Institute of Biochemical Engineering, Technische Universität Braunschweig, Gaussstraße 17, 38106 Braunschweig, Germany.
Biotechnol Bioeng. 2012 Feb;109(2):462-71. doi: 10.1002/bit.23313. Epub 2011 Sep 2.
The present study describes the design of bio-pellet morphologies of the industrial working horse Aspergillus niger strains in submerged culture. The novel approach recruits the intended addition of titanate microparticles (TiSiO(4), 8 µm) to the growth medium. As tested for two recombinant strains producing fructofuranosidase and glucoamylase, the enzyme titer by the titanate-enhanced cultures in shake flasks was increased 3.7-fold to 150 U/mL (for fructofuranosidase) and 9.5-fold to 190 U/mL (for glucoamylase) as compared to the control. This could be successfully utilized for improved enzyme production in stirred tank reactors. Stimulated by the particles, the achieved final glucoamylase activity of 1,080 U/mL (fed-batch) and 320 U/mL (batch) was sevenfold higher as compared to the conventional processes. The major reason for the enhanced production was the close association between the titanate particles and the fungal cells. Already below 2.5 g/L the micromaterial was found inside the pellets, including single particles embedded as 50-150 µm particle aggregates in the center resulting in core shell pellets. With increasing titanate levels the pellet size decreased from 1,700 µm (control) to 300 µm. Fluorescence based resolution of GFP expression revealed that the large pellets of the control were only active in a 200 µm surface layer. This matches with the critical penetration depth for nutrients and oxygen typically observed for fungal pellets. The biomass within the titanate derived fungal pellets, however, was completely active. This was due a reduced thickness of the biomass layer via smaller pellets as well as the core shell structure. Moreover, also the created loose inner pellet structure enabled a higher mass transfer and penetration depths for up to 500 µm. The creation of core-shell pellets has not been achieved previously by the addition of microparticles, for example, made of talc or alumina. Due to this, the present work opens further possibilities to use microparticles for tailor-made morphology design of filamentous fungi, especially for pellet based processes which have a long and strong industrial relevance for industrial production.
本研究描述了在浸没培养中工业用马 Aspergillus niger 菌株的生物颗粒形态设计。该新方法利用向生长培养基中添加钛酸盐微粒（TiSiO(4)，8 µm）。作为对两种生产果聚糖酶和葡萄糖淀粉酶的重组菌株的测试，与对照相比，在摇瓶中进行的钛酸盐增强培养的酶滴度增加了 3.7 倍，达到 150 U/mL（果聚糖酶）和 9.5 倍，达到 190 U/mL（葡萄糖淀粉酶）。这可成功用于在搅拌罐反应器中提高酶的产量。受颗粒刺激，实现的最终葡萄糖淀粉酶活性分别为 1,080 U/mL（分批补料）和 320 U/mL（分批），是常规工艺的七倍。产量提高的主要原因是钛酸盐颗粒与真菌细胞之间的紧密结合。即使在低至 2.5 g/L 的浓度下，也发现微米材料在颗粒内部，包括嵌入在中心的 50-150 µm 颗粒聚集体的单个颗粒，导致核壳颗粒。随着钛酸盐水平的增加，颗粒尺寸从 1,700 µm（对照）减小到 300 µm。基于 GFP 表达的荧光分辨率表明，对照的大颗粒仅在 200 µm 的表面层中具有活性。这与通常观察到的真菌颗粒的营养物质和氧气的临界穿透深度相匹配。然而，通过更小的颗粒和核壳结构，钛酸盐衍生真菌颗粒中的生物量层厚度减小，使生物量完全具有活性。此外，创建的松散的内部颗粒结构还为高达 500 µm 的更高传质和穿透深度提供了可能性。通过添加例如滑石或氧化铝制成的微粉，以前尚未实现核壳颗粒的形成。由于这一点，本工作为使用微粉来定制丝状真菌的形态设计开辟了更多可能性，特别是对于基于颗粒的工艺，这些工艺在工业生产中具有悠久而强大的工业相关性。
