CN116970658A - 一种基于生物炭发酵产丁二酸的方法 - Google Patents
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Abstract
本发明公开了一种基于生物炭发酵产丁二酸的方法,包括菌种活化、种子培养和发酵培养,菌种为大肠埃希氏菌(Escherichiacoli)BER208,保藏编号为CCTCCNO:M2012351;所述发酵培养的发酵培养基的配方为:0~70g/L生物炭、0.12g/L甜菜碱、20~80g/L葡萄糖、2.6g/L(NH4)HPO4、0.87g/LNH4H2PO4、0.15g/LKCl、0.37g/LMgSO4·7H2O、2.4g/LFeCl3·6H2O、0.1g/LH3BO3、0.3g/LCoCl2·6H2O、0.15g/LCuCl2·2H2O、0.5g/LZnCl2·4H2O、0.5g/LNaMoO4·2H2O、0.5g/LMnCl2·4H2O,溶剂为水。本方法丁二酸的产量最高达到78.90g/L,收率为0.98g/g且本发明的方法能够有效降低发酵产物中甲酸等副产物的含量。与传统的化学法生产的丁二酸相比,生物法所得的天然丁二酸具有高效、环保等优点,具有广阔的市场前景。
Description
技术领域
本发明属于微生物发酵领域,具体涉及一种基于生物炭的发酵产丁二酸的方法。
背景技术
琥珀酸,又名丁二酸,是一种具有重要应用价值的四碳平台化合物,被广泛应用于化学化工、食品和农业等行业。首先,琥珀酸被用作为抗菌剂,芳香剂和酸味调节剂。在食品行业中,琥珀酸钠盐可以调节并改善酱味调料的质量,也可以取代谷氨酸钠的使用。琥珀酸已被应用于合成许多改善健康相关的产品,包括:药物、抗生素、维他命和氨基酸等。在医药领域,琥珀酸还可以用来合成解毒剂、利尿剂、镇静剂、止血药、合成抗生素以及维生素A、维生素B等。其次,在化学工业中,琥珀酸是一种优良的表面活性剂、清洁剂、起泡剂,以及离子螯合剂用于电镀行业防止金属的溶蚀和点蚀。另外,在农业上,琥珀酸可以用于合成低毒高效的植物生长抑制剂,控制植物生长,调节养分,增加抗旱、抗病、抗冻能力,在水果蔬菜等经济作物中广泛应用。同时,琥珀酸作为重要的有机合成中间体,也是用于合成一系列具有更高附加值的专用化学品的前体物质,例如:1,4-丁二醇、四氢呋喃、γ-丁内酯、N-甲基吡咯烷酮、己二酸等化工产品以及聚丁烯琥珀酸酯(PBS)、聚酰胺等生物聚合物。目前全球琥珀酸的产能达到10~12万吨/年,生物基琥珀酸产量约为2~4万吨/年。
传统工艺生产丁二酸主要是催化加氢法:该方法是以顺酐为原料,通过加氢、水解来制备丁二酸。此方法生成的丁二酸稳定性较高,但加氢所用的催化剂价格较高,且对中间产物顺烯丁二酸对管道有一定腐蚀性,对设备材质要求较高。生物发酵法是利用细菌或其他微生物发酵的方法,以淀粉、糖或其他微生物能够利用的废料为原料生产丁二酸及其衍生物。该方法工艺流程长,设备复杂,但可实现用生物质资源替代化石资源。生物发酵法合成丁二酸能以可再生的生物质为原料,具有反应条件温和、产品纯度高、过程绿色环保等优势;且生物发酵合成的丁二酸,可以完全替代目前以石油为原料的电解顺酐法工艺。
生物炭介导的生物发酵产丁二酸是一种基于可逆氧化机制调控的可持续性手段,可以有效解决发酵过程中生物体内酸的积累问题,可以降低厌氧发酵过程中中间产物的抑制作用,促进发酵进程。生物炭具有较大的比表面积和多孔结构,为微生物定植提供了更多的场所,有利于生物膜的形成,还可作为细胞生长的载体,并且在发酵过程中生物炭释放的矿物质和金属可以作为细胞生长和溶剂生产的营养素。
目前,国内外研究人员已开展了生物发酵法合成丁二酸的工作,基于合成途径分为三类:
1)还原三羧酸途径合成丁二酸,还原三羧酸途径是厌氧条件下微生物进行琥珀酸合成的主要途径,该途径首先经过糖酵解过程生成磷酸烯醇式丙酮酸;随后磷酸烯醇式丙酮酸或丙酮酸通过PEP羧化酶或丙酮酸羧化酶转化成草酰乙酸,再通过苹果酸脱氢酶、富马酸酶和富马酸还原酶在胞质中进一步还原生成丁二酸;
2)有氧三羧酸循环途径是第二个琥珀酸积累途径。在有氧条件下,草酰乙酸和乙酰辅酶A在线粒体中首先被催化成柠檬酸,再经多个氧化反应继而生成琥珀酸;
3)乙醛酸循环是另外一条用于合成琥珀酸的生物合成途径。其合成过程如下:乙酰辅酶A与草酰乙酸在柠檬酸合成酶的作用下缩合生成柠檬酸,再经顺乌头酸酶催化形成异柠檬酸。随后,异柠檬酸裂解酶将异柠檬酸分解为琥珀酸和乙醛酸。生成的乙醛酸在苹果酸合酶的作用下与另一分子乙酰辅酶A结合生成苹果酸,继而生成草酰乙酸用于下一轮的乙醛酸循环。
然而,无论何种合成途径,均不可避免地产生甲酸、乙酸等副产物,在实际应用中,还需要进一步对副产物进行分离去除,这使得生物发酵法合成丁二酸的使用受到限制。
发明内容
针对上述问题,本发明提出一种基于生物炭发酵产丁二酸的方法,不仅将生物质资源农林废弃物进行合理化利用,将其制备的生物炭应用在微生物厌氧发酵生产丁二酸,有效保护生态环境的同时创造经济效益,且本发明的方法能够有效降低发酵产物中甲酸等副产物的含量。
为实现上述目的,本发明采用如下的技术方案:
一种基于生物炭发酵产丁二酸的方法,包括菌种活化、种子培养和发酵培养:
菌种为大肠埃希氏菌(Escherichiacoli)BER208,保藏编号为CCTCCNO:M2012351;
所述发酵培养的发酵培养基的配方为:5~70g/L生物炭、0.12g/L甜菜碱、20~80g/L葡萄糖、2.6g/L(NH4)HPO4、0.87g/LNH4H2PO4、0.15g/LKCl、0.37g/LMgSO4·7H2O、2.4g/LFeCl3·6H2O、0.1g/LH3BO3、0.3g/LCoCl2·6H2O、0.15g/LCuCl2·2H2O、0.5g/LZnCl2·4H2O、0.5g/LNaMoO4·2H2O、0.5g/LMnCl2·4H2O,溶剂为水。
优选的,所述发酵培养的条件为:35~39℃发酵60~168h,pH为5.0~7.0,转速0~200rpm。
更优选的,所述发酵培养的条件为:37℃发酵120h,pH为6.8,转速180rpm。
优选的,所述种子培养的过程为:将活化后的菌种转接到种子培养基中,37℃、180rpm活化12h。
优选的,所述种子培养基的配方为0.12g/L甜菜碱、6.54g/LK2HPO4·3H2O、3.5g/LKH2PO4、3.5g/L(NH4)2HPO4、0.25g/LMgSO4·7H2O、1.6g/LFeCl3·6H2O、0.05g/LH3BO3、0.2g/LCoCl2·6H2O、0.1g/LCuCl2·2H2O、0.2g/LZnCl2·4H2O、0.2g/LNaMoO4·2H2O、0.2g/LZnCl2·4H2O,溶剂为水。
优选的,活化的菌种的接种量为所述发酵培养基的体积的1~10%。
优选的,所述的生物炭为果木生物炭。
优选的,发酵培养基中葡萄糖添加量为50g/L。
优选的,所述发酵培养中,当葡萄糖浓度低于20g/L时,补加葡萄糖。
优选的,所述发酵培养基中,生物炭的添加量为5~10g/L。
有益效果:与现有技术相比,本发明的技术优势如下:
(1)本发明通过运用生物炭介导发酵的手段,将农林废弃物制备的生物炭应用在厌氧发酵高产丁二酸中,在合理利用农林废弃物的同时创造经济效益。
(2)本发明通过添加农林废弃物生物炭,可以发酵生产丁二酸,将微生物发酵与生物炭特性进行结合应用,发酵完成后的发酵液较为澄清,很大程度上降低了后续的脱色分离成本。同时相应的生物炭也可回收利用。
(3)该发明利用生物炭的吸附作用,将生物炭作为固定化载体,提高了丁二酸的产量和收率,且使用的是可再生和可持续原料的天然底物组分制成的生物炭,具有重要的应用价值。
(4)该发明成果将丰富和发展丁二酸生物合成的理论和实践,实现丁二酸的绿色和高效合成;同时也为生物发酵法合成其它有机酸,如戊二酸、苹果酸等提供新的思路和借鉴,在理论和实际应用方面均具有重要价值。
(5)本发明的方法能够在提高产物丁二酸产量的同时有效降低副产物甲酸等的产量。
附图说明
图1是不同生物炭对最终丁二酸产量及副产物产量的影响对比图。
图2是不同果木生物炭添加量对最终丁二酸产量的影响对比图。
图3是实施例3中添加果木生物炭组的5L生物反应器放大培养结果。
图4是实施例3中不添加生物炭组的5L生物反应器放大培养结果。
具体实施方式
实施例1
(1)将大肠埃希氏菌(Escherichiacoli)BER208,保藏编号为CCTCCNO:M2012351,以接种量2%v/v接种到种子培养基中,37℃、180rpm培养化12h;
种子培养基配方为:0.12g/L甜菜碱、6.54g/LK2HPO4·3H2O、3.5g/LKH2PO4、3.5g/L(NH4)2HPO4、0.25g/LMgSO4·7H2O、1.6g/LFeCl3·6H2O、0.05g/LH3BO3、0.2g/LCoCl2·6H2O、0.1g/LCuCl2·2H2O、0.2g/LZnCl2·4H2O、0.2g/LNaMoO4·2H2O、0.2g/LZnCl2·4H2O,溶剂为水,调节pH为7.0。
(2)将菌种以接种量10%v/v接种到含生物炭的发酵培养基中,发酵培养基配方为:50g/L生物炭、0.12g/L甜菜碱、2.6g/L(NH4)HPO4、0.87g/LNH4H2PO4、0.15g/LKCl、0.37g/LMgSO4·7H2O、2.4g/LFeCl3·6H2O、0.1g/LH3BO3、0.3g/LCoCl2·6H2O、0.15g/LCuCl2·2H2O、0.5g/LZnCl2·4H2O、0.5g/LNaMoO4·2H2O、0.5g/LMnCl2·4H2O,溶剂为水。
分别考察以椰子壳生物炭、玉米秸秆生物炭、竹子生物炭、芦苇生物炭、水稻壳生物炭和果木生物炭作为实验组,以及一组不添加生物炭的空白对照进行发酵,每组做三个平行,37℃、180rpm发酵120h。
生物炭表面含有极为丰富的官能团如芳香型C=C、COOH、酚型C-OH、芳型CH和脂肪族CH,大多为负电荷并且比较密集,这加强了生物炭的极性且加强了生物炭与外界物质上阳离子的交换能力。培养过程中,发酵0h与发酵120h测定发酵液中的底物消耗量,当添加果木生物炭时,丁二酸产量最高,达到47.95g/L,收率为79.9%(图1),相较于未添加生物炭的对照组,发酵体系中副产物甲酸和乙酸的含量都有减少,甲酸含量降低12.07%,乙酸含量降低26.61%。
实施例2
方法同实施例1,不同的是步骤(2)配制的发酵培养基中,设置五组不同果木生物炭添加量,其中果木生物炭添加量浓度分别为0g/L,16.7g/L,33.4g/L,50.0g/L,66.7g/L,共5组,每组3个平行。
培养过程中,发酵0h与发酵120h测定发酵液中的底物消耗量,当果木生物炭添加量为16.7g/L时,丁二酸产量最高,达到50.33g/L,收率为85%(图2)。
继续对果木生物炭添加量进行优化,设置0g/L,3.34g/L,8.33g/L,16.67g/L,33.33g/L,共5组,每组3个平行。
培养过程中,发酵0h与发酵120h测定发酵液中的底物消耗量,当果木生物炭添加量为8.33g/L时,丁二酸产量最高,达到55.51g/L,收率为88.02%(图2),相较于不添加生物炭的对照组,丁二酸产量增加了31.45%,同时副产物甲酸减少14.16%,乙酸减少22.76%。
实施例3
方法同实施例1,不同的是生物炭为果木炭,添加量为8.33g/L。每隔12h测量发酵液中葡萄糖浓度和丁二酸浓度,当发酵液中葡萄糖含量消耗到0~20g/L时,补加葡萄糖浓度至40~60g/L。结果显示,使用5-L发酵罐进行放大培养时,添加生物炭的实验组最终丁二酸累积量为78.9g/L,收率达到96%,副产物甲酸减少20.59%,乙酸积累量减少16.08%。经图3、4对比分析可得,生物炭可以提高丁二酸的产量,对副产物甲酸与乙酸的产量有一定的抑制效果,且添加生物炭延长了菌种的存活时间。
生物炭作为固定化载体,可以将菌株固定在生物炭孔隙中,对发酵后期的不利环境有一定抗逆作用,经图3、4分析比较,可以看出在发酵后期,添加生物炭的实验组仍具备一定的代谢生产能力。
与现有技术相比,此发明将生物炭与应用在大肠杆菌厌氧发酵高产丁二酸中,在合理利用农林废弃物的同时创造经济效益。将微生物发酵与生物炭特性进行结合应用,发酵完成后的发酵液较为澄清,很大程度上降低了后续的脱色分离成本。且目前发酵方式为分批发酵,后期通过流加补料发酵方式,可进一步大大提高丁二酸产量。
Claims (10)
1.一种基于生物炭发酵产丁二酸的方法,包括菌种活化、种子培养和发酵培养,其特征在于:
菌种为大肠埃希氏菌(Escherichiacoli)BER208,保藏编号为CCTCCNO:M2012351;
所述发酵培养的发酵培养基的配方为:0~70g/L生物炭、0.12g/L甜菜碱、20~80g/L葡萄糖、2.6g/L(NH4)HPO4、0.87g/LNH4H2PO4、0.15g/LKCl、0.37g/LMgSO4·7H2O、2.4g/LFeCl3·6H2O、0.1g/LH3BO3、0.3g/LCoCl2·6H2O、0.15g/LCuCl2·2H2O、0.5g/LZnCl2·4H2O、0.5g/LNaMoO4·2H2O、0.5g/LMnCl2·4H2O,溶剂为水。
2.根据权利要求1所述的方法,其特征在于,所述发酵培养的条件为:35~39℃发酵60~168h,pH为5.0~7.0,转速0~200rpm。
3.根据权利要求2所述的方法,其特征在于,所述发酵培养的条件为:37℃发酵120h,pH为6.8,转速180rpm。
4.根据权利要求1所述的方法,其特征在于,所述种子培养的过程为:将活化后的菌种转接到种子培养基中,37℃、180rpm活化12h。
5.根据权利要求4所述的方法,其特征在于,所述种子培养基的配方为0.12g/L甜菜碱、6.54g/LK2HPO4·3H2O、3.5g/LKH2PO4、3.5g/L(NH4)2HPO4、0.25g/LMgSO4·7H2O、1.6g/LFeCl3·6H2O、0.05g/LH3BO3、0.2g/LCoCl2·6H2O、0.1g/LCuCl2·2H2O、0.2g/LZnCl2·4H2O、0.2g/LNaMoO4·2H2O、0.2g/LZnCl2·4H2O,溶剂为水。
6.根据权利要求1所述的方法,其特征在于,活化的菌种的接种量为所述发酵培养基的体积的1~10%。
7.根据权利要求1所述的方法,其特征在于,所述的生物炭为果木生物炭。
8.根据权利要求1所述的方法,其特征在于,发酵培养基中葡萄糖添加量为50g/L。
9.根据权利要求1所述的方法,其特征在于,所述发酵培养中,当葡萄糖浓度低于20g/L时,补加葡萄糖。
10.根据权利要求1所述的方法,其特征在于,所述发酵培养基中,生物炭的添加量为5~10g/L。
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