~4h。
4.根据权利要求1所述的方法,其特征在于,所述步骤(2)中,向经预处理的玉米芯粉末、1,3-二甲基咪唑磷酸二甲酯盐、γ-戊内酯的共混溶液中加入等体积的等离子水反溶剂。
5. 根据权利要求1所述的方法,其特征在于,所述步骤(3)中,将玉米芯残渣按照50 g/L的底物浓度与乙酸盐缓冲液混合;乙酸盐缓冲液浓度为50 mM,pH 4.8。
6.根据权利要求1所述的方法,其特征在于,所述步骤(3)中,在50℃、150rpm条件下酶解。
7. 根据权利要求1所述的方法,其特征在于,所述步骤(3)中,纤维素酶添加量为45FPU/g 干燥玉米芯残渣。
8.根据权利要求1所述的方法,其特征在于,所述步骤(4)中,加入盐酸缓慢酸化至pH调节至2.0,之后在4℃下冷藏沉淀木质素。
9. 根据权利要求1所述的方法,其特征在于,所述步骤(5)中,混合溶液中含质量分数2%海藻酸钠,0.5%木质素和5%碳酸钙;氯化钙溶液浓度为11 mg/mL。
10. 根据权利要求1所述的方法,其特征在于,所述步骤(6)中,发酵培养基的组成如下:可发酵糖液50 g/L,酵母粉5 g/L,蛋白胨5 g/L,NaCl 6 g/L,(NH4)2SO4 3 g/L,K2HPO4 1.5 g/L, MgSO4·7H2O 0.6 g/L,FeSO4·7H2O 0.03 g/L,L-半胱氨酸盐酸盐0.3 g/L,0.1%刃天青,pH 6.0。
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