Mixing and In situ product removal in micro-bioreactors

Summary Of the thesis :’ Mixing and In-situ product removal in micro bioreactors’ by Xiaonan Li The work presented in this thesis is a part of a large cluster project, which was formed between DSM, Organon, Applikon and two university groups (TU Delft and University of Twente), under the ACTS and IBOS program. The aim of this cluster project was to develop a system consisting of parallel bioreactors of 30 to 200 microliter working volume for the cultivation of micro-organisms under well controlled industrially relevant condition (T, pH, DO etc.), and operated as fed-batch reactor in long term (>200h). This platform has the potential to be used for high throughput screening applications for gene identification or the related small scale protein fermentation to increase the protein production process development rate and to reduce the research cost. The development of the platform starts with the design of a single micro-reactor; the single micro-reactor is the integration of well developed sensing system, control system, mixing system and other accessories, like, pumps, valves, adaptors, vessels etc. However many components are not available or not suitable for our application. In this thesis several novel mixing methods, which can provide sufficient mixing in a micro-reactor to satisfy the need of micro-organism fermentation, are developed. Furthermore, microfluidic components are important to facilitate substrate feeding as well as by product removing. An ISPR concept was experimentally demonstrated in this thesis to distinguish a wider scope of micro-reactor applications. One of the main reasons to apply micro systems technology is that compared with traditional reaction (fermentation) technologies, a superior, rapid and sufficient mixing can easily be achieved using micro technologies, especially for those microfluidic devices, which integrated with passive micro structures, with working volume tens of nanoliters. However, the mixing in the microreactor, which with working volme around hundreds microliter, is still be considered as a bottleneck for the high biomass concentration fermentation. In chapter 2, recycle flow mixing (RFM) method was presented. By continuously moving liquid solution from high oxygen concentration area to low oxygen concentration area via multiple fluxes, the system obtains maximum oxygen transfer, which is considered as the bottleneck for high cell density fermentation. Meanwhile, the recycled flows create vigorous convectionin the micro-reactor and obtain good mixing. The mixing performance was experimentally verified with a prototype reactor (with working volume 30 microliter). Under a small recycle flow rate (20 microliter/min), the measured well mixing time was around 800s. However, after taken into account the influence of the recycle tubes (50 microliter), the mixing in the microreactor was considered as comparable as an ideal mixed reactor. The impact of various oxygen transfer abilities on high cell density fermentation was estimated by 2D / 3D CFD simulations. With recycle flow rate 0.001 m/s, the kla value of the microreactor was around 0.023 s-1, which was in the same order of magnitude as a regular stirred tank. This oxygen transfer was sufficient for a high biomass concentration fermentation (Max. biomass concentration > 20 g/l). The mixing performance of RFM method is dependent on the value of the recycle fluxes, therefore, a strong internal micro-pump plays an essential role in the system. To avoid the dependence on the micro-pump development, an alternative micro-mixing method was presented in chapter 3. Oscillation flows, which are created by a central actuator, induce vigorous convection in the micro-reactor(s) to obtain good mixing. The mixing performance within a single reactor was estimated by CFD calculations, a simplified micro-mixing correlation and validated experimentally. With an oscillation frequency (f) of 8.33 Hz, oscillation flow rate (fv) 1000 microliter/min, the experimental well mixing time was 45s; the CFD simulated well mixing time was 37 s; the model calculated well mixing time was 35s. With a stronger oscillation (f=8.33Hz, fv=3000 microliter/min) the well mixing time dropped to 4s.(CFD simulated result & model correlative result) The oscillation mixing method has the potential to be easily integrated with parallel reactorsrrelative This concept has been proven experimentally using a 96-wells micro titer plate and one oscillation pump (f=2.22 Hz, fv=2000 microliter/min). Three parallel reactors followed the same trend and reached to well mixing time at 120s, 122s and 128s, respectively. The comparison of dye distribution results between various tubes indicated a similar mixing behavior in different reactors. Hence, the result show the possibility of using one central actuator to create oscillation fluids to achieve mixing on multi-reactors. Additional experiments have been done with oscillation mixing method to test the influence of the mixing methods on cells viability and influence of the oscillation mixing method on cells suspension. The experiment clearly indicated that compared to the magnetic stirrer mixing method, oscillation mixing method showed less damage on the cells during cell viability test. Homogeneous cell suspension was maintained in the micro bioreactor during overnight oscillation mixing. The characteristics of microfluidic channels for mass transfer were explored in chapter 4. When two liquid streams join into one microchannel with diameter around 150 mm, both streams will behave as laminar flows and run parallel to each other with a stable interface in between. If for certain components there are concentration differences between two streams, over the interface, components can transfer from one stream to another via diffusion. In this chapter the quantitative transfer of glucose between two cocurrent streams was estimated by CFD and experimentally verified. A microchannel has a large surface to volume ratio; therefore, within a short time significant amount of glucose can be transferred from one stream to another. The transfer rate of glucose was measured to be 2.4 - 11.9 nmol /min at a residence time of 54 - 857ms and glucose concentration in the feed stream of a modest 10.4mM. If this transfer would be applied for a fed-batch cultivations in a 100ml microbioreactor, glucose feed rates ranging from 0.26 to 1.3 g/Lreactor/h could be achieved, which is sufficient to perform industrial fermentation processes of fed-batch cultivations at high biomass concentrations. This microfluidic channel also could be used for by-product removal application. The reason by-product needs to be removed is because of the potential risk of the product inhibition, which may cause a decrease in microorganism activity. An implementaed In Situ Product Removal (ISPR) method can circumvent this risk by keeping the dissolved product concentration low in the reactor. Chapter 5 focuses on demonstrating the feasibility of applying a suitable ISPR method on micro-scale bioreactor. Lactic acid was selected as the target chemical. Extraction was selected as the separation method. By pushing a selected extractant (trioctylamine / decanol / dodecane) through a hydrophobic micro hollow fiber, lactic acid is extracted from the aqueous phase into organic phase, and then removed from the microreactor. The micro hollow fiber has the sole task to be the barrier to isolate microorganism from organic phase. The extraction ability was estimated by a model and then validated experimentally. The high specific interfacial area in the micro ISPR system (13.3E+3 m2/m3) shows the advantage of the microextraction for ISPR processes. High ISPR removal rate (1.92e-6 mol/l/s) was obtained experimentally. This removal rate was in the same order of magnitude as the reported lactic acid production rates in mammalian cell cultures (7.09e-7 to 3.7 e-5 mol/l/s). In conclusion, this thesis presents the development of novel micro-mixing methods and the preliminary application of possible In Situ Production Removal (ISPR) methods, leading to the increased applicability of (fed-) batch micro bioreactor for long-term high-biomass concentration fermentation. However, a combined microbioreactor (including sensor, ISPR design and novel mixing design) has yet not been tested experimentally. A number of present challenges is discussed in Chapter 6