Comparison of bioreactor systems operated at high bacterial cell density for the production of lactic acid: Batch – CSTR – CSTR cascade – Tubular reactor

Subject of the work was the microbial conversion of lactose, an abundant processing by-product in the dairy industry, to lactic acid. Lactic acid serves as a preservative in many areas in various product sectors, but its potential applications go far beyond this. In industrial applications lactic acid as a renewable material can be applied as a building block for novel technical materials such as biodegradable films for packaging purposes in replacement of materials based on fossile raw materials. Purpose of the work was to compare different bioreactor systems with regard to achievable lactic acid concentrations and volumetric productivities. The goal was to quantitatively assess the standard batch stirred tank reactor (STR) in comparison to various continuous reaction systems. Emphasis was put on a continuous stirred tank reactor (CSTR), a CSTR cascade comprised of seven individual stages, and a tubular reactor (TR) (d = 50 mm, L = 600 m). The continuous systems were equipped with a dynamic microfiltration (MF) cell retention system using rotating membranes to prevent extreme deposit formation or centrifugal separation and cell recirculation to the reactor front. In STR systems the reaction product is produced as a function of time and the lactic acid concentration reaches high levels at the end of the fermentation, which may take 8-14 h, depending on cell density inoculated. In a CSTR, operated such that the substrate lactose is fully converted in steady state mode, high lactic acid concentrations are also reached. In comparison to a STR, however, the CSTR is affected by the product inhibition anywhere in the reactor and anytime. Thus, the CSTR volumetric productivity was shown to be considerably lower despite the fact that the cell density reached in steady state was much higher than in the STR. Therefore, in a CSTR high levels of end product concentrations cannot be achieved simultaneously with high volumetric productivities. The CSTR cascade and the TR systems are distinctively different from a CSTR in terms of their concentration profiles over time and reactor length. In comparison to the CSTR, the CSTR cascade and the TR are affected by high lactic acid concentrations only in the rear sections of the reactors. Therefore, volumetric productivities in these systems were drastically higher than in the CSTR. The work also included the screening of lactic acid bacteria cultures, the optimization of the medium composition to achieve high end product concentrations and volumetric productivities. Special emphasis was put on the cell retention system ensuring high flux levels, small effects of deposit formation and long term stability of high flux levels. Thus, it was possible to recirculate the mactic acid culture with little aqueous phase and lactic acid contained therein. It was shown to be of great importance to realize a low recirculation ratio of the aqueous phase including lactic acid and very low carbohydrate concentrations in order to maintain the spatial effects, i.e. the reduced impact of product inhibition described above. In other words, the challenge for cell retention systems in such reactor systems with spatial distribution of concentrations is to achieve high cell concentration factors so that only small amounts of already fermented medium are recirculated to the reactor front. Dynamic membrane systems were found to be capable to achieve this, because they do not require high crossflow volume throughputs and they can cope with high viscosities of the cell concentrates produced for recirculation. Volumetric productivities were thus increased by a factor of 10+

Fouling mitigation in membrane based perfusion systems by oscillating tangential flow

Good scalability and robust handling have promoted the application of membrane based cell retention devices. A physical barrier, i.e. a filter, retains cells and cell debris. One major drawback of these devices is their ten-dency to foul and clog. One form of fouling is deposit layer formation on the filter surface, consisting of cells, cell debris and other fermentation broth constituents. This leads to the build-up of a secondary membrane, which can alter the permeation profile. Furthermore, deposit layers lead to an increased filtration resistance and thus negatively affect permeate flux, filtration efficiency and process robustness. In tangential flow filtration, the tan-gential flow velocity is increased in order to enhance shear forces that can promote deposit layer removal. A new approach to mitigate fouling is oscillation, i.e. pulsation or alternation of the tangential flow. Alternating tan-gential flow filtration (also known as ATF) is already used as a cell retention device. Thereby, the alternating flow is triggered by a pressurized air driven diaphragm pump, which is placed at the retentate side of a hollow fiber module (HFM). If vacuum is applied, the diaphragm moves down and fermentation broth is pulled into the HFM. The exhaust phase is followed by a pressure phase. Pressurized air moves the diaphragm up, thus expel-ling the broth from the HFM back to the bioreactor [1]. Although this alternating stress mitigates deposit layer formation, long residence times in the HFM can lead to nutrient shortage and negatively affect cell viability. With a customized test filtration plant we aim at reaching comparable deposit layer mitigation, while drastically reduc-ing mean residence time in the HFM and, compared to common tangential flow filtration, at reduced tangential flow velocities. To reach this goal, not only alternating, but also pulsating tangential flow is examined. This poster will report on the methodology established in order to understand the mechanisms of deposit layer re-moval in both ATF and oscillating mode. First results from a systematic study on the influence of frequency and amplitude of the oscillation will also be reported. Please click Additional Files below to see the full abstract

Novel concepts for efficient and predictable membrane separation in continuous cell retention and downstream processing

Membranes are applied in biotechnological operations for sterile filtration, cell retention during continuous oper-ation, and cell separation as the first step after fermentation. Membranes are also in use in various steps during purification and isolation of certain target components. In all applications the retained substances, mainly bio-genic material such as cells, protein or polysaccharides, form a deposited layer at the membrane surface. This layer acts as an often dominating secondary membrane, which affects the permeability of the whole system more than the membrane as such. Thus, predictability, efficiency and consistency of all affected processing steps are impaired, which might create issues especially in GMP processes. Therefore, a deeper understanding and a better control of deposit formation would be beneficial for biotechnological operations in general and membrane filtrations in continuous processes in particular. This presentation reports on recent work on a better understanding of deposit formation on membrane surfaces. It was the aim to intensify processes by minimizing the effect of deposit formation and, in turn, increasing flux and permeation of target substances. Success factor in all related projects was a better control of deposit for-mation on membrane surfaces, which in particular was enabled by assessing deposit formation along the mem-brane flow path using special membrane module constructions. These modules allow for the measurement of flux, solutes permeation, structure and amount of deposited material as a function of position in an industrially sized membrane system. Ceramic and polymeric membrane materials as well as tubular and spiralwound mod-ule (SWM) configurations are compared. Please click Additional Files below to see the full abstrac

Novel concepts for efficient and predictable membrane separation in continuous cell retention and downstream processing

Membranes are applied in biotechnological operations for sterile filtration, cell retention during continuous oper-ation, and cell separation as the first step after fermentation. Membranes are also in use in various steps during purification and isolation of certain target components. In all applications the retained substances, mainly bio-genic material such as cells, protein or polysaccharides, form a deposited layer at the membrane surface. This layer acts as an often dominating secondary membrane, which affects the permeability of the whole system more than the membrane as such. Thus, predictability, efficiency and consistency of all affected processing steps are impaired, which might create issues especially in GMP processes. Therefore, a deeper understanding and a better control of deposit formation would be beneficial for biotechnological operations in general and membrane filtrations in continuous processes in particular. This presentation reports on recent work on a better understanding of deposit formation on membrane surfaces. It was the aim to intensify processes by minimizing the effect of deposit formation and, in turn, increasing flux and permeation of target substances. Success factor in all related projects was a better control of deposit for-mation on membrane surfaces, which in particular was enabled by assessing deposit formation along the mem-brane flow path using special membrane module constructions. These modules allow for the measurement of flux, solutes permeation, structure and amount of deposited material as a function of position in an industrially sized membrane system. Ceramic and polymeric membrane materials as well as tubular and spiralwound mod-ule (SWM) configurations are compared. Please click Additional Files below to see the full abstrac

Influence of thermomechanical treatment and ratio of β-lactoglobulin and α-lactalbumin on the denaturation and aggregation of highly concentrated whey protein systems

The influence of thermomechanical treatment (temperature 60 °C–100 °C and shear rate 0.06 s$^{−1}$–50 s$^{−1}$) and mixing ratio of β-lactoglobulin (βLG) and α-lactalbumin (αLA) (5:2 and 1:1) on the denaturation and aggregation of whey protein model systems with a protein concentration of 60% and 70% (w/w) was investigated. An aggregation onset temperature was determined at approx. 80 °C for both systems (5:2 and 1:1 mixing ratio) with a protein concentration of 70% at a shear rate of 0.06 s$^{−1}$. Increasing the shear rate up to 50 s$^{−1}$ led to a decrease in the aggregation onset temperature independent of the mixing ratio. By decreasing the protein concentration to 60% in unsheared systems, the aggregation onset temperature decreased compared to that at a protein concentration of 70%. Furthermore, two significantly different onset temperatures were determined when the shear rate was increased to 25 s$^{−1}$ and 50 s$^{−1}$, which might result from a shear-induced phase separation. Application of combined thermal and mechanical treatment resulted in overall higher degrees of denaturation independent of the mixing ratio and protein concentration. At the conditions applied, the aggregation of the βLG and αLA mixtures was mainly due to the formation of non-covalent bonds. Although the proportion of disulfide bond aggregation increased with treatment temperature and shear rate, it was higher at a mixing ratio of 5:2 compared to that at 1:1

Structural characterisation of deposit layer during milk protein microfiltration by means of in-situ mri and compositional analysis

Milk protein fractionation by microfiltration membranes is an established but still growing field in dairy technology. Even under cross-flow conditions, this filtration process is impaired by the formation of a deposit by the retained protein fraction, mainly casein micelles. Due to deposition formation and consequently increased overall filtration resistance, the mass flow of the smaller whey protein fraction declines within the first few minutes of filtration. Currently, there are only a handful of analytical techniques available for the direct observation of deposit formation with opaque feed media and membranes. Here, we report on the ongoing development of a non-invasive and non-destructive method based on magnetic resonance imaging (MRI), and its application to characterise deposit layer formation during milk protein fractionation in ceramic hollow fibre membranes as a function of filtration pressure and temperature, temporally and spatially resolved. In addition, the chemical composition of the deposit was analysed by reversed phase high pressure liquid chromatography (RP-HPLC). We correlate the structural information gained by in-situ MRI with the protein amount and composition of the deposit layer obtained by RP-HPLC. We show that the combination of in-situ MRI and chemical analysis by RP-HPLC has the potential to allow for a better scientific understanding of the pressure and temperature dependence of deposit layer formation

The uORF-containing thrombopoietin mRNA escapes nonsense-mediated decay (NMD)

Platelet production is induced by the cytokine thrombopoietin (TPO). It is physiologically critical that TPO expression is tightly regulated, because lack of TPO causes life-threatening thrombocytopenia while an excess of TPO results in thrombocytosis. The plasma concentration of TPO is controlled by a negative feedback loop involving receptor-mediated uptake of TPO by platelets. Furthermore, TPO biosynthesis is limited by upstream open reading frames (uORFs) that curtail the translation of the TPO mRNA. uORFs are suggested to activate RNA degradation by nonsense-mediated decay (NMD) in a number of physiological transcripts. Here, we determine whether NMD affects TPO expression. We show that reporter mRNAs bearing the seventh TPO uORF escape NMD. Importantly, endogenously expressed TPO mRNA from HuH7 cells is unaffected by abrogation of NMD by RNAi. Thus, regulation of TPO expression is independent of NMD, implying that mRNAs bearing uORFs cannot generally be considered to represent NMD targets