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Permanent URI for this collectionhttps://hohpublica.uni-hohenheim.de/handle/123456789/16626

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Changes of microorganism composition in fresh and stored bee pollen from Southern Germany
(2021) Friedle, Carolin; D’Alvise, Paul; Schweikert, Karsten; Wallner, Klaus; Hasselmann, Martin
Analysis of plant pollen can provide valuable insights into the existing spectrum of microorganisms in the environment. When harvesting bee-collected pollen as a dietary supplement for human consumption, timely preservation of the freshly collected pollen is fundamental for product quality. Environmental microorganisms contained in freshly collected pollen can lead to spoilage by degradation of pollen components. In this study, freshly collected bee pollen was sampled at different locations and stored under various storage conditions to examine the hypothesis that storage conditions may have an effect on the composition of microorganisms in pollen samples. The samples were analyzed using 16S and 18S amplicon sequencing and characterized by palynological analysis. Interestingly, the bacterial communities between pollen samples from different locations varied only slightly, whereas for fungal community compositions, this effect was substantially increased. Further, we noticed that fungal communities in pollen are particularly sensitive to storage conditions. The fungal genera proportion Cladosporium and Mycosphaerella decreased, while Zygosaccharomyces and Aspergillus increased during storage. Aspergillus and Zygosaccharomyces fractions increased during storage at 30 °C, which could negatively impact the pollen quality if it is used as a dietary supplement.
Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis
(2020) Vahidinasab, Maliheh; Lilge, Lars; Reinfurt, Aline; Pfannstiel, Jens; Henkel, Marius; Morabbi Heravi, Kambiz; Hausmann, Rudolf
Background: Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production. Results: The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition. Conclusions: This study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.
Diacetoxyscirpenol, a Fusarium exometabolite, prevents efficiently the incidence of the parasitic weed Striga hermonthica
(2022) Anteyi, Williams Oyifioda; Klaiber, Iris; Rasche, Frank
Background: Certain Fusarium exometabolites have been reported to inhibit seed germination of the cereal-parasitizing witchweed, Striga hermonthica , in vitro . However, it is unknown if these exometabolites will consistently prevent S. hermonthica incidence in planta . The study screened a selection of known, highly phytotoxic Fusarium exometabolites, in identifying the most potent/efficient candidate (i.e., having the greatest effect at minimal concentration) to completely hinder S. hermonthica seed germination in vitro and incidence in planta , without affecting the host crop development and yield. Results: In vitro germination assays of the tested Fusarium exometabolites (i.e., 1,4-naphthoquinone, equisetin, fusaric acid, hymeglusin, neosolaniol (Neo), T-2 toxin (T-2) and diacetoxyscirpenol (DAS)) as pre- Striga seed conditioning treatments at 1, 5, 10, 20, 50 and 100 µM, revealed that only DAS, out of all tested exometabolites, completely inhibited S. hermonthica seed germination at each concentration. It was followed by T-2 and Neo, as from 10 to 20 µM respectively. The remaining exometabolites reduced S. hermonthica seed germination as from 20 µM ( P < 0. 0001). In planta assessment (in a S. hermonthica -sorghum parasitic system) of the exometabolites at 20 µM showed that, although, none of the tested exometabolites affected sorghum aboveground dry biomass ( P > 0.05), only DAS completely prevented S. hermonthica incidence. Following a 14-d incubation of DAS in the planting soil substrate, bacterial 16S ribosomal RNA (rRNA) and fungal 18S rRNA gene copy numbers of the soil microbial community were enhanced; which coincided with complete degradation of DAS in the substrate. Metabolic footprinting revealed that the S. hermonthica mycoherbicidal agent, Fusarium oxysporum f. sp. strigae (isolates Foxy-2, FK3), did not produce DAS; a discovery that corresponded with underexpression of key genes (Tri5, Tri4) necessary for Fusarium trichothecene biosynthesis ( P < 0.0001). Conclusions: Among the tested Fusarium exometabolites, DAS exhibited the most promising herbicidal potential against S. hermonthica . Thus, it could serve as a new biocontrol agent for efficient S. hermonthica management. Further examination of DAS specific mode of action against the target weed S. hermonthica at low concentrations (≤ 20 µM), as opposed to non-target soil organisms, is required.
Fed-batch bioreactor cultivation of Bacillus subtilis using vegetable juice as an alternative carbon source for lipopeptides production: a shift towards a circular bioeconomy
(2024) Gugel, Irene; Vahidinasab, Maliheh; Benatto Perino, Elvio Henrique; Hiller, Eric; Marchetti, Filippo; Costa, Stefania; Pfannstiel, Jens; Konnerth, Philipp; Vertuani, Silvia; Manfredini, Stefano; Hausmann, Rudolf; Gudiña, Eduardo
In a scenario of increasing alarm about food waste due to rapid urbanization, population growth and lifestyle changes, this study aims to explore the valorization of waste from the retail sector as potential substrates for the biotechnological production of biosurfactants. With a perspective of increasingly contributing to the realization of the circular bioeconomy, a vegetable juice, derived from unsold fruits and vegetables, as a carbon source was used to produce lipopeptides such as surfactin and fengycin. The results from the shake flask cultivations revealed that different concentrations of vegetable juice could effectively serve as carbon sources and that the fed-batch bioreactor cultivation strategy allowed the yields of lipopeptides to be significantly increased. In particular, the product/substrate yield of 0.09 g/g for surfactin and 0.85 mg/g for fengycin was obtained with maximum concentrations of 2.77 g/L and 27.53 mg/L after 16 h, respectively. To conclude, this study provides the successful fed-batch cultivation of B. subtilis using waste product as the carbon source to produce secondary metabolites. Therefore, the consumption of agricultural product wastes might be a promising source for producing valuable metabolites which have promising application potential to be used in several fields of biological controls of fungal diseases.
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.
Investigating the environmental Kuznets curve between economic growth and chemical fertilizer surpluses in China: a provincial panel cointegration approach
(2022) Yu, Xiaomin; Schweikert, Karsten; Doluschitz, Reiner
This study investigated the relationship between fertilizer nitrogen (N) and phosphate (P) surpluses and economic development on the regional level in China. With a balanced panel dataset covering 30 provinces of mainland China from 1988 to 2019, we employed panel cointegrating polynomial regression (CPR) analysis using fully modified OLS (FM-OLS) estimators. Our results suggested that all provinces exhibit a long-run cointegrated relationship between fertilizer surpluses and real per capita gross regional product (GRP). A total of 22 provinces out of 30 showed a significant inverted U-shaped environmental Kuznets curve (EKC). Among those, 14 provinces are considered to have reached the peak and 8 provinces are considered to be before the peak. The group-mean turning points on the EKC are CNY 7022, CNY 9726, CNY 4697, CNY 3749, and CNY 5588 per capita GRP (1978 = 100) for the Northeast, Northcentral, Middle, and lower reaches of the Yangtze River, Southwest and Northwest China, respectively. The overall turning point of China is CNY 6705 per capita real gross domestic product (GDP), which was reached in circa 2012. This shows a general improvement of chemical fertilizer management in China. However, six provinces still exhibit linear growth in fertilizer surpluses when the economy grows. These regions are characterized by high cash-crop ratios and are mostly located along the southeast coast. Therefore, more effort and attention should be given to these regions to promote further fertilizer reduction. At the same time, nutrient use efficiencies should be improved, especially for cash crops such as fruit and vegetables.
Modeling the time course of ComX: towards molecular process control for Bacillus wild-type cultivations
(2021) Treinen, Chantal; Magosch, Olivia; Hoffmann, Mareen; Klausmann, Peter; Würtz, Berit; Pfannstiel, Jens; Morabbi Heravi, Kambiz; Lilge, Lars; Hausmann, Rudolf; Henkel, Marius
Wild-type cultivations are of invaluable relevance for industrial biotechnology when it comes to the agricultural or food sector. Here, genetic engineering is hardly applicable due to legal barriers and consumer’s demand for GMO-free products. An important pillar for wild-type cultivations displays the genus Bacillus. One of the challenges for Bacillus cultivations is the global ComX-dependent quorum sensing system. Here, molecular process control can serve as a tool to optimize the production process without genetic engineering. To realize this approach, quantitative knowledge of the mechanism is essential, which, however, is often available only to a limited extent. The presented work provides a case study based on the production of cyclic lipopeptide surfactin, whose expression is in dependence of ComX, using natural producer B. subtilis DSM 10 T. First, a surfactin reference process with 40 g/L of glucose was performed as batch fermentation in a pilot scale bioreactor system to gain novel insights into kinetic behavior of ComX in relation to surfactin production. Interestingly, the specific surfactin productivity did not increase linearly with ComX activity. The data were then used to derive a mathematic model for the time course of ComX in dependence of existing biomass, biomass growth as well as a putative ComX-specific protease. The newly adapted model was validated and transferred to other batch fermentations, employing 20 and 60 g/L glucose. The applied approach can serve as a model system for molecular process control strategies, which can thus be extended to other quorum sensing dependent wild-type cultivations.
The need for consumer-focused household food waste reduction policies using dietary patterns and socioeconomic status as predictors: a study on wheat bread waste in Shiraz, Iran
(2022) Ghaziani, Shahin; Ghodsi, Delaram; Schweikert, Karsten; Dehbozorgi, Gholamreza; Rasekhi, Hamid; Faghih, Shiva; Doluschitz, Reiner
Current household food waste (HFW) reduction plans usually focus on raising consumer awareness, which is essential but insufficient because HFW is predominantly attributed to unconscious behavioral factors that vary across consumer groups. Therefore, identifying such factors is crucial for predicting HFW levels and establishing effective plans. This study explored the role of dietary patterns (DP) and socioeconomic status (SES) as predictors of HBW using linear and non-linear regression models. Questionnaire interviews were performed in 419 households in Shiraz during 2019. A multilayer sampling procedure including stratification, clustering, and systematic sampling was used. Three main DPs, i.e., unhealthy, Mediterranean, and traditional, were identified using a food frequency questionnaire. Results indicated that a one-unit rise in the household’s unhealthy DP score was associated with an average increase in HBW of 0.40%. Similarly, a one-unit increase in the unhealthy DP score and the SES score increased the relative likelihood of bread waste occurrence by 25.6% and 14.5%, respectively. The comparison of findings revealed inconsistencies in HFW data, and therefore the necessity of studying HFW links to factors such as diet and SES. Further investigations that explore HFW associations with household characteristics and behavioral factors will help establish contextual and effective consumer-focused plans.
A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii
(2024) Ahmed, Uzair; Stadelmann, Tobias; Heid, Daniel; Würtz, Berit; Pfannstiel, Jens; Ochsenreither, Katrin; Eisele, Thomas
A novel peptidyl-lys metalloendopeptidase ( Tc -LysN) from Tramates coccinea was recombinantly expressed in Komagataella phaffii using the native pro-protein sequence. The peptidase was secreted into the culture broth as zymogen (~38 kDa) and mature enzyme (~19.8 kDa) simultaneously. The mature Tc -LysN was purified to homogeneity with a single step anion-exchange chromatography at pH 7.2. N-terminal sequencing using TMTpro Zero and mass spectrometry of the mature Tc- LysN indicated that the pro-peptide was cleaved between the amino acid positions 184 and 185 at the Kex2 cleavage site present in the native pro-protein sequence. The pH optimum of Tc -LysN was determined to be 5.0 while it maintained ≥60% activity between pH values 4.5—7.5 and ≥30% activity between pH values 8.5—10.0, indicating its broad applicability. The temperature maximum of Tc -LysN was determined to be 60 °C. After 18 h of incubation at 80 °C, Tc -LysN still retained ~20% activity. Organic solvents such as methanol and acetonitrile, at concentrations as high as 40% (v/v), were found to enhance Tc -LysN’s activity up to ~100% and ~50%, respectively. Tc -LysN’s thermostability, ability to withstand up to 8 M urea, tolerance to high concentrations of organic solvents, and an acidic pH optimum make it a viable candidate to be employed in proteomics workflows in which alkaline conditions might pose a challenge. The nano-LC-MS/MS analysis revealed bovine serum albumin (BSA)’s sequence coverage of 84% using Tc -LysN which was comparable to the sequence coverage of 90% by trypsin peptides. Key points • A novel LysN from Trametes coccinea (Tc-LysN) was expressed in Komagataella phaffii and purified to homogeneity • Tc-LysN is thermostable, applicable over a broad pH range, and tolerates high concentrations of denaturants • Tc-LysN was successfully applied for protein digestion and mass spectrometry fingerprinting
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.

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