Browsing by Person "Perino, Elvio Henrique Benatto"

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Glucoselipid biosurfactant biosynthesis operon of Rouxiella badensis DSM 100043T: screening, identification, and heterologous expression in Escherichia coli
(2025) Harahap, Andre Fahriz Perdana; Treinen, Chantal; Van Zyl, Leonardo Joaquim; Williams, Wesley Trevor; Conrad, Jürgen; Pfannstiel, Jens; Klaiber, Iris; Grether, Jakob; Hiller, Eric; Vahidinasab, Maliheh; Perino, Elvio Henrique Benatto; Lilge, Lars; Burger, Anita; Trindade, Marla; Hausmann, Rudolf; Seo, Myung-Ji
Rouxiella badensis DSM 100043T had been previously proven to produce a novel glucoselipid biosurfactant which has a very low critical micelle concentration (CMC) as well as very good stability against a wide range of pH, temperature, and salinity. In this study, we performed a function-based library screening from a R. badensis DSM 100043T genome library to identify responsible genes for biosynthesis of this glucoselipid. The identified open reading frames (ORFs) were cloned into several constructs in Escherichia coli for gene permutation analysis and the individual products were analyzed using high-performance thin-layer chromatography (HPTLC). Products of interest from positive expression strains were purified and analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and nuclear magnetic resonance (NMR) for further structure elucidation. Function-based screening of 5400 clones led to the identification of an operon containing three ORFs encoding acetyltransferase GlcA (ORF1), acyltransferase GlcB (ORF2), and phosphatase/HAD GlcC (ORF3). E. coli pCAT2, with all three ORFs, resulted in the production of identical R. badensis DSM 100043T glucosedilipid with Glu-C10:0-C12:1 as the main congener. ORF2-deletion strain E. coli pAFP1 primarily produced glucosemonolipids, with Glu-C10:0,3OH and Glu-C12:0 as the major congeners, predominantly esterified at the C-2 position of the glucose moiety. Furthermore, fed-batch bioreactor cultivation of E. coli pCAT2 using glucose as the carbon source yielded a maximum glucosedilipid titer of 2.34 g/L after 25 h of fermentation, which is 55-fold higher than that produced by batch cultivation of R. badensis DSM 100043T in the previous study.
Model-based process design for surfactin production with Bacillus subtilis
(2025) Hiller, Eric; Off, Manuel; Dittmann, Holger; Perino, Elvio Henrique Benatto; Lilge, Lars; Hausmann, Rudolf; Hiller, Eric; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Off, Manuel; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Dittmann, Holger; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Perino, Elvio Henrique Benatto; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Lilge, Lars; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Hausmann, Rudolf; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany
Bacillus subtilis is one of the most important production organisms in industrial biotechnology. However, there is still limited knowledge about the kinetics of fed-batch processes in bioreactors, as well as a lack of biological performance indicators, such as production yields, particularly regarding their variation over time. Understanding these kinetics and changes is crucial for optimizing the productivity in fed-batch processes. Fed-batch bioreactor cultures of Bacillus subtilis BMV9 in high cell density processes for surfactin production have been characterized with a kinetic model composed of first-order ordinary differential equations, describing the time course of biomass, substrate, surfactin and acetate. This model contributes to understanding critical restrictions and the knowledge gained was used to design and implement a model-based process. The model integrates biomass growth based on Monod kinetics, substrate consumption, surfactin synthesis and formation of the by-product acetate. After the model was parameterized for B. subtilis BMV9 using 12 different fed-batch bioreactor experiments, the kinetic model was able to accurately describe biomass accumulation, substrate consumption, product formation rates and, to some extent, the overflow metabolism involving acetate. Based on this, the kinetic model was used for a process design, in which the batch was omitted, which led to a product titre of 46.33 g/L and a space–time-yield of 2.11 g/(L*h) was achieved. The kinetic model developed in this study enables the description of the time course of biomass growth, substrate consumption and product formation and thus significantly improves process understanding. The computation of process parameters, which are not analytically accessible at any time, could be realized. A sensitivity analysis identified the maximum specific growth rate, substrate-related maintenance and the maximum acetate formation rate as key parameters influencing model outputs.
Structure elucidation and characterization of novel glycolipid biosurfactant produced by Rouxiella badensis DSM 100043T
(2025) Harahap, Andre Fahriz Perdana; Conrad, Jürgen; Wolf, Mario; Pfannstiel, Jens; Klaiber, Iris; Grether, Jakob; Hiller, Eric; Vahidinasab, Maliheh; Salminen, Hanna; Treinen, Chantal; Perino, Elvio Henrique Benatto; Hausmann, Rudolf; Serianni, Anthony S.
Microbial biosurfactants have become increasingly attractive as promising ingredients for environmentally friendly products. The reasons for this are their generally good performance and biodegradability, low toxicity, production from renewable raw materials, and benefits for the environment perceived by consumers. In this study, we investigated the chemical structure and properties of a novel glycolipid from a new biosurfactant-producing strain, Rouxiella badensis DSM 100043 T . Bioreactor cultivation was performed at 30 °C and pH 7.0 for 28 h using 15 g/L glycerol as a carbon source. The glycolipid was successfully purified from the ethyl acetate extract of the supernatant using medium pressure liquid chromatography (MPLC). The structure of the glycolipid was determined by one- and two-dimensional ( 1 H and 13 C) nuclear magnetic resonance (NMR) and confirmed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI/MS). NMR analysis revealed the hydrophilic moiety as a glucose molecule and the hydrophobic moieties as 3-hydroxy-5-dodecenoic acid and 3-hydroxydecanoic acid, which are linked with the glucose by ester bonds at the C2 and C3 positions. Surface tension measurement with tensiometry indicated that the glucose–lipid could reduce the surface tension of water from 72.05 mN/m to 24.59 mN/m at 25 °C with a very low critical micelle concentration (CMC) of 5.69 mg/L. Moreover, the glucose–lipid demonstrated very good stability in maintaining emulsification activity at pH 2–8, a temperature of up to 100 °C, and a NaCl concentration of up to 15%. These results show that R. badensis DSM 100043 T produced a novel glycolipid biosurfactant with excellent surface-active properties, making it promising for further research or industrial applications.