CN114703072B - 利用3d打印构建好氧真菌和兼性或厌氧微生物共存的方法 - Google Patents
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Abstract
本发明公开了利用3D打印构建好氧真菌和兼性或厌氧微生物共存的方法,将好氧真菌接种至发酵培养基,在发酵培养基上放置带孔隙的支撑材料,使好氧真菌在支撑材料上形成致密的生物膜;通过3D打印将兼性或厌氧微生物制成栅栏结构的活体材料;将所述活体材料放入发酵培养基,与好氧真菌共存并发酵反应。本发明还提供了利用3D打印的容器和活体材料构建的生物反应器。本发明通过好氧真菌在支撑材料和在活体材料上形成的生物膜来实现氧气的消耗,为活体材料内部的兼性或厌氧微生物创造了适宜的生长和生产条件,并通过活体材料和3D打印设备设计了一个生物反应器,利用好氧菌的氧气消耗在生物反应器中产生氧梯度,满足以CBP系统为基础的化学品生产的氧气需求。
Description
技术领域
本发明属于生物材料和合成生物学领域,具体涉及利用3D打印构建好氧真菌和兼性或厌氧微生物共存的方法。
背景技术
随着合成生物学的快速发展,重组微生物已经生产出各种大宗和精细化学品。然而,在构建工程菌株过程中,外源基因受到的排他性和基因沉默途径的存在以及发酵过程需要的严格培养条件等因素,制约了生物制造产业的发展。通过将代谢模块划分为不同的微生物成员,微生物组合为生物化学生产的单一培养提供了一个有希望的选择。一个典型的例子是一体化生物加工过程(Consolidated bioprocessing, CBP)人工混菌系统,它在一个反应器结合了水解酶生产、木质纤维素水解、和微生物发酵。与单一培养相比,微生物组合可以提高生产效率。然而,由于难以维持不同菌株群体之间的最佳平衡,以及需要创造不同菌株所需的特定微环境条件,稳定的人工微生物共培养体系仍然困难。例如,构建这种自下而上的CBP系统的一个主要挑战是好氧真菌和兼性或厌氧微生物之间的氧浓度要求。
发明内容
本发明的第一目的在于提供利用3D打印构建好氧真菌和兼性或厌氧微生物共存的方法。
为实现上述技术目的,本发明采用如下技术方案:
利用3D打印构建好氧真菌和兼性或厌氧微生物共存的方法,包括:
将好氧真菌接种至发酵培养基,在发酵培养基上放置带孔隙的支撑材料,使好氧真菌在支撑材料上形成致密的生物膜;
通过3D打印将兼性或厌氧微生物制成栅栏结构的活体材料;
将所述活体材料放入发酵培养基,好氧真菌在栅栏结构的活体材料形成生物膜,实现混菌共存并发酵反应。
本发明通过好氧真菌在支撑材料和在活体材料上形成的生物膜来实现氧气的消耗,为活体材料内部的兼性和厌氧微生物创造了适宜的生长和生产条件。
作为一种优选的实施方式,所述3D打印使用的生物墨水为双键改性的聚乙烯醇和苯硼酸功能化的海藻酸钠双交联生物墨水。生物打印活体材料应用成功的关键是生物墨水的开发。生物墨水应具有适合目标微生物的机械、流变和生物学特性。生物墨水应满足细胞在3D打印凝胶基质内均匀分布的特性,并且能满足在发酵过程中的长期使用。此外,为了在生物打印过程中有利于微生物存活,3D打印的总体方案应该具有最小的细胞毒性,包括温和的温度和没有有机溶剂的条件。目前,生物聚合物(如明胶和藻酸盐)制备的可交联水凝胶常用作生物墨水。然而,此类凝胶墨水不能满足长期稳定性的需求。例如,生物相容性的海藻酸钙凝胶它们会随着时间的推移而降解。为了克服这些缺点,一种方法是使用基因修饰工具来对目标微生物进行改造,使微生物不能降解这些生物相容的材料。然而,在长时间的发酵过程中,凝胶结合多余的水分也会促进凝胶破裂。因此,具有更好的可加工性、更强的机械强度和对发酵介质的化学惰性功能性水凝胶亟待开发。为了满足生物墨水适合目标微生物的机械、流变和生物学特性,我们对生物墨水设计了双级交联(图1)。第一阶段的交联决定了材料在印刷过程中的流变行为,而第二阶段的交联决定了材料最终的力学性能。交联的第一阶段可以通过聚合物本身的物理作用力或加入可逆键或少量共价交联而形成。第一阶段交联技术的例子包括离子交联、主客相互作用、动态共价化学和非特异性物理作用力。当对注射器施加作用力时,物理或动态交联可以断开,凝胶将产生和流动。3D打印后,这些交联可以重新组装,材料快速自愈合,再次形成弱凝胶。然后我们在第二阶段引入更强交联机制。然而,由于交联的动态特性会导致膨胀、侵蚀和蠕变行为,使这些材料既可打印又稳定还面临许多挑战。双键改性的聚乙烯醇(PVA-GMA)和苯硼酸功能化的海藻酸钠(PBA-SA)具有较高的生物相容性。因此,微生物的活性和发酵能力可得以维持。PVA-GMA和PBA-SA之间的可逆共价交联(硼酸酯键)允许细胞在打印结构内均匀分布。此外,PVA-GMA/PBA-SA是剪切稀释的,可以通过挤压型的3D打印机通过注射器和喷嘴挤压,并通过计算机辅助打印成栅栏结构。随后的光聚合提高了印刷的保真度和长期稳定性。
作为一种优选的实施方式,所述3D打印使用的生物墨水中加入蓝光引发剂苯基-2,4,6-三甲基苯甲酰磷酸盐。生物墨水内部加入了蓝光引发剂苯基-2,4,6-三甲基苯甲酰磷酸盐(LAP),在蓝光条件下光聚合提高了活体材料长期稳定性并用于后续发酵。
作为一种优选的实施方式,通过3D打印将兼性或厌氧微生物制成栅栏结构的活体材料的方式为:
将兼性或厌氧微生物过夜培养后,与蓝光引发剂一并加入PVA-GMA溶液中,之后将混合液与PBA-SA溶液混合震荡,制成凝胶;
将凝胶转移至注射筒中打印沉积成连续的丝状,每层印刷后光固化,获得打印好的栅栏结构的活体材料。
作为一种优选的实施方式,所述孔隙直径为1-2mm。支撑材料中1-2mm直径的孔隙可以满足真菌生物膜充分接触培养基。
本发明通过特定的计算机辅助设计,利用3D打印创造出以聚合物基质和包埋微生物为主的三维个性化和高传质的活体材料。并且,通过生物材料的尺寸、空间构型和结构形态的定制,设计的微环境可以维持微生物在生物发酵过程中的长期生存能力和代谢活动。具体的,3D打印技术可定制特定栅栏结构的活体材料,该结构具备高的比表面积,可以提高传质,以实现最大的生物转化效率。
活体材料内部可打印包埋S. cerevisiae、L. paracasei、Escherichia coli、 Actinobacillus succinogenes等不同微生物作为化学品生产菌株。
本发明的另一目的在于提供一种利用3D打印构建好氧真菌和兼性或厌氧微生物共存的生物反应器。
所述生物反应器由容器、好氧真菌、兼性或厌氧微生物构成;
所述容器上部设有进气口,用于供氧,下部设有取样口;容器中部固定发酵培养基,发酵培养基上放置带孔隙的支撑材料;
所述好氧真菌接种至发酵培养基,在支撑材料上形成致密的生物膜;所述兼性或厌氧微生物通过3D打印制成栅栏结构的活体材料后放入发酵培养基;好氧真菌在容器中以生物膜的形式与兼性或厌氧微生物制成的活体材料共存。
作为一种优选的实施方式,所述容器由3D打印制成。
作为一种优选的实施方式,所述容器为ABS材质。
作为一种优选的实施方式,所述支撑材料可拆卸的放置于发酵培养基上。
本发明的生物反应器中容器设计简单,价格低,体积小,方便维修,方便清洗。
本发明通过计算机辅助设计设计3D打印的容器,3D打印的容器和3D打印活体材料表面可形成真菌生物膜(T. asperellum、T. reesei等),该生物膜可分泌纤维素酶并利用氧气,为活体材料内部提供适宜的厌氧环境和底物实现目标化学品的生产。
本发明的构建的生物反应器可应用于CBP体系,CBP系统的一个主要挑战是好氧真菌和兼性或厌氧微生物之间的氧浓度需求差异。本发明通过3D打印的容器和3D打印活体材料设计了一个生物反应器,再利用好氧菌的氧气消耗在生物反应器中产生氧梯度。从而,活体材料内部设计的强制空间生态位可以满足以CBP系统为基础的化学品生产的氧气需求。利用所述生物反应器进行棘孢木霉(Trichoderma asperellum)和酿酒酵母(Saccharomyces cerevisiae)混菌体系反应,在未优化发酵条件的情况下,可利用40 g/L的微晶生成12.5 g/L的乙醇,是悬浮液发酵的3.2倍。这表明设计的生物反应器实现了真菌酵母共存并能完成复杂的底物转化为化学品的生产。
有益效果:
(1)本发明开发的活体材料能有效得把微生物限制在其限域空间内。并且,栅栏结构使活体材料具备高的比表面积,以提高传质和实现最大的生物转化效率。活体材料适用于绝大多数微生物,只需要将不同微生物进行3D打印,即可实现不同化学品的生产。
(2)通过生物材料和3D打印设备设计了一个生物反应器,再利用好氧菌的氧气消耗在生物反应器中产生氧梯度。从而,活体材料内部设计的强制空间生态位可以满足以CBP系统为基础的化学品生产的氧气需求。
附图说明
图1 3D打印栅栏结构活体材料。
图2 活体材料内部微生物可视化生物量积累。
图3 3D打印活体材料用于乙醇发酵。
图4 生物反应器结构示意图。
图5 生物反应器运用于CBP体系效果图。
具体实施方式
实施例中使用的T. asperellum为棘孢木霉(Trichoderma asperellum)LYS1,已公开于申请人在先申请CN114214206A,保藏编号为CCTCC NO:M 20211179;S. cerevisiae购买自ATCC,收录号为9763。
实施例使用的PVA-GMA材料制备方式参见Crispim, Edson G et.al. "Functionalization of poly(vinyl alcohol) by addition of methacryloyl groups:characterization by FTIR and NMR and optimization of reaction conditions byRSM" e-Polymers, vol. 6, no. 1, 2006, pp. 062.
实施例使用的PBA-SA材料制备方式参见Guangfeng Wu et.al. "Rapid self-healing hydrogel based on PVA and sodium alginate with conductive and cold-resistant property" Soft Matter,2020,16, 3319-3324.
实施例1 3D打印栅栏结构活体材料
栅栏结构的活体材料是使用基于挤压的3D打印机(EFL-BP66)打印的。首先利用计算机辅助系统设计具有高传质的三维栅栏结构,然后制备3D打印凝胶。3D打印凝胶制备方式如下:
将1 vol.%过夜培养的S. cerevisiae和1 wt.%苯基-2,4,6-三甲基苯甲酰磷酸盐(LAP)光引发剂加入5 wt.% PVA-GMA中。将溶解的溶液与2 wt.% PBA-SA按1:2的比例混合后用涡旋振荡器振荡。在此之后,在冰上孵育10分钟,使凝胶无气泡。然后将凝胶转移到体积为5 mL、喷嘴长度为20 mm的注射筒中,以50-70 mm/s的打印速度沉积成连续的丝状。每层印刷后30秒光固化(405 nm)。最后,将打印好的栅栏结构的活体材料立即用磷酸缓冲液漂洗6次,去除表面残留的细胞,以备后续使用。
实施例2 活体材料内部微生物可视化生物量积累
用上述活体材料的3D时空设计的方法打印表达绿色荧光蛋白的S. cerevisiae。打印后,立即用PBS漂洗6次。在荧光显微镜下拍摄活体材料的图像,然后将栅栏结构的活体材料转移到培养基中(磷酸二氢钾:3 g/L,硫酸铵:5 g/L,七水硫酸镁:0.5 g/L,尿嘧啶:0.15 g/L,葡萄糖:40 g/L)进行培养。每12小时拍摄活体材料荧光图像。荧光显微镜使用相同的参数,以确保荧光读数的一致性。使用ImageJ对荧光进行量化,以每个打印样品在所有时间点收集的6张图像的平均荧光强度用作生物量随时间累积的参数(图2(A))。如图2(B)所示,封装在活体材料中的微生物在0 h呈现出小的、离散的菌落,随着发酵时间的延长,微生物生长为更大的相互重叠的菌落,这表明打印的活体材料可以与外界发酵培养基交换营养物质维持微生物的生长。
实施例3 3D打印活体材料用于乙醇发酵
为了验证3D打印活体材料的生产乙醇能力,以3D打印的栅栏结构活体材料和游离细胞作为种子进行发酵动力学的研究。发酵在50 mL的厌氧瓶中进行,厌氧瓶中含有10 mL发酵培养基,温度30℃,转速200rpm。初始葡萄糖浓度为40 g/L。如图3所示,与悬浮发酵相比,3D打印的栅栏结构活体材料酿酒酵母在24 h内表现出较低的生产效率。这可能是3D打印过程影响了细胞的活性,在发酵开始时细胞需要适应新的环境。但是在发酵后期,3D打印的栅栏结构活体材料中乙醇的产量达到16.87 g/L,是悬浮发酵的1.3倍。并且,3D打印活体材料的发酵培养基未检测到细胞,表明了3D打印活体材料能将酿酒酵母限制在其限域空间内并能维持和提高细胞的代谢活性。
实施例4 生物反应器容器的构建
生物反应器的容器由3D打印机打印得到,其材质为丙烯腈-丁二烯-苯乙烯(ABS)。3D打印设备的模型由Creo Parametric软件设计。将ABS加热融化后,由喷嘴挤压成型。冷却后,ABS逐层固化粘合在一起。打印机喷嘴温度设置为225℃,打印平台温度设置为105℃。打印沉积速率为60mm/s。如图4所示,容器包括进气口1,取样口2,生物膜支撑材料3,发酵培养基可放置于容器中段,支撑材料可拆卸的放置于发酵培养基上,在需要向发酵培养基中接种菌种或投入活体材料时,将支撑材料拆卸后进行相应操作。
实施例5生物反应器运用于CBP体系
首先将T. asperellum接种到发酵培养基(蛋白胨:0.75 g/L,尿素:0.3 g/L,硫酸铵:1.4 g/L,七水硫酸镁:0.3 g/L,磷酸二氢钾:2 g/L,酵母粉:0.15 g/L,氯化钙:0.35 g/L,微晶纤维素:40 g/L)培养48小时,T. asperellum在发酵培养基中分泌纤维素酶并在支撑材料上形成致密的生物膜,之后将3D打印的栅栏结构活体材料和游离S. cerevisiae(悬浮发酵)分别接入发酵培养基。如图5所示,在未优化发酵条件的情况下,T. asperellum和包含S. cerevisiae活体材料混菌体系可利用40 g/L的微晶生成12.5 g/L的乙醇,是悬浮液发酵的3.2倍。这表明设计的生物反应器实现了真菌酵母共存并能完成复杂的底物转化为化学品的生产。
Claims (8)
1.利用3D打印构建好氧真菌和兼性或厌氧微生物共存的方法,其特征在于,包括:
将好氧真菌接种至发酵培养基,在发酵培养基上放置带孔隙的支撑材料,使好氧真菌在支撑材料上形成致密的生物膜;
通过3D打印将兼性或厌氧微生物制成栅栏结构的活体材料;所述3D打印使用的生物墨水为双键改性的聚乙烯醇(PVA-GMA)和苯硼酸功能化的海藻酸钠(PBA-SA)双交联生物墨水;
将所述活体材料放入发酵培养基,好氧真菌在栅栏结构的活体材料形成生物膜,实现混菌共存并发酵反应。
2.根据权利要求1所述的方法,其特征在于,所述3D打印使用的生物墨水中加入蓝光引发剂苯基-2,4,6-三甲基苯甲酰磷酸盐。
3.根据权利要求2所述的方法,其特征在于,通过3D打印将兼性或厌氧微生物制成栅栏结构的活体材料的方式为:
将兼性或厌氧微生物过夜培养后,与蓝光引发剂一并加入PVA-GMA溶液中,之后将混合液与PBA-SA溶液混合震荡,制成凝胶;
将凝胶转移至注射筒中打印沉积成连续的丝状,每层印刷后光固化,获得打印好的栅栏结构的活体材料。
4.根据权利要求1所述的方法,其特征在于,所述孔隙直径为1-2mm。
5.利用3D打印构建好氧真菌和兼性或厌氧微生物共存的生物反应器,其特征在于,包括容器、好氧真菌、兼性或厌氧微生物;
所述容器上部设有进气口,用于供氧,下部设有取样口;容器中部固定发酵培养基,发酵培养基上放置带孔隙的支撑材料;
所述好氧真菌接种至发酵培养基,在支撑材料上形成致密的生物膜;所述兼性或厌氧微生物通过3D打印制成栅栏结构的活体材料后放入发酵培养基;3D打印使用的生物墨水为双键改性的聚乙烯醇(PVA-GMA)和苯硼酸功能化的海藻酸钠(PBA-SA)双交联生物墨水;
好氧真菌在容器中以生物膜的形式与兼性或厌氧微生物制成的活体材料共存。
6.根据权利要求5所述的生物反应器,其特征在于,所述容器由3D打印制成。
7.根据权利要求6所述的生物反应器,其特征在于,所述容器为ABS材质。
8.根据权利要求5所述的生物反应器,其特征在于,所述支撑材料可拆卸的放置于发酵培养基上。
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