Browsing by Author "Zibek, Susanne"

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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
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
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
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.
Open Access
Growth behavior of selected Ustilaginaceae fungi used for mannosylerythritol lipid (MEL) biosurfactant production - evaluation of a defined culture medium
(2020) Beck, Alexander; Zibek, Susanne
Fungi of the Ustilaginaceae family are a promising source for many biotechnologically relevant products. Among these, mannosylerythritol lipid (MEL) biosurfactants have drawn a special interested over the last decades due to their manifold application possibilities. Nevertheless, there is still a knowledge gap regarding process engineering of MEL production. As an example, no reports on the use of a chemically defined culture medium have been published yet, although such a defined medium might be beneficial for scaling-up the production process toward industrial scale. Our aim therefore was to find a mineral medium that allows fast biomass growth and does not negatively affect the successive MEL production from plant oils. The results showed comparable growth performance between the newly evaluated mineral medium and the established yeast extract medium for all seven investigated Ustilaginaceae species. Final biomass concentrations and specific growth rates of 0.16-0.25 h-1 were similar for the two media. Oxygen demand was generally higher in the mineral medium than in the yeast extract medium. It was shown that high concentrations of vitamins and trace elements were necessary to support the growth. Increasing starting concentrations of the media by a factor of 10 resulted in proportionally increasing final biomass concentrations and up to 2.3-times higher maximum growth rates for all species. However, it could also lead to oxygen limitation and stagnant growth rates when too high medium concentrations were used, which was observed for Ustilago siamensis and Moesziomyces aphidis. Successive MEL production from rapeseed oil was effectively shown for 4 out of 7 organisms when the mineral medium was used for cell growth, and it was even enhanced for two organisms, M. aphidis and Pseudozyma hubeiensis pro tem., as compared to the established yeast extract medium. Conversion of rapeseed oil into MEL was generally improved when higher biomass concentrations were achieved during the initial growth phase, indicating a positive relationship between biomass concentration and MEL production. Overall, this is the first report on the use of a chemically defined mineral medium for the cell growth of Ustilaginaceae fungi and successive MEL production from rapeseed oil, as an alternative to the commonly employed yeast extract medium.
Open Access
Life cycle assessment as a driver for process optimisation of cellobiose lipids fermentation and purification
(2024) Oraby, Amira; Briem, Ann-Kathrin; Bippus, Lars; Rupp, Steffen; Zibek, Susanne
Purpose: Cellobiose lipids (CL) are biosurfactants produced by various Ustilaginaceae species in aerobic fermentations. They show high potential for application as alternatives to conventional oleochemical- or petrochemical surfactants. To ensure their environmentally friendly performance, we aimed to assess CL production from a life cycle perspective at an early developmental stage to identify process steps that have the highest impact on the environment. With this information, optimisation approaches can be derived.
Materials and methods: Following a cradle-to-gate approach, we modelled the CL fermentation and purification process based on experimental data from the lab scale and process simulation data at a 10 m 3 scale. For LCA, the impact categories (IC) abiotic depletion potential (ADP), eutrophication potential, photochemical ozone creation potential, global warming potential, acidification potential, and the primary energy demand were calculated for all process steps. Based on the obtained results, process bottlenecks were identified, and alternative process scenarios varying the related process parameters were simulated. These were used to assess the environmental impact reduction potential (EIRP) of an optimised process and draw recommendations for experimental process optimisation.
Results and discussion: The obtained results showed that the fermentation caused ~ 73% of ADP and more than 85% of all other ICs. The major contributor was the electricity consumption for continuous fermenter aeration. Thus, reducing the fermentation duration from the initial 14 to 5 days would result in a decrease in all investigated ICs of up to ~ 27-52%. An increase in CL concentration results in a decrease in all ICs of a similar magnitude due to the higher yield per batch at comparable energy and material consumption. Although the share of purification process steps to all ICs is overall relatively small, implementing foam fractionation for in situ product recovery showed an additional EIRP of 18-27% in all purification IC shares.
Conclusions: The conducted LCA showed that overall, more EIRP can be achieved by optimising fermentation process parameters compared to purification process steps. This is mainly due to the long fermentation duration and large energy consumption for fermenter aeration. This highlights the importance of using LCA as a driver for process optimisation to identify process steps with high EIRP. While some of the results are specific to CL, other obtained results can be transferred to other fermentations.
Open Access
Life cycle assessment for early-stage process optimization of microbial biosurfactant production using kinetic models : a case study on mannosylerythritol lipids (MEL)
(2024) Bippus, Lars; Briem, Ann-Kathrin; Beck, Alexander; Zibek, Susanne; Albrecht, Stefan
Introduction: This study assesses the environmental impacts of mannosylerythritol lipids (MELs) production for process optimization using life cycle assessment (LCA). MELs are glycolipid-type microbial biosurfactants with many possible applications based on their surface-active properties. They are generally produced by fungi from the family of Ustilaginaceae via fermentation in aerated bioreactors. The aim of our work is to accompany the development of biotechnological products at an early stage to enable environmentally sustainable process optimization. Methods: This is done by identifying hotspots and potentials for improvement based on a reliable quantification of the environmental impacts. The production processes of MELs are evaluated in a cradle-to-gate approach using the Environmental Footprint (EF) 3.1 impact assessment method. The LCA model is based on upscaled experimental data for the fermentation and purification, assuming the production at a 10 m³ scale. In the case analyzed, MELs are produced from rapeseed oil and glucose, and purified by separation, solvent extraction, and chromatography. Results: The results of the LCA show that the provision of substrates is a major source of environmental impacts and accounts for 20% of the impacts on Climate Change and more than 70% in the categories Acidification and Eutrophication. Moreover, 33% of the impacts on Climate Change is caused by the energy requirements for aeration of the bioreactor, while purification accounts for 42% of the impacts respectively. For the purification, solvents are identified as the main contributors in most impact categories. Discussion: The results illustrate the potentials for process optimization to reduce the environmental impacts of substrate requirements, enhanced bioreactor aeration, and efficient solvent use in downstream processing. By a scenario analysis, considering both experimental adaptations and prospective variations of the process, the laboratory development can be supported with further findings and hence efficiently optimized towards environmental sustainability. Moreover, the presentation of kinetic LCA results over the fermentation duration shows a novel way of calculating and visualizing results that corresponds to the way of thinking of process engineers using established environmental indicators and a detailed system analysis. Altogether, this LCA study supports and demonstrates the potential for further improvements towards more environmentally friendly produced surfactants.
Open Access
Mannosylerythritollipide : mikrobielle Biotenside aus dem Bioreaktor
(2020) Beck, Alexander; Zibek, Susanne
Mannosylerythritol lipids (MEL) are microbial biosurfactants belonging to the class of glycolipids. They can be produced biotechnologically by smut fungi and have the potential to replace current chemical surfactant products, for example in household detergents or cosmetics. In this article, we highlight our latest research on the various producer organisms as well as the genetics and regulation of MEL biosynthesis, aiming for the development of an industrial production process in the future.
Open Access
Ökobilanz für die Bioprozessoptimierung : Herstellung des Biotensids MEL
(2024) Bippus, Lars; Briem, Ann-Kathrin; Beck, Alexander; Zibek, Susanne; Albrecht, Stefan
This study evaluates the environmental impacts of producing mannosylerythritol lipids (MELs) using life cycle assessment (LCA) and kinetic models. MELs are microbial biosurfactants with various applications produced from biobased sources. The LCA results indicate that substrate provision, bioreactor aeration and solvent use for purification are major environmental impact sources. The findings highlight areas for improving the environmental sustainability of the production processes.
Open Access
Optimization and kinetic modeling of a fed-batch fermentation for mannosylerythritol lipids (MEL) production with moesziomyces aphidis
(2022) Beck, Alexander; Vogt, Franziska; Hägele, Lorena; Rupp, Steffen; Zibek, Susanne
Mannosylerythritol lipids are glycolipid biosurfactants with many interesting properties. Despite the general interest in those molecules and the need for a robust process, studies on their production in bioreactors are still scarce. In the current study, the fermentative production of MEL in a bioreactor with Moesziomyces aphidis was performed using a defined mineral salt medium. Several kinetic process parameters like substrate consumption rates and product formation rates were evaluated and subsequently enhanced by increasing the biomass concentration through an exponential fed-batch strategy. The fed-batch approaches resulted in two to three fold increased dry biomass concentrations of 10.9-15.5 g/L at the end of the growth phase, compared with 4.2 g/L in the batch process. Consequently, MEL formation rates were increased from 0.1 g/Lh up to around 0.4 g/Lh during the MEL production phase. Thus, a maximum concentration of up to 50.5 g/L MEL was obtained when oil was added in excess, but high concentrations of residual fatty acids were also present in the broth. By adjusting the oil feeding to biomass-specific hydrolysis and MEL production rates, a slightly lower MEL concentration of 34.3 g/L was obtained after 170 h, but at the same time a very pure crude lipid extract with more than 90% MEL and a much lower concentration of remaining fatty acids. With rapeseed oil as substrate, the ideal oil-to-biomass ratio for full substrate conversion was found to be around 10 goil/gbiomass. In addition, off-gas analysis and pH trends could be used to assess biomass growth and MEL production. Finally, kinetic models were developed and compared to the experimental data, allowing for a detailed prediction of the process behavior in future experiments.
Open Access
Past, present and future of glycolipids from Ustilaginaceae : a review on cellobiose lipids and mannosylerythritol lipids
(2024) Münßinger, Sini; Beck, Alexander; Oraby, Amira; Zibek, Susanne
The glycolipids cellobiose lipids (CL) and mannosylerythritol lipids (MEL) are biosurfactants mainly synthesized by microorganisms of the Ustilaginaceae family. They have a large structural diversity, varying in their sugar moieties and the attached fatty acids, resulting in a prospectively broad range of applications. This literature review provides a detailed overview of known microbial producers of CL and MEL, and their respective metabolic pathways that result in different molecular structures. Further, current advances in the aerobic fermentative synthesis of the glycolipids and their purification methods are illustrated. All influencing factors identified to date with regard to the fermentation are highlighted in detail: For CL synthesis usually hydrophilic carbon sources are used as substrate, whereas hydrophobic carbon sources are usually metabolized to MEL. Nitrogen limitation was described as a major trigger for glycolipid synthesis and an acidic pH range was favored for increased CL production. An overview of applied fermentation parameters in recent publications (e.g., substrate‐concentrations, feeding approaches) demonstrates the future potential of CL and MEL production optimization. Foaming during fermentation is either combated or exploited by foam fractionation as the first purification step. The current purification processes focus on solvent extractions and chromatography in the laboratory scale and a need for development was identified for future scale‐up. Finally, environmental hotspots during CL and MEL production are presented and future optimization potentials are highlighted.
Open Access
Purification of chitin from pupal exuviae of the black soldier fly
(2021) Hahn, Thomas; Tafi, Elena; Seggern, Nils von; Falabella, Patrizia; Salvia, Rosanna; Thomä, Jannik; Febel, Eva; Fijalkowska, Malgorzata; Schmitt, Eric; Stegbauer, Linus; Zibek, Susanne
Chitin purification from remains (pupal exuviae after metamorphosis to adult flies) of Hermetia illucens farming was optimized performing demineralization, deproteinization and bleaching under different conditions. The optimal parameters to obtain high-purity chitin were determined. Dried and ground pupal exuviae, whose composition was initially determined, were demineralized using six different acids. Proteins were removed with a NaOH treatment in which temperature, molarity and duration were varied in a randomized experiment. Bleaching was carried out testing ten different chemicals, including NaOCl, H2O2, solvent mixtures and enzymes. The efficiency of each step was determined to assess the optimal conditions for each of them. The resulting chitin was subjected to spectroscopic characterization. The highest demineralization efficiency (90%) was achieved using 0.5 M formic acid for 2 h at 40 °C, confirming the validity of organic acids as a more sustainable alternative to inorganic acids. The treatment with 1.25 M NaOH at 90 °C for 4 h showed the highest deproteinization efficiency, removing 96% of the proteins. Temperature and NaOH concentration were the significant parameters for deproteinization efficiency. The most efficient bleaching treatment was with 6% NaOCl at 60 °C for 1 h (67% efficiency). H2O2 could also be a valid alternative to avoid environmental risk related to chlorine-containing compounds. At the end of the purification process 17% of the original biomass was retained with a chitin content of 85%, corresponding to a chitin yield of 14% related to the initial biomass. Solid-state nuclear magnetic resonance showed that the purified chitin had a degree of acetylation of 96% and X-ray powder diffraction gave a crystallinity index of 74%. This investigation shows an optimized method for extraction of high-purity chitin from H. illucens pupal exuviae, supporting the validity of insect-farming remains as source of this versatile biopolymer.
Open Access
Techno-economic analysis as a driver for optimisation of cellobiose lipid fermentation and purification
(2022) Oraby, Amira; Rupp, Steffen; Zibek, Susanne
Cellobiose lipids (CL) are glycolipids synthesized by Ustilaginaceae species with potential application as detergents or in cosmetics. This study identified process optimisation potential for CL fermentation based on process modelling and techno-economic analysis. Using a stoichiometric equation based on laboratory data, we calculated the maximum possible CL yield YP/S of 0.45 gCL·gglucose -1 at the biomass yield of 0.10 gBiomass·gglucose -1 with an Ustilago maydis strain. Due to substrate inhibition that may occur at high glucose concentrations, a fed-batch process to increase biomass and CL concentrations was considered in our model. Simulation of different process scenarios showed that the choice of aeration units with high oxygen transfer rates and adaptation of power input to oxygen uptake can significantly decrease electricity consumption. We further assessed scenarios with different fermentation media and CL purification methods, suggesting additional process optimisation potential. Here the omission of vitamins from the fermentation medium proved to be a possible mean to enhance process economy, without compromising CL productivity.
Open Access
Transmembrane chemical absorption process for recovering ammonia as an organic fertilizer using citric acid as the trapping solution
(2024) Reyes Alva, Ricardo; Mohr, Marius; Zibek, Susanne
Membrane contactors are among the available technologies that allow a reduction in the amount of ammoniacal nitrogen released into the environment through a process called transmembrane chemical absorption (TMCA). This process can be operated with different substances acting as trapping solutions; however, strong inorganic acids have been studied the most. The purpose of this study was to demonstrate, at laboratory scale, the performance of citric acid as a capturing solution in TMCA processes for recovering ammonia as an organic fertilizer from anaerobic digestor reject water using membrane contactors in a liquid-liquid configuration and to compare it with the most studied solution, sulfuric acid. The experiments were carried out at 22 °C and 40 °C and with a feed water pH of 10 and 10.5. When the system was operated at pH 10, the rates of recovered ammonia from the feed solution obtained with citric acid were 10.7-16.5 percentage points (pp) lower compared to sulfuric acid, and at pH 10.5, the difference decreased to 5-10 pp. Under all tested conditions, the water vapor transport in the system was lower when using citric acid as the trapping solution, and at pH 10 and 40 °C, it was 5.7 times lower. When estimating the operational costs for scaling up the system, citric acid appears to be a better option than sulfuric acid as a trapping solution, but in both cases, the process was not profitable under the studied conditions.