04 Fakultät Energie-, Verfahrens- und Biotechnik

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Open Access
Acid catalyzed cross‐linking of polyvinyl alcohol for humidifier membranes
(2021) Michele, Andre; Paschkowski, Patrick; Hänel, Christopher; Tovar, Günter E. M.; Schiestel, Thomas; Southan, Alexander
Polyvinyl alcohol (PVA) is a hydrophilic polymer well known for good film forming properties, high water vapor permeance JW, and low nitrogen permeance. However, depending on molar mass and temperature, PVA swells strongly in water until complete dissolution. This behavior affects the usability of PVA in aqueous environments and makes cross‐linking necessary if higher structural integrity is envisaged. In this work, PVA networks are formed by thermal cross‐linking in the presence of p‐toluenesulfonic acid (TSA) and investigated in a design of experiments approach. Experimental parameters are the cross‐linking period tc, temperature ϑ and the TSA mass fraction wTSA. Cross‐linking is found to proceed via ether bond formation at all reaction conditions. Degradation is promoted especially by a combination of high wTSA, tc and ϑ. Thermal stability of the networks after preparation is strongly improved by neutralizing residual TSA. Humidification membranes with a JW of 6423 ± 63.0 gas permeation units (GPU) are fabricated by coating PVA on polyvinyliden fluoride hollow fibers and cross‐linking with TSA. Summarizing, the present study contributes to a clearer insight into the cross‐linking of PVA in presence of TSA, the thermal stability of the resulting networks and the applicability as selective membrane layers for water vapor transfer.
Open Access
Active ester containing surfmer for one-stage polymer nanoparticle surface functionalization in mini-emulsion polymerization
(2018) Albernaz, Vanessa L.; Bach, Monika; Weber, Achim; Southan, Alexander; Tovar, Günter E. M.
Functional surface active monomers (surfmers) are molecules that combine the functionalities of surface activity, polymerizability and reactive groups. This study presents an improved pathway for the synthesis of the active ester containing surfmer p-(11-acrylamido)undecanoyloxyphenyl dimethylsulfonium methyl sulfate (AUPDS). Further, the preparation of poly(methyl methacrylate) and polystyrene nanoparticles (NPs) by miniemulsion polymerization using AUPDS is investigated, leading to NPs with active ester groups on their surface. By systematically varying reaction parameters and reagent concentrations, it was found that AUPDS feed concentrations between 2-4 mol-% yielded narrowly distributed and stable spherical particles with average sizes between 83 nm and 134 nm for non-cross-linked NPs, and up to 163 nm for cross-linked NPs. By basic hydrolysis of the active ester groups in aqueous dispersion, the positive ζ-potential (ZP) was converted into a negative ZP and charge quantities determined by polyelectrolyte titrations before and after hydrolysis were in the same range, indicating that the active ester groups were indeed accessible in aqueous suspension. Increasing cross-linker amounts over 10 mol-% also led to a decrease of ZP of NPs, probably due to internalization of the AUPDS during polymerization. In conclusion, by using optimized reaction conditions, it is possible to prepare active ester functionalized NPs in one stage using AUPDS as a surfmer in miniemulsion polymerization.
Open Access
Adsorption on inkjet-printable polyelectrolyte hydrogels allows refractive index sensing of diclofenac and metoprolol in aqueous solution
(2024) Southan, Alexander; Tan, Jennifer; Schuster, Fabian; Rotenberger, Julia; Tovar, Günter E. M.
Polyelectrolyte hydrogels containing negatively charged sulfonate groups or positively charged ammonium groups are characterized by their adsorption behavior toward the pharmaceuticals metoprolol (cationic) and diclofenac (anionic) in an aqueous solution. Additionally, the change in the hydrogel refractive index with metoprolol and diclofenac concentrations inside the hydrogel is investigated. Both metoprolol adsorption on sulfonate group containing hydrogels as well as diclofenac adsorption on the ammonium group containing hydrogels can be described using a modified Langmuir-type adsorption isotherm with Ks values around 0.1 and 10 mL μmol-1, respectively. In both cases, the adsorption capacities are close to the concentration of charged groups in the hydrogels. Thus, diclofenac concentrations inside the hydrogels are enhanced by a factor of approximately 1000 and metoprolol concentrations by a factor of approximately 10 compared to their concentrations in solution. In contrast, metoprolol was completely excluded from the ammonium group containing hydrogels, and diclofenac showed weak adsorption on the poly(ethylene glycol) fraction of the sulfonate group containing hydrogels, resulting in lower concentration enhancements. Hydrogel refractive indices increased linearly with the concentration of metoprolol and diclofenac inside the hydrogels. Thus, monitoring the refractive index of sulfonate group containing hydrogels is shown to be efficient in measuring the solution concentrations of metoprolol up to 10 μmol mL-1 and of ammonium group containing hydrogels for diclofenac concentrations up to 0.1 μmol mL-1, both values corresponding to the reciprocal of the Ks values. In the case of Langmuir-type adsorption, maximizing Ks values therefore leads to the best refractive index sensor sensitivities at low analyte concentrations, whereas lower Ks values lead to lower sensitivities at low concentrations but to superior sensitivities at rather high analyte concentrations. Inkjet printing of the hydrogel formulations is demonstrated to facilitate their future use as spatially resolved coatings on sensor surfaces.
Open Access
Azido-functionalized gelatin via direct conversion of lysine amino groups by diazo transfer as a building block for biofunctional hydrogels
(2020) Keller, Silke; Bakker, Tomke; Kimmel, Benjamin; Rebers, Lisa; Götz, Tobias; Tovar, Günter E. M.; Kluger, Petra J.; Southan, Alexander
Gelatin is one of the most prominent biopolymers in biomedical material research and development. It is frequently used in hybrid hydrogels, which combine the advantageous properties of bio-based and synthetic polymers. To prevent the biological component from leaching out of the hydrogel, the biomolecules can be equipped with azides. Those groups can be used to immobilize gelatin covalently in hydrogels by the highly selective and specific azide-alkyne cycloaddition. In this contribution, we functionalized gelatin with azides at its lysine residues by diazo transfer, which offers the great advantage of only minimal side-chain extension. Approximately 84-90% of the amino groups are modified as shown by 1H-NMR spectroscopy, 2,4,6-trinitrobenzenesulfonic acid assay as well as Fourier-transform infrared spectroscopy, rheology, and the determination of the isoelectric point. Furthermore, the azido-functional gelatin is incorporated into hydrogels based on poly(ethylene glycol) diacrylate (PEG-DA) at different concentrations (0.6, 3.0, and 5.5%). All hydrogels were classified as noncyctotoxic with significantly enhanced cell adhesion of human fibroblasts on their surfaces compared to pure PEG-DA hydrogels. Thus, the new gelatin derivative is found to be a very promising building block for tailoring the bioactivity of materials.
Open Access
Beta-glucan production of Phaeodactylum tricornutum, Monodopsis subterranea and Cylindrotheca fusiformis during nitrogen depletion
(2023) Frick, Konstantin; Ebbing, Tobias; Yeh, Yen-Cheng; Schmid-Staiger, Ulrike; Tovar, Günter E. M.
AbstractBeta-glucans are polysaccharides that can be used for different applications, for example as an immunomodulator in food or feed or for managing high cholesterol levels. Certain microalgae species use beta-glucans as energy storage, accumulating them during nutrient depletion. In this study, we examined and compared beta-glucan production during nitrogen depletion in three different algae species, Phaeodactylum tricornutum, Monodopsis subterranea and Cylindrotheca fusiformis, grown in artificially illuminated flat panel airlift reactors, in order to determine the most promising microalgae species for beta-glucan production. Co-products such as fatty acids (especially eicosapentaenoic acid) and the carotenoid fucoxanthin (not produced by M. subterranea) were also considered. Biomass analysis showed that P. tricornutum cultures reached a maximal beta-glucan content of 317 ± 9 mg gDW-1, M. subterranea cultures reached 188 ± 6 mg gDW-1 and C. fusiformis cultures reached 129 ± 13 mg gDW-1. Furthermore, beta-glucan production was faster in P. tricornutum cultures. However, the maximum volumetric beta-glucan concentration reached was higher in M. subterranea cultures compared to P. tricornutum cultures as M. subterranea cultures produced more biomass during nitrogen depletion. In terms of possible co-products, P. tricornutum produced fucoxanthin and EPA, whereas M. subterranea did not produce fucoxanthin. However, M. subterranea exhibited a higher EPA content, which remained above 45 mg g-1 even after several days of nitrogen depletion. Overall, our results suggest that P. tricornutum and M. subterranea are both suitable species for beta-glucan production in flat panel airlift reactors.
Open Access
Charged triazole cross-linkers for hyaluronan-based hybrid hydrogels
(2016) Martini, Maike; Hegger, Patricia S.; Schädel, Nicole; Minsky, Burcu B.; Kirchhof, Manuel; Scholl, Sebastian; Southan, Alexander; Tovar, Günter E. M.; Boehm, Heike; Laschat, Sabine
Polyelectrolyte hydrogels play an important role in tissue engineering and can be produced from natural polymers, such as the glycosaminoglycan hyaluronan. In order to control charge density and mechanical properties of hyaluronan-based hydrogels, we developed cross-linkers with a neutral or positively charged triazole core with different lengths of spacer arms and two terminal maleimide groups. These cross-linkers react with thiolated hyaluronan in a fast, stoichiometric thio-Michael addition. Introducing a positive charge on the core of the cross-linker enabled us to compare hydrogels with the same interconnectivity, but a different charge density. Positively charged cross-linkers form stiffer hydrogels relatively independent of the size of the cross-linker, whereas neutral cross-linkers only form stable hydrogels at small spacer lengths. These novel cross-linkers provide a platform to tune the hydrogel network charge and thus the mechanical properties of the network. In addition, they might offer a wide range of applications especially in bioprinting for precise design of hydrogels.
Open Access
The choice of biopolymer is crucial to trigger angiogenesis with vascular endothelial growth factor releasing coatings
(2020) Claaßen, Christiane; Dannecker, Miriam; Grübel, Jana; Kotzampasi, Maria-Elli; Tovar, Günter E. M.; Stanzel, Boris V.; Borchers, Kirsten
Bio-based coatings and release systems for pro-angiogenic growth factors are of interest to overcome insufficient vascularization and bio-integration of implants. This study compares different biopolymer-based coatings on polyethylene terephthalate (PET) membranes in terms of coating homogeneity and stability, coating thickness in the swollen state, endothelial cell adhesion, vascular endothelial growth factor (VEGF) release and pro-angiogenic properties. Coatings consisted of carbodiimide cross-linked gelatin type A (GelA), type B (GelB) or albumin (Alb), and heparin (Hep), or they consisted of radically cross-linked gelatin methacryloyl-acetyl (GM5A5) and heparin methacrylate (HepM5). We prepared films with thicknesses of 8–10 µm and found that all coatings were homogeneous after washing. All gelatin-based coatings enhanced the adhesion of primary human endothelial cells compared to the uncoated membrane. The VEGF release was tunable with the loading concentration and dependent on the isoelectric points and hydrophilicities of the biopolymers used for coating: GelA-Hep showed the highest releases, while releases were indistinguishable for GelB-Hep and Alb-Hep, and lowest for GM5A5-HepM5. Interestingly, not only the amount of VEGF released from the coatings determined whether angiogenesis was induced, but a combination of VEGF release, metabolic activity and adhesion of endothelial cells. VEGF releasing GelA-Hep and GelB-Hep coatings induced angiogenesis in a chorioallantoic membrane assay, so that these coatings should be considered for further in vivo testing.
Open Access
Comparing three different Phaeodactylum tricornutum strains for the production of chrysolaminarin in flat panel airlift photobioreactors
(2022) Frick, Konstantin; Yeh, Yen-Cheng; Schmid-Staiger, Ulrike; Tovar, Günter E. M.
In recent years, various applications for algae-based ß-1,3-glucans have been postulated, including animal feed and human nutrition. Chrysolaminarin is a ß-1,3-1,6-glucan produced by diatoms such as Phaeodactylum tricornutum for energy storage. It is accumulated under nutrient-depleted cultivation conditions. In this study, the production of chrysolaminarin in artificially illuminated scalable flat panel airlift photobioreactors (FPA) was investigated by using P. tricornutum in a two-stage production process. In the growth stage primarily biomass is produced, and the subsequent nitrogen-depleted stage induces the accumulation of chrysolaminarin. Three P. tricornutum strains (SAG 1090-1a, SAG 1090-1b, SAG 1090-6) were cultured at laboratory scale in 6 L-FPA reactors under controlled light conditions to characterize the process and identify a production strain. The chrysolaminarin content of the algae strains was analysed and additionally their contents of eicosapentaenoic acid and fucoxanthin, both of which could be involved in a possible co-production. Strain SAG 1090-1b exhibited the highest biomass productivity and chrysolaminarin content (317 ± 9 mg gDW-1) after nitrogen depletion, and thus stood out as the most suitable for chrysolaminarin production in a two-stage process. A co-production of the three compounds is possible. However, during nitrogen depletion there occurred trade-offs between the compounds. As chrysolaminarin was produced, the amount of fucoxanthin in the culture stagnated or even decreased depending on the selected strain.
Open Access
Comprehensive characterization and evaluation of the process chain and products from Euphausia superba exocuticles to chitosan
(2023) Hahn, Thomas; Egger, Jeannine; Krake, Simon; Dyballa, Michael; Stegbauer, Linus; Seggern, Nils von; Bruheim, Inge; Zibek, Susanne
Antarctic krill (Euphausia superba) is a source for compounds of high nutritive value. Within that process of extraction, exocuticles (shells) accumulate which are currently disposed. A valorization of the compounds of the exocuticle such as chitosan would be beneficial to avoid waste and to obtain a versatile polymer at the same time. In contrast to previous investigations focusing on chitosan production from whole krill, we applied and optimized process stages of the chitosan production from the exocuticles, performing a comprehensive analytical evaluation of the whole process, the side streams and the products for the first time. Degreasing was the first step resulting in a krill oil yield of 6.2% using ethanol. The fatty acid profile exhibited high contents of phospholipids (21.2%). Citric acid offered a demineralization efficiency of 93%. Deproteinization investigation revealed 2 M NaOH and 90°C for 2.5 h to be the best parameters, resulting in a deproteinization efficiency of 99.9% and a chitin content of 92.8%. The spectroscopic investigation indicated that the chitin has a crystallinity index of 76% and an acetylation degree of 88%. The deacetylation degrees of the resulting chitosans is determined to be 74%-88%, the molecular weight ranges from 102 to 126 kDa.
Open Access
Coumarin-4-ylmethyl- and p-hydroxyphenacyl-based photoacid generators with high solubility in aqueous media: synthesis, stability and photolysis
(2020) Adatia, Karishma K.; Halbritter, Thomas; Reinfelds, Matiss; Michele, Andre; Tran, Michael; Laschat, Sabine; Heckel, Alexander; Tovar, Günter E. M.; Southan, Alexander
(Coumarin‐4‐yl)methyl (c4m) and p‐hydroxyphenacyl (pHP)‐based compounds are well known for their highly efficient photoreactions, but often show limited solubility in aqueous media. To circumvent this, we synthesized and characterized the two new c4m and pHP‐based photoacid generators (PAGs), 7‐[bis(carboxymethyl)amino]‐4‐(acetoxymethyl)coumarin (c4m‐ac) and p‐hydroxyphenacyl‐2,5,8,11‐tetraoxatridecan‐13‐oate (pHP‐t), and determined their solubilities, stabilities and photolysis in aqueous media. The two compounds showed high solubilities in water of 2.77 mmol L−1±0.07 mmol L−1 (c4m‐ac) and 124.66 mmol L−1±2.1 mmol L−1 (pHP‐t). In basic conditions at pH 9, solubility increased for c4m‐ac to 646.46 mmol L−1±0.63 mmol L−1, for pHP‐t it decreased to 34.68 mmol L−1±0.62 mmol L−1. Photochemical properties of the two PAGs, such as the absorption maxima, the maximum molar absorption coefficients and the quantum yields, were found to be strongly pH‐dependent. Both PAGs showed high stabilities s24h ≥95 % in water for 24 h, but decreasing stability with increasing pH value due to hydrolysis. The present study contributes to a clearer insight into the synthesis, solubilities, stabilities, and photolysis of c4m and pHP‐based PAGs for further photochemical applications when high PAG concentrations are required, such as in polymeric foaming.
Open Access
Covalent incorporation of tobacco mosaic virus increases the stiffness of poly(ethylene glycol) diacrylate hydrogels
(2018) Southan, Alexander; Lang, Tina; Schweikert, Michael; Tovar, Günter E. M.; Wege, Christina; Eiben, Sabine
Hydrogels are versatile materials, finding applications as adsorbers, supports for biosensors and biocatalysts or as scaffolds for tissue engineering. A frequently used building block for chemically cross-linked hydrogels is poly(ethylene glycol) diacrylate (PEG-DA). However, after curing, PEG-DA hydrogels cannot be functionalized easily. In this contribution, the stiff, rod-like tobacco mosaic virus (TMV) is investigated as a functional additive to PEG-DA hydrogels. TMV consists of more than 2000 identical coat proteins and can therefore present more than 2000 functional sites per TMV available for coupling, and thus has been used as a template or building block for nano-scaled hybrid materials for many years. Here, PEG-DA (Mn = 700 g/mol) hydrogels are combined with a thiol-group presenting TMV mutant (TMVCys). By covalent coupling of TMVCys into the hydrogel matrix via the thiol-Michael reaction, the storage modulus of the hydrogels is increased compared to pure PEG-DA hydrogels and to hydrogels containing wildtype TMV (wt-TMV) which is not coupled covalently into the hydrogel matrix. In contrast, the swelling behaviour of the hydrogels is not altered by TMVCys or wt-TMV. Transmission electron microscopy reveals that the TMV particles are well dispersed in the hydrogels without any large aggregates. These findings give rise to the conclusion that well-defined hydrogels were obtained which offer the possibility to use the incorporated TMV as multivalent carrier templates e.g. for enzymes in future studies.
Open Access
Current state of chitin purification and chitosan production from insects
(2020) Hahn, Thomas; Tafi, Elena; Paul, Aman; Salvia, Rosanna; Falabella, Patrizia; Zibek, Susanne
Chitin, and especially its deacetylated variant chitosan, has many applications, e.g. as carrier material for pharmaceutical drugs or as a flocculant in wastewater treatment. Despite its versatility and accessibility, chitin, the second most abundant polysaccharide on Earth, has so far been commercially extracted only from crustaceans and to a minor extent from fungi. Insects are a viable alternative source of chitin, but they have not been exploited in the past due to limited availability. Today however, for the sustainable production of animal feed, insect farming is being developed substantially. The availability of large quantities of insect biomass and chitin‐rich side products such as exuviae and exoskeletons has been increasing. This review provides an overview of recently published studies of chitin extraction from insects, its subsequent conversion into chitosan and the primary analytical methods used to characterize insect‐based chitin and chitosan. We have discovered a large number of research articles published over the past 20 years, confirming the increased attention being received by chitin and chitosan production from insects. Despite numerous publications, we identified several knowledge gaps, such as a lack of data concerning chitin purification degree and chitosan yield. Furthermore, analytical methods used to obtain physicochemical characteristics, structural information and chemical composition meet basic qualitative requirements but do not satisfy the need for a more quantitative evaluation. Despite the current shortcomings that need to be overcome, this review presents encouraging data on the use of insects as an alternative source of chitin and chitosan in the future.
Open Access
Differentiation of physical and chemical cross-linking in gelatin methacryloyl hydrogels
(2021) Rebers, Lisa; Reichsöllner, Raffael; Regett, Sophia; Tovar, Günter E. M.; Borchers, Kirsten; Baudis, Stefan; Southan, Alexander
Gelatin methacryloyl (GM) hydrogels have been investigated for almost 20 years, especially for biomedical applications. Recently, strengthening effects of a sequential cross-linking procedure, whereby GM hydrogel precursor solutions are cooled before chemical cross-linking, were reported. It was hypothesized that physical and enhanced chemical cross-linking of the GM hydrogels contribute to the observed strengthening effects. However, a detailed investigation is missing so far. In this contribution, we aimed to reveal the impact of physical and chemical cross-linking on strengthening of sequentially cross-linked GM and gelatin methacryloyl acetyl (GMA) hydrogels. We investigated physical and chemical cross-linking of three different GM(A) derivatives (GM10, GM2A8 and GM2), which provided systematically varied ratios of side-group modifications. GM10 contained the highest methacryloylation degree (DM), reducing its ability to cross-link physically. GM2 had the lowest DM and showed physical cross-linking. The total modification degree, determining the physical cross-linking ability, of GM2A8 was comparable to that of GM10, but the chemical cross-linking ability was comparable to GM2. At first, we measured the double bond conversion (DBC) kinetics during chemical GM(A) cross-linking quantitatively in real-time via near infrared spectroscopy-photorheology and showed that the DBC decreased due to sequential cross-linking. Furthermore, results of circular dichroism spectroscopy and differential scanning calorimetry indicated gelation and conformation changes, which increased storage moduli of all GM(A) hydrogels due to sequential cross-linking. The data suggested that the total cross-link density determines hydrogel stiffness, regardless of the physical or chemical nature of the cross-links.
Open Access
Discrimination of pancreato-biliary cancer and pancreatitis patients by non-invasive liquid biopsy
(2024) Hartwig, Christina; Müller, Jan; Klett, Hagen; Kouhestani, Dina; Mittelstädt, Anke; Anthuber, Anna; David, Paul; Brunner, Maximilian; Jacobsen, Anne; Glanz, Karolina; Swierzy, Izabela; Roßdeutsch, Lotta; Klösch, Bettina; Grützmann, Robert; Wittenberger, Timo; Sohn, Kai; Weber, Georg F.
Background. Current diagnostics for the detection of pancreato-biliary cancers (PBCs) need to be optimized. We therefore propose that methylated cell-free DNA (cfDNA) derived from non-invasive liquid biopsies serves as a novel biomarker with the ability to discriminate pancreato-biliary cancers from non-cancer pancreatitis patients. Methods. Differentially methylated regions (DMRs) from plasma cfDNA between PBCs, pancreatitis and clinical control samples conditions were identified by next-generation sequencing after enrichment using methyl-binding domains and database searches to generate a discriminatory panel for a hybridization and capture assay with subsequent targeted high throughput sequencing. Results. The hybridization and capture panel, covering around 74 kb in total, was applied to sequence a cohort of 25 PBCs, 25 pancreatitis patients, 25 clinical controls, and seven cases of Intraductal Papillary Mucinous Neoplasia (IPMN). An unbiased machine learning approach identified the 50 most discriminatory methylation markers for the discrimination of PBC from pancreatitis and controls resulting in an AUROC of 0.85 and 0.88 for a training ( n = 45) and a validation ( n = 37) data set, respectively. The panel was also able to distinguish high grade from low grade IPMN samples. Conclusions. We present a proof of concept for a methylation biomarker panel with better performance and improved discriminatory power than the current clinical marker CA19-9 for the discrimination of pancreato-biliary cancers from non-cancerous pancreatitis patients and clinical controls. This workflow might be used in future diagnostics for the detection of precancerous lesions, e.g. the identification of high grade IPMNs vs. low grade IPMNs.
Open Access
Eclectic characterisation of chemically modified cell-derived matrices obtained by metabolic glycoengineering and re-assessment of commonly used methods
(2020) Keller, Silke; Liedek, Anke; Shendi, Dalia; Bach, Monika; Tovar, Günter E. M.; Kluger, Petra J.; Southan, Alexander
Azide-bearing cell-derived extracellular matrices (“clickECMs”) have emerged as a highly exciting new class of biomaterials. They conserve substantial characteristics of the natural extracellular matrix (ECM) and offer simultaneously small abiotic functional groups that enable bioorthogonal bioconjugation reactions. Despite their attractiveness, investigation of their biomolecular composition is very challenging due to the insoluble and highly complex nature of cell-derived matrices (CDMs). Yet, thorough qualitative and quantitative analysis of the overall material composition, organisation, localisation, and distribution of typical ECM-specific biomolecules is essential for consistent advancement of CDMs and the understanding of the prospective functions of the developed biomaterial. In this study, we evaluated frequently used methods for the analysis of complex CDMs. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and (immune)histochemical staining methods in combination with several microscopic techniques were found to be highly eligible. Commercially available colorimetric protein assays turned out to deliver inaccurate information on CDMs. In contrast, we determined the nitrogen content of CDMs by elementary analysis and converted it into total protein content using conversion factors which were calculated from matching amino acid compositions. The amount of insoluble collagens was assessed based on the hydroxyproline content. The Sircol™ assay was identified as a suitable method to quantify soluble collagens while the Blyscan™ assay was found to be well-suited for the quantification of sulphated glycosaminoglycans (sGAGs). Eventually, we propose a series of suitable methods to reliably characterise the biomolecular composition of fibroblast-derived clickECM.
Open Access
Extrusion-based 3D printing of poly(ethylene glycol) diacrylate hydrogels containing positively and negatively charged groups
(2018) Joas, Sebastian; Tovar, Günter E. M.; Celik, Oguz; Bonten, Christian; Southan, Alexander
Hydrogels are an interesting class of materials used in extrusion-based 3D printing, e.g., for drug delivery or tissue engineering. However, new hydrogel formulations for 3D printing as well as a detailed understanding of crucial formulation properties for 3D printing are needed. In this contribution, hydrogels based on poly(ethylene glycol) diacrylate (PEG-DA) and the charged monomers 3-sulfopropyl acrylate and [2-(acryloyloxy)ethyl]trimethylammonium chloride are formulated for 3D printing, together with Poloxamer 407 (P407). Chemical curing of formulations with PEG-DA and up to 5% (w/w) of the charged monomers was possible without difficulty. Through careful examination of the rheological properties of the non-cured formulations, it was found that flow properties of formulations with a high P407 concentration of 22.5% (w/w) possessed yield stresses well above 100 Pa together with pronounced shear thinning behavior. Thus, those formulations could be processed by 3D printing, as demonstrated by the generation of pyramidal objects. Modelling of the flow profile during 3D printing suggests that a plug-like laminar flow is prevalent inside the printer capillary. Under such circumstances, fast recovery of a high vicosity after material deposition might not be necessary to guarantee shape fidelity because the majority of the 3D printed volume does not face any relevant shear stress during printing.
Open Access
Factors affecting the synthesis of cellobiose lipids by Sporisorium scitamineum
(2020) Oraby, Amira; Werner, Nicole; Sungur, Zehra; Zibek, Susanne
Cellobiose lipids (CL) are extracellular glycolipids that are produced by many microorganisms from the family Ustilaginaceae. The sugarcane smut fungus Sporisorium scitamineum has been long known as a producer of the glycolipids mannosylerythritol lipids (MEL) and was recently described to additionally secrete CL as a byproduct. In fact, we identified 11 homologous genes in S. scitamineum by in silico analysis sharing a high similarity to the CL biosynthesis gene cluster of Ustilago maydis. We here report the first systematic cultivation of S. scitamineum targeting the synthesis of CL with high product titers and its transfer to the bioreactor. In an initial screening we examined different fermentation media compositions, consisting of a mineral salts solution with vitamins and/or trace elements, three carbon sources (glucose, fructose, sucrose), three pH values (2.5, 4.0, 6.7) and three levels of C/N values (42.2, 83.8, 167.2 molC molN -1) with urea as nitrogen source. A pH of 2.5 proved to result in the highest product titers. An increase of urea concentration from 0.6 to 1.2 g L-1 had a positive effect on biomass formation, however the glycolipid formation was favored at a C/N ratio of 83.8 molC molN -1, using 0.6 g L-1 urea. Amongst the examined carbon sources, sucrose resulted in an increase in the secretion of cellobiose lipids, compared to glucose. Comparing different media compositions, vitamins were identified as not necessary for CL synthesis. We obtained a concentration of cellobiose lipids of 8.3 1.0 g L-1 in shaking flasks. This increased to 17.6 g L-1 in the 1 L bioreactor with additional feeding of carbon source, with a final purity of 85-93%. As a side product, erythritol and mannosylerythritol lipids (MEL) were also synthesized. Via HPTLC coupled MALDI-TOF MS we were able to analyze the secreted CL structures. S. scitamineum produces a mixture of acylated low molecular weight D-glucolipids, linked to a 2,15,16-trihydroxy-hexadecanoic acid via their &-hydroxyl group (CL-B). The produced cellobiose lipids precipitate as needle like crystals at an acidic pH value of 2.5.
Open Access
Fast and non-invasive evaluation of yeast viability in fermentation processes using Raman spectroscopy and machine learning
(2023) Heese, Raoul; Wetschky, Jens; Rohmer, Carina; Bailer, Susanne M.; Bortz, Michael
Fermentation processes used for producing alcoholic beverages such as beer, wine, and cider have a long history, having been developed early on across different civilizations. In most instances, yeast strains are used for fermentation processes, e.g., at breweries and wineries. Monitoring of yeast viability, cell count, and growth behavior is essential to ensure a controlled fermentation process. However, classical microbiological techniques to monitor fermentation process parameters are time-consuming and require sampling, along with the risk of contamination. Nowadays, industries are moving toward automation and digitalization. This necessitates state-of-the-art process analytical technologies to ensure an efficient and controlled process to obtain high-quality product outputs. Hence, there is a strong need for a fast, non-invasive, and generally applicable method to evaluate the viability of yeast cells during fermentation to warrant the standardization and purity of produced products in industrial applications. The aim of our study is to discriminate between viable and non-viable yeast in various culture media using Raman spectroscopy (RS) followed by data analysis with machine learning (ML) tools. These techniques allow for rapid, non-invasive analysis addressing the limitations of traditional methods. The present work primarily focuses on the evaluation of RS combined with predictive ML models in a non-real-time setting. Our goal is to adapt these techniques for future application in real-time monitoring and determination of yeast viability in biotechnological processes. We demonstrate that RS, in combination with ML, is a promising tool for non-invasive inline monitoring of fermentation processes.
Open Access
Fermentation and recovery of cellobiose lipids using foam fractionation
(2023) Oraby, Amira; Hug, Daniel; Weickardt, Isabell; Maerz, Lea; Nebel, Sabrina; Kurmann, Jasper; Rupp, Steffen; Tovar, Günter E. M.; Zibek, Susanne
Cellobiose lipids (CL) are glycolipids secreted by many Ustilaginaceae species in aerobic fermentations characterised by excessive foaming. While increasing CL concentrations remains an aim for its industrial production, excessive foaming during fermentation presents a challenge even at laboratory scale. Foam fractionation (FF) provides a solution to the foaming problem and facilitates the proceeding purification of CL. Here, we present a first CL fermentation process applying FF. With our set-up, we manage to exploit the excessive foaming for continuous product separation. The set-up includes a foam collecting vessel (FCV) with inserts for CL accumulation and foamate recirculation to minimise biomass and nutrient loss. Integrating a foam column (FC) into the fermenter headspace enabled foam enrichment, resulting in the recovery of > 90% of the produced CL from the separated fractions consisting of foam depositions in the fermenter headspace and the FCV. We also increased the fermenter filling volume and thus achieved a higher fermentation capacity. The separated CL fraction was purified via ethanol extraction to obtain CL with purities > 90%. We further examined the effects of different culture media constituents, including biomass and CL, on foam generation and decay and assessed the effect of FC geometries on product enrichment and recovery. In this work, a FF set-up is presented that enables a stable CL fermentation without additional foam mitigation methods. At the same time, the application of FF separated a fraction that was highly enriched in CL during fermentation, resulting in highly pure CL after a simple ethanol extraction.
Open Access
Foam fractionation methods in aerobic fermentation processes
(2022) Oraby, Amira; Weickardt, Isabell; Zibek, Susanne
Inherently occurring foam formation during aerobic fermentation of surface‐active compounds can be exploited by fractionating the foam. This also serves as the first downstream processing step for product concentration and is used for in situ product recovery. Compared to other foam prevention methods, it does not interfere with fermentation parameters or alter broth composition. Nevertheless, parameters affecting the foaming behavior are complex. Therefore, the specific foam fractionation designs need to be engineered for each fermentation individually. This still hinders a widespread industrial application. However, few available commercial approaches demonstrate the applicability of foam columns on an industrial scale. This systematic literature review highlights relevant design aspects and process demands that need to be considered for an application to fermentations and proposes a classification of foam fractionation designs and methods. It further analyses substance‐specific characteristics associated with foam fractionation. Finally, solutions for current challenges are presented, and future perspectives are discussed.