Development and application of a milliliter-scale bioreactor for continuous microbial cultivations

The workhorses of process development, optimization and characterization in the biotech and the pharmaceutical industry are microtiter plates, shake flasks and bench scale bioreactors. They are widely used in academia as well, and with a good reason. Beside inherent benefits, they have standardized properties and have been studied extensively, and thus they offer the possibility to compare research results and to rely on an already collected knowledge base.

However, they also have shortcomings, which were emphasized in a recent past and today by development of genetic engineering techniques that enable genetic manipulation of microorganisms, producing more strains and thereby creating a need for even more processes that need to be evaluated than ever before.

In order to provide a high-throughput solution to this issue and cut the cost, time spent and the general labor intensity per experiment, a new experimental approach is necessary. Micro- and milliliter scale bioreactors are considered as an adequate solution to address this experimental challenge, since they unite the possibility of parallelization with better control and sensing performance.

Current state of the art research on micro and milliliter scale bioreactors shows a spectrum of different approaches in providing an adequate environment for microbial cultivations with small footprint. Currently, there is no consensus on the choice of best suited working volume for the small scale bioreactors, ranging from nanoliters, over microliters to milliliter scale, which raises a question of potential application and what is the aim or purpose of the developed tool.

Examining commercial solutions, it is clear that two design directions are adopted for submerged microbial cultivations: (1) microtiter plate modifications to gain a more controlled environment and better sensing performance in each well (usually up to 2 mL volume); and (2) small scale stirred tank bioreactors for better scale up performance (10-15 mL volumes).

Adequate sensing and mixing with an impeller that enables a good oxygen transfer rate at 1-2 mL scale was investigated and addressed in this thesis, by designing and fabricating two prototypes of milliliter scale bioreactors (MSBR). The engineering design process methodology was utilized to answer the question: “How to go from idea to prototype?” and to find ways to evaluate and materialize ideas.

The designed milliliter scale bioreactors aimed to provide the middle ground between the two established bioreactor design directions mentioned above and explore benefits and drawbacks of milliliter scalebioreactors during batch and continuous microbial cultivations. The first prototype (MSBR I) consisted of a reusable platform containing heater, gasconnections, temperature sensor and three optical fiber bundles, and a milliliter scalebioreactor with special stirrer and sensors for measurement of dissolve oxygen, pH andscattered light intensity.

A modular approach in design and fabrication provided highflexibility in the choice of working volume (0.5 – 2 mL), aeration type (sparger or surfaceaeration) and mixing possibilities (one- and bi-directional). The MSBR I exhibited short mixing times and a high oxygen transfer rate at higher mixing speeds.

On-linemeasurement of the scattered light intensity was based on a transflectance measurementwhere light was sent through the MSBR bottom and sample to a mirror-like surface in the MSBR and returned back to a fiber bundle. Aerobic and anaerobic batch cultivations were performed with Saccharomyces cerevisiae and Lactobacillus paracasei, respectively.

A high evaporation rate was experienced during cultivations as a penalty for the lack of a proper humidifier and control of gas flow rates.The second prototype (MSBR II) had a similar modular concept to the previous one, however heater, temperature sensor and gas connections were moved from the platformto the bioreactor, while the three optical fiber bundles and the heating element that was incontact with the heater were part of the platform.

The MSBR II also had a sensor for dissolved oxygen and a small stainless steel element that was used for acquiring ascattered light intensity measurement via transflectance. The stirrer had four impellerblades and a simplified structure compared to the stirrer in the previous prototype. The mixing time was longer than in the MSBR I, but efficient mixing was still obtained.

A humidifier was developed for this platform and evaporation was reduced substantially. The interaction between end user and small scale bioreactor platforms is usually challenging if not automated, due to practical issues that the small scale brings along. Connectivity between the small bioreactor and the macro world is troublesome without standardized solutions.

Furthermore, any additional equipment required to complete bioreactor functionality usually comes in regular lab size, which then transforms a smallscale bioreactor platform to a regular size experimental set up. To address this issue, effort was placed in developing 2 push/pull pumps that were able to deliver gas and medium ina controlled manner as a part of the MSBR II platform design.

Cultivations with Saccharomyces cerevisiae as model organism were performed in the MSBRII where batch mode produced sustainable and reproducible results and displayed the expected growth profile while continuous mode cultivations were performed with limited success. With few further design improvements, the MSBR II platform has the potential to become an experimental tool that will sustainably support microbial cultivations at milliliter scale.

Afterwards, implementation of parallelization should be relatively straightforward.