Kittler Stefan, Müller Fabian, Elshazly Mohamed, Wandrey Georg Benjamin, Klein Tobias, Daub Andreas, Spadiut Oliver, Kopp Julian
Research Division Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Gumpendorfer Strasse 1a, Vienna, 1060, Austria.
White Biotechnology Research, BASF SE, 67063, Ludwigshafen am Rhein, Germany.
BMC Biotechnol. 2025 Jan 31;25(1):13. doi: 10.1186/s12896-025-00947-9.
Proteases are essential in various industries due to their unique substrate specificities and robustness in different operational conditions. Bacillus strains consist of a genotype favorable for rapid growth whilst secreting enzymes extracellularly, thereby simplifying recombinant protease production. Despite the widespread use of batch and fed-batch fermentations for their ease and robustness, these cultivation types are often marred by significant energy requirements and prolonged downtimes. The switch towards continuous cultivation methods promises reduced carbon footprints and improved equipment efficiency. Yet, research focusing on Bacillus strains is limited, therefore we aimed to establish a continuous cultivation as a competitive alternative to fed-batch.
Therefore, this study aimed to explore the potential of chemostat cultivations for producing a protease from Bacillus licheniformis utilizing a derepressed induction system, and comparing specific productivities and space-time yields to fed-batch cultivations. The continuous cultivations were described in a hybrid model, considering the effect of productivity as function of the applied dilution rate as well as the generation time. The workflow of this study demonstrates that screenings in a fed-batch mode and chemostat cultivations conducted at the same growth rate, result in different specific productivities for derepressible systems.
The results of this study highlight that the feeding rate's impact on specific productivity varies significantly between fed-batch and chemostat cultivations. These differences suggest that fed-batch screenings may not be adequate for developing a continuous process using a derepressed promoter system in B. licheniformis. Although the space-time yield of fed-batch cultivations has not been surpassed by stable continuous operations-achieving only a third of the highest space-time yield observed in fed-batch-valuable mechanistic insights have been gained. This knowledge could facilitate the transition towards a more sustainable mode of cultivation for industrial protease production.
蛋白酶因其独特的底物特异性和在不同操作条件下的稳定性，在各个行业中都至关重要。芽孢杆菌菌株具有有利于快速生长的基因型，同时能在细胞外分泌酶，从而简化了重组蛋白酶的生产过程。尽管分批发酵和补料分批发酵因其简便性和稳定性而被广泛使用，但这些培养方式往往存在能源需求大以及停机时间长的问题。转向连续培养方法有望减少碳足迹并提高设备效率。然而，针对芽孢杆菌菌株的研究有限，因此我们旨在建立一种连续培养方式，作为补料分批发酵的一种有竞争力的替代方法。
因此，本研究旨在探索利用解除阻遏诱导系统，通过恒化器培养从地衣芽孢杆菌生产蛋白酶的潜力，并将比生产率和时空产率与补料分批发酵进行比较。连续培养在一个混合模型中进行描述，该模型考虑了生产率作为所应用稀释率以及世代时间的函数的影响。本研究的工作流程表明，在补料分批模式下进行的筛选以及在相同生长速率下进行的恒化器培养，对于可解除阻遏的系统会产生不同的比生产率。
本研究结果突出表明，补料分批培养和恒化器培养中，进料速率对比生产率的影响存在显著差异。这些差异表明，补料分批筛选可能不足以开发一种使用地衣芽孢杆菌中解除阻遏启动子系统的连续工艺。尽管补料分批培养的时空产率尚未被稳定的连续操作所超越——连续操作仅达到补料分批中观察到的最高时空产率的三分之一——但已获得了有价值的机理见解。这些知识有助于向更可持续的工业蛋白酶生产培养模式转变。
