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

Performance assessment of membrane distillation for skim milk and whey processing

AbstractMembrane distillation is an emerging membrane process based on evaporation of a volatile solvent. One of its often stated advantages is the low flux sensitivity toward concentration of the processed fluid, in contrast to reverse osmosis. In the present paper, we looked at 2 high-solids applications of the dairy industry: skim milk and whey. Performance was assessed under various hydrodynamic conditions to investigate the feasibility of fouling mitigation by changing the operating parameters and to compare performance to widespread membrane filtration processes. Whereas filtration processes are hydraulic pressure driven, membrane distillation uses vapor pressure from heat to drive separation and, therefore, operating parameters have a different bearing on the process. Experimental and calculated results identified factors influencing heat and mass transfer under various operating conditions using polytetrafluoroethylene flat-sheet membranes. Linear velocity was found to influence performance during skim milk processing but not during whey processing. Lower feed and higher permeate temperature was found to reduce fouling in the processing of both dairy solutions. Concentration of skim milk and whey by membrane distillation has potential, as it showed high rejection (>99%) of all dairy components and can operate using low electrical energy and pressures (<10kPa). At higher cross-flow velocities (around 0.141m/s), fluxes were comparable to those found with reverse osmosis, achieving a sustainable flux of approximately 12kg/h·m2 for skim milk of 20% dry matter concentration and approximately 20kg/h·m2 after 18h of operation with whey at 20% dry matter concentration

Towards recombinantly produced milk proteins: Physicochemical and emulsifying properties of engineered whey protein beta-lactoglobulin variants

DFG, 273937032, SPP 1934: Dispersitäts-, Struktur- und Phasenänderungen von Proteinen und biologischen Agglomeraten in biotechnologischen ProzessenBMBF, 031B0222, Basistechnologie Nachwuchsgruppe "Multiskalige Modellierung und Modifikation von Multienzymkomplexen als Basistechnologie für zellfreie Reaktionskaskaden" (II

Genome Sequencing of SHH Medulloblastoma Predicts Genotype-Related Response to Smoothened Inhibition

SummarySmoothened (SMO) inhibitors recently entered clinical trials for sonic-hedgehog-driven medulloblastoma (SHH-MB). Clinical response is highly variable. To understand the mechanism(s) of primary resistance and identify pathways cooperating with aberrant SHH signaling, we sequenced and profiled a large cohort of SHH-MBs (n = 133). SHH pathway mutations involved PTCH1 (across all age groups), SUFU (infants, including germline), and SMO (adults). Children >3 years old harbored an excess of downstream MYCN and GLI2 amplifications and frequent TP53 mutations, often in the germline, all of which were rare in infants and adults. Functional assays in different SHH-MB xenograft models demonstrated that SHH-MBs harboring a PTCH1 mutation were responsive to SMO inhibition, whereas tumors harboring an SUFU mutation or MYCN amplification were primarily resistant

Sarcoma classification by DNA methylation profiling

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications

The German National Registry of Primary Immunodeficiencies (2012-2017)

Introduction: The German PID-NET registry was founded in 2009, serving as the first national registry of patients with primary immunodeficiencies (PID) in Germany. It is part of the European Society for Immunodeficiencies (ESID) registry. The primary purpose of the registry is to gather data on the epidemiology, diagnostic delay, diagnosis, and treatment of PIDs. Methods: Clinical and laboratory data was collected from 2,453 patients from 36 German PID centres in an online registry. Data was analysed with the software Stata® and Excel. Results: The minimum prevalence of PID in Germany is 2.72 per 100,000 inhabitants. Among patients aged 1–25, there was a clear predominance of males. The median age of living patients ranged between 7 and 40 years, depending on the respective PID. Predominantly antibody disorders were the most prevalent group with 57% of all 2,453 PID patients (including 728 CVID patients). A gene defect was identified in 36% of patients. Familial cases were observed in 21% of patients. The age of onset for presenting symptoms ranged from birth to late adulthood (range 0–88 years). Presenting symptoms comprised infections (74%) and immune dysregulation (22%). Ninety-three patients were diagnosed without prior clinical symptoms. Regarding the general and clinical diagnostic delay, no PID had undergone a slight decrease within the last decade. However, both, SCID and hyper IgE- syndrome showed a substantial improvement in shortening the time between onset of symptoms and genetic diagnosis. Regarding treatment, 49% of all patients received immunoglobulin G (IgG) substitution (70%—subcutaneous; 29%—intravenous; 1%—unknown). Three-hundred patients underwent at least one hematopoietic stem cell transplantation (HSCT). Five patients had gene therapy. Conclusion: The German PID-NET registry is a precious tool for physicians, researchers, the pharmaceutical industry, politicians, and ultimately the patients, for whom the outcomes will eventually lead to a more timely diagnosis and better treatment