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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.
Orotic acid production by Yarrowia lipolytica under conditions of limited pyrimidine
(2021) Swietalski, Paul; Hetzel, Frank; Klaiber, Iris; Pross, Eva; Seitl, Ines; Fischer, Lutz
Orotic acid (OA) is an intermediate of the pyrimidine biosynthesis with high industrial relevance due to its use as precursor for production of biochemical pyrimidines or its use as carrier molecule in drug formulations. It can be produced by fermentation of microorganisms with engineered pyrimidine metabolism. In this study, we surprisingly discovered the yeast Yarrowia lipolytica as a powerful producer of OA. The overproduction of OA in the Y. lipolytica strain PO1f was found to be caused by the deletion of the URA3 gene which prevents the irreversible decarboxylation of OA to uridine monophosphate. It was shown that the lack of orotidine‐5′‐phosphate decarboxylase was the reason for the accumulation of OA inside the cell since a rescue mutant of the URA3 deletion in Y. lipolytica PO1f completely prevented the OA secretion into the medium. In addition, pyrimidine limitation in the cell massively enhanced the OA accumulation followed by secretion due to intense overflow metabolism during bioreactor cultivations. Accordingly, supplementation of the medium with 200 mg/L uracil drastically decreased the OA overproduction by 91%. OA productivity was further enhanced in fed‐batch cultivation with glucose and ammonium sulfate feed to a maximal yield of 9.62 ± 0.21 g/L. Y. lipolytica is one of three OA overproducing yeasts described in the literature so far, and in this study, the highest productivity was shown. This work demonstrates the potential of Y. lipolytica as a possible production organism for OA and provides a basis for further metabolic pathway engineering to optimize OA productivity.
Evaluation of the importance of ornamental plants for pollinators in urban and suburban areas in Stuttgart, Germany
(2021) Marquardt, Melanie; Kienbaum, Lydia; Kretschmer, Lea Annina; Penell, Anja; Schweikert, Karsten; Ruttensperger, Ute; Rosenkranz, Peter
Urban landscapes are often characterized by a wide range of diverse flowering plants consisting of native and exotic plants. These flower-rich habitats have proven to be particularly valuable for urban pollinating insects. However, the ability of ornamental plants in supporting urban pollinator communities is still not well documented. For this study, we established flower beds at 13 different urban testing sites, which were planted with identical sets of ornamental garden plants. The pollinator visitation patterns were then observed throughout the summer seasons. Over a two-year period, a total of 10,565 pollinators were recorded with wild bees (> 50%, excluding bumblebees) being the most abundant pollinator group. Our results revealed that (I) the assortment of ornamental plants was visited by a high number of urban pollinators for collecting pollen and nectar, and (II) the pollinator abundance and composition varied significantly within the tested ornamental plants. These differences occurred not only among plant species but to the same extent among cultivars, whereby the number of pollinators was positively correlated with number of flowering units per plant. By using a generalized linear mixed model (GLMM) and redundancy analysis (RDA) we identified further significant impacts of the two variables year and location on the insect pollinator abundance and richness. Despite of the local and yearly variations, our approach provided a good and field-applicable method to evaluate the pollinator friendliness in ornamental plants. Such tools are urgently required to validate labels like ‘bee friendly’ or ‘pollinator friendly’ used by plant breeding companies.
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.
Comparison of floral traits in Calibrachoa cultivars and assessment of their impacts on attractiveness to flower-visiting insects
(2021) Marquardt, Melanie; Kienbaum, Lydia; Losert, Dominik; Kretschmer, Lea Annina; Rigling, Marina; Zhang, Yanyan; Schweikert, Karsten; Westermann, Nils; Ruttensperger, Ute; Rosenkranz, Peter
Ornamental plants are appreciated by humans for their colorfulness, beauty, abundant flowering and long blooming periods. Many ornamental plants can also constitute an additional foraging resource for flower-visiting insects. However, the ability of the popular ornamental plant Calibrachoa to support urban insect communities is not well documented. In this study, 20 different Calibrachoa cultivars were selected and tested in regard to their insect friendliness based on standardized observations (I) in flight tents using the large earth bumble bee Bombus terrestris as a model species and (II) in open field trials. To investigate what floral characteristics might constitute attractiveness to bumble bees, various floral traits were recorded and compared across all tested Calibrachoa cultivars. Over a two-year period, a total of 6,327 foraging bumble bees were recorded in the tent observations. In the open field observations, we counted 4,188 flower-visiting insects. Our results revealed that (I) all Calibrachoa cultivars were visited by insects for foraging, (II) the number of insect visitors varied significantly among the 20 tested cultivars and (III) the cultivars displayed different floral traits. For the morphometric floral traits and the aroma profiles of Calibrachoa, only the mean nectar quantity and a few identified compounds could be correlated with attractiveness to the model species B. terrestris. We also found that the petal color of the tested cultivars had a significant impact on the number of visitors. Therefore, B. terrestris clearly preferred red or blue Calibrachoa cultivars over those with other petal colors. However, as the cultivar preferences in the different insect groups differed, it is highly recommended to use various cultivars in urban plantings. Nevertheless, efforts must be made to explain what additional floral traits make Calibrachoa and other ornamental plants generally attractive to flower visitors. This information can then be used for breeding purposes to increase the insect friendliness of ornamental plants.
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.
New approaches to manage Asian soybean rust (Phakopsora pachyrhizi) using Trichoderma spp. or their antifungal secondary metabolites
(2022) El-Hasan, Abbas; Walker, Frank; Klaiber, Iris; Schöne, Jochen; Pfannstiel, Jens; Voegele, Ralf T.
Attempts have been made to determine the in vitro and in planta suppressive potential of particular Trichoderma strains (T16 and T23) and their secondary metabolites (SMs) against Asian soybean rust (ASR) incited by Phakopsora pachyrhizi. Aside from the previously identified SMs 6-pentyl-α-pyrone (6PAP) and viridiofungin A (VFA), the chemical structures of harzianic acid (HA), iso-harzianic acid (iso-HA), and harzianolide (HZL) were characterized in this study. Our results indicate that exposure of urediospores to 200 ppm 6PAP completely inhibits germination. A slightly higher dosage (250 ppm) of HZL and VFA reduces germination by 53.7% and 44%, respectively. Germ tube elongation seems more sensitive to 6PAP than urediospore germination. On detached leaves, application of conidia of T16 and T23 results in 81.4% and 74.3% protection, respectively. Likewise, 200 ppm 6PAP recorded the highest ASR suppression (98%), followed by HZL (78%) and HA (69%). Treatment of undetached leaves with 6PAP, HA, or HZL reduces ASR severity by 84.2%, 65.8%, and 50.4%, respectively. Disease reduction on the next, untreated trifoliate by T23 (53%), T16 (41%), HZL (42%), and 6PAP (32%) suggests a translocation or systemic activity of the SMs and their producers. To our knowledge, this study provides the first proof for controlling ASR using antifungal SMs of Trichoderma. Our findings strongly recommend the integration of these innovative metabolites, particularly 6PAP and/or their producers in ASR management strategies.
Household food waste quantification and cross-examining the official figures: A study on household wheat bread waste in Shiraz, Iran
(2022) Ghaziani, Shahin; Ghodsi, Delaram; Schweikert, Karsten; Dehbozorgi, Gholamreza; Faghih, Shiva; Mohabati, Shabnam; Doluschitz, Reiner
The global consumer food waste (FW) estimates are mainly based on modeling data obtained from governments. However, a major data gap exists in FW at the household level, especially in developing countries. Meanwhile, the reliability of the existing data is questionable. This study aimed to quantify wheat bread waste (HBW) in Shiraz, Iran, and cross-examine the governmental HBW data. Face-to-face waste recall questionnaire interviews were conducted in 419 households from December 2018 to August 2019. A multistage sampling strategy consisting of stratification, clustering, and systematic sampling was employed. Moreover, we carried out a comprehensive document review to extract and analyze the official HBW data. The results revealed that the HBW in Shiraz is 1.80%—the waste amounts for traditional bread and non-traditional bread were 1.70% and 2.50%, respectively. The survey results were compared with the previous official data, revealing a substantial contradiction with the 30% HBW reported between 1991 and 2015. Possible reasons for this disparity are explored in this paper. Although our results cannot be generalized to other food commodities and locations, our findings suggest that considering the substantial likelihood of bias in the official data, policymakers should conduct more FW measurements and re-evaluate the accuracy of the existing data.
Surfactin shows relatively low antimicrobial activity against Bacillus subtilis and other bacterial model organisms in the absence of synergistic metabolites
(2022) Lilge, Lars; Ersig, Nadine; Hubel, Philipp; Aschern, Moritz; Pillai, Evelina; Klausmann, Peter; Pfannstiel, Jens; Henkel, Marius; Morabbi Heravi, Kambiz; Hausmann, Rudolf
Surfactin is described as a powerful biosurfactant and is natively produced by Bacillus subtilis in notable quantities. Among other industrially relevant characteristics, antimicrobial properties have been attributed to surfactin-producing Bacillus isolates. To investigate this property, stress approaches were carried out with biotechnologically established strains of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli and Pseudomonas putida with the highest possible amounts of surfactin. Contrary to the popular opinion, the highest growth-reducing effects were detectable in B. subtilis and E. coli after surfactin treatment of 100 g/L with 35 and 33%, respectively, while P. putida showed no growth-specific response. In contrast, other antimicrobial biosurfactants, like rhamnolipids and sophorolipids, showed significantly stronger effects on bacterial growth. Since the addition of high amounts of surfactin in defined mineral salt medium reduced the cell growth of B. subtilis by about 40%, the initial stress response at the protein level was analyzed by mass spectrometry, showing induction of stress proteins under control of alternative sigma factors σB and σW as well as the activation of LiaRS two-component system. Overall, although surfactin is associated with antimicrobial properties, relatively low growth-reducing effects could be demonstrated after the surfactin addition, challenging the general claim of the antimicrobial properties of surfactin.
Characterization of Bacillus velezensis UTB96, demonstrating improved lipopeptide production compared to the strain B. velezensis FZB42
(2022) Vahidinasab, Maliheh; Adiek, Isabel; Hosseini, Behnoush; Akintayo, Stephen Olusanmi; Abrishamchi, Bahar; Pfannstiel, Jens; Henkel, Marius; Lilge, Lars; Vögele, Ralf ; Hausmann, Rudolf
Bacillus strains can produce various lipopeptides, known for their antifungal properties. This makes them attractive metabolites for applications in agriculture. Therefore, identification of productive wild-type strains is essential for the development of biopesticides. Bacillus velezensis FZB42 is a well-established strain for biocontrol of plant pathogens in agriculture. Here, we characterized an alternative strain, B. velezensis UTB96, that can produce higher amounts of all three major lipopeptide families, namely surfactin, fengycin, and iturin. UTB96 produces iturin A. Furthermore, UTB96 showed superior antifungal activity towards the soybean fungal pathogen Diaporthe longicolla compared to FZB42. Moreover, the additional provision of different amino acids for lipopeptide production in UTB96 was investigated. Lysine and alanine had stimulatory effects on the production of all three lipopeptide families, while supplementation of leucine, valine and isoleucine decreased the lipopeptide bioproduction. Using a 45-litre bioreactor system for upscaling in batch culture, lipopeptide titers of about 140 mg/L surfactin, 620 mg/L iturin A, and 45 mg/L fengycin were achieved. In conclusion, it becomes clear that B. velezensis UTB96 is a promising strain for further research application in the field of agricultural biological controls of fungal diseases.
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.
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.
Exploration of surfactin production by newly isolated Bacillus and Lysinibacillus strains from food‐related sources
(2022) Akintayo, Stephen Olusanmi; Treinen, Chantal; Vahidinasab, Maliheh; Pfannstiel, Jens; Bertsche, Ute; Fadahunsi, I.; Oellig, Claudia; Granvogl, Michael; Henkel, Marius; Lilge, Lars; Hausmann, Rudolf
As a lipopeptide (LP), surfactin exhibits properties, such as emulsifying and dispersing ability, which are useful in food industry. Discovery of new LP‐producing strains from food sources is an important step towards possible application of surfactin in foods. A total of 211 spore‐forming, Gram‐positive, and catalase‐positive bacterial strains were isolated from fermented African locust beans (iru) and palm oil mill effluents in a screening process and examined for their ability to produce surfactin. This was achieved by a combination of methods, which included microbiological and molecular classification of strains, along with chemical analysis of surfactin production. Altogether, 29 isolates, positive for oil spreading and emulsification assays, were further identified with 16S rDNA analysis. The strains belonged to nine species including less commonly reported strains of Lysinibacillus, Bacillus flexus, B. tequilensis, and B. aryabhattai. The surfactin production was quantitatively and qualitatively analysed by high‐performance thin‐layer chromatography and liquid chromatography‐mass spectrometry (LC–MS). Confirmation of surfactin by MS was achieved in all the 29 strains. Highest surfactin production capability was found in B. subtilis IRB2‐A1 with a titre of 1444·1 mg L−1.
Dynamic changes in O-GlcNAcylation regulate osteoclast differentiation and bone loss via nucleoporin 153
(2022) Li, Yi-Nan; Chen, Chih-Wei; Trinh-Minh, Thuong; Zhu, Honglin; Matei, Alexandru-Emil; Györfi, Andrea-Hermina; Kuwert, Frederic; Hubel, Philipp; Ding, Xiao; Manh, Cuong Tran; Xu, Xiaohan; Liebel, Christoph; Fedorchenko, Vladyslav; Liang, Ruifang; Huang, Kaiyue; Pfannstiel, Jens; Huang, Min-Chuan; Lin, Neng-Yu; Ramming, Andreas; Schett, Georg; Distler, Jörg H. W.
Bone mass is maintained by the balance between osteoclast-induced bone resorption and osteoblast-triggered bone formation. In inflammatory arthritis such as rheumatoid arthritis (RA), however, increased osteoclast differentiation and activity skew this balance resulting in progressive bone loss. O-GlcNAcylation is a posttranslational modification with attachment of a single O-linked β-D-N-acetylglucosamine (O-GlcNAc) residue to serine or threonine residues of target proteins. Although O-GlcNAcylation is one of the most common protein modifications, its role in bone homeostasis has not been systematically investigated. We demonstrate that dynamic changes in O-GlcNAcylation are required for osteoclastogenesis. Increased O-GlcNAcylation promotes osteoclast differentiation during the early stages, whereas its downregulation is required for osteoclast maturation. At the molecular level, O-GlcNAcylation affects several pathways including oxidative phosphorylation and cell-cell fusion. TNFα fosters the dynamic regulation of O-GlcNAcylation to promote osteoclastogenesis in inflammatory arthritis. Targeted pharmaceutical or genetic inhibition of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) arrests osteoclast differentiation during early stages of differentiation and during later maturation, respectively, and ameliorates bone loss in experimental arthritis. Knockdown of NUP153, an O-GlcNAcylation target, has similar effects as OGT inhibition and inhibits osteoclastogenesis. These findings highlight an important role of O-GlcNAcylation in osteoclastogenesis and may offer the potential to therapeutically interfere with pathologic bone resorption.
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.
Characterization ofantifungal properties of lipopeptide-producing Bacillus velezensis strains and their proteome-based response to the phytopathogens, Diaporthe spp
(2023) Akintayo, Stephen Olusanmi; Hosseini, Behnoush; Vahidinasab, Maliheh; Messmer, Marc; Pfannstiel, Jens; Bertsche, Ute; Hubel, Philipp; Henkel, Marius; Hausmann, Rudolf; Vögele, Ralf; Lilge, Lars
Introduction: B. velezensis strains are of interest in agricultural applications due to their beneficial interactions with plants, notable through their antimicrobial activity. The biocontrol ability of two new lipopeptides-producing B. velezensis strains ES1-02 and EFSO2-04, against fungal phytopathogens of Diaporthe spp., was evaluated and compared with reference strains QST713 and FZB42. All strains were found to be effective against the plant pathogens, with the new strains showing comparable antifungal activity to QST713 and slightly lower activity than FZB42. Methods: Lipopeptides and their isoforms were identified by high-performance thin-layer chromatography (HPTLC) and mass spectrometric measurements. The associated antifungal influences were determined in direct in vitro antagonistic dual culture assays, and the inhibitory growth effects on Diaporthe spp. as representatives of phytopathogenic fungi were determined. The effects on bacterial physiology of selected B. velezensis strains were analyzed by mass spectrometric proteomic analyses using nano-LC-MS/MS. Results and Discussion: Lipopeptide production analysis revealed that all strains produced surfactin, and one lipopeptide of the iturin family, including bacillomycin L by ES1-02 and EFSO2-04, while QST713 and FZB42 produced iturin A and bacillomycin D, respectively. Fengycin production was however only detected in the reference strains. As a result of co-incubation of strain ES1-02 with the antagonistic phytopathogen D. longicolla, an increase in surfactin production of up to 10-fold was observed, making stress induction due to competitors an attractive strategy for surfactin bioproduction. An associated global proteome analysis showed a more detailed overview about the adaptation and response mechanisms of B. velezensis, including an increased abundance of proteins associated with the biosynthesis of antimicrobial compounds. Furthermore, higher abundance was determined for proteins associated with oxidative, nitrosative, and general stress response. In contrast, proteins involved in phosphate uptake, amino acid transport, and translation were decreased in abundance. Altogether, this study provides new insights into the physiological adaptation of lipopeptide-producing B. velezensis strains, which show the potential for use as biocontrol agents with respect to phytopathogenic fungi.
The antioxidant potential of various wheat crusts correlates with AGE content independently of acrylamide
(2023) Wächter, Kristin; Longin, Carl Friedrich H.; Winterhalter, Patrick R.; Bertsche, Ute; Szabó, Gábor; Simm, Andreas
Epidemiological studies have indicated that the consumption of whole-grain products is associated with a reduced risk of cardiovascular diseases, type II diabetes, and cancer. In the case of bread, high amounts of antioxidants and advanced glycation end products (AGEs) are formed during baking by the Maillard reaction in the bread crust; however, the formation of potentially harmful compounds such as acrylamide also occurs. This study investigated the antioxidant responses of different soluble extracts from whole-grain wheat bread crust extracts (WBCEs) in the context of the asparagine, AGE, and acrylamide content. For that, we analyzed nine bread wheat cultivars grown at three different locations in Germany (Hohenheim, Eckartsweier, and Oberer Lindenhof). We determined the asparagine content in the flour of the 27 wheat cultivars and the acrylamide content in the crust, and measured the antioxidant potential using the induced expression of the antioxidant genes GCLM and HMOX1 in HeLa cells. Our study uncovered, for the first time, that the wheat crust’s antioxidant potential correlates with the AGE content, but not with the acrylamide content. Mass spectrometric analyses of WBCEs for identifying AGE-modified proteins relevant to the antioxidant potential were unsuccessful. However, we did identify the wheat cultivars with a high antioxidant potential while forming less acrylamide, such as Glaucus and Lear. Our findings indicate that the security of BCEs with antioxidative and cardioprotective potential can be improved by choosing the right wheat variety.
Comparison of aqueous and lactobacterial-fermented Mercurialis perennis L. (Dog’s Mercury) extracts with respect to their immunostimulating activity
(2023) Lorenz, Peter; Zilkowski, Ilona; Mailänder, Lilo K.; Klaiber, Iris; Nicolay, Sven; Garcia-Käufer, Manuel; Zimmermann-Klemd, Amy M.; Turek, Claudia; Stintzing, Florian C.; Kammerer, Dietmar R.; Gründemann, Carsten
Lactic acid (LA) fermentation of dog’s mercury (M. perennis L.) herbal parts was investigated in samples inoculated with either Lactobacteria (Lactobacillus plantarum and Pediococcus pentosaceus, LBF) or whey (WF). Depending on fermentation time, LA concentrations were monitored in a range of 3.4–15.6 g/L with a concomitant pH decline from 6.5 to 3.9. A broad spectrum of cinnamic acids depsides containing glucaric, malic and 2-hydroxyglutaric acids along with quercetin and kaempferol glycosides were detected by LC-DAD-ESI-MSn. Moreover, in this study novel constituents were also found both in unfermented and fermented extracts. Furthermore, amino acids and particular Lactobacteria metabolites such as biogenic amines (e.g., putrescine, 4-aminobutyric acid, cadaverine) and 5-oxoproline were assigned in WF extracts by GC-MS analysis after silylation. Enhanced NFκB and cytokine expression (IL-6, TNFα, IL-8 and IL-1β) was induced by all extracts, both non-fermented and fermented, in NFκB-THP-1 reporter cells, showing a concentration-dependent immunostimulatory effect. The WF extracts were tested for micronuclei formation in THP-1 cells and toxicity in luminescent bacteria (V. fischeri), whereby no mutagenic or toxic effects could be detected, which corroborates their safe use in pharmaceutical remedies.
Mineral and phytic acid content as well as phytase activity in flours and breads made from different wheat species
(2023) Longin, C. Friedrich H.; Afzal, Muhammad; Pfannstiel, Jens; Bertsche, Ute; Melzer, Tanja; Ruf, Andrea; Heger, Christoph; Pfaff, Tobias; Schollenberger, Margit; Rodehutscord, Markus
Wheat is of high importance for a healthy and sustainable diet for the growing world population, partly due to its high mineral content. However, several minerals are bound in a phytate complex in the grain and unavailable to humans. We performed a series of trials to compare the contents of minerals and phytic acid as well as phytase activity in several varieties from alternative wheat species spelt, emmer and einkorn with common wheat. Additionally, we investigated the potential of recent popular bread making recipes in German bakeries to reduce phytic acid content, and thus increase mineral bioavailability in bread. For all studied ingredients, we found considerable variance both between varieties within a species and across wheat species. For example, whole grain flours, particularly from emmer and einkorn, appear to have higher mineral content than common wheat, but also a higher phytic acid content with similar phytase activity. Bread making recipes had a greater effect on phytic acid content in the final bread than the choice of species for whole grain flour production. Recipes with long yeast proofing or sourdough and the use of whole grain rye flour in a mixed wheat bread minimized the phytic acid content in the bread. Consequently, optimizing food to better nourish a growing world requires close collaboration between research organizations and practical stakeholders ensuring a streamlined sustainable process from farm to fork.
Chemometric approach for profiling of metabolites of potential antioxidant activity in Apiaceae species based on LC-PDA-ESI-MS/MS and FT-NIR
(2023) Atta, Noha H.; Handoussa, Heba; Klaiber, Iris; Hitzmann, Bernd; Hanafi, Rasha S.
Chemometrics is a tool for data mining and unlocking the door for solving big data queries. Apiaceae is a family species which is commonly cultivated worldwide. Although members of this species are widely used as antioxidant, antibacterial, antifungal, and anti-inflammatory agents, their metabolites profiling remains ambiguous. Based on WHO support, chemometrics has been used in evaluating the quality and authenticity of the herbal products. The objective of this study is to profile and characterize phenolic metabolites in nine species from Egyptian cultivars and three different species of German cultivars from the Apiaceae family using multivariate analysis after LC-PDA-ESI-MS/MS and near infrared spectroscopy data are generated. Principal component analysis was successfully applied to distinguish between the nine Egyptian cultivars and the three German cultivars, and hierarchical cluster analysis also confirmed this distinctive clustering. Partial least square regression (PLS-R) models showed a relationship between phytochemicals and antioxidant activities. The metabolites responsible for the clustering pattern and variables important for projection (VIP) were identified, being twelve amongst nine Egyptian cultivar samples and thirteen amongst the Egyptian cultivar and the German cultivar comparison. The identified VIPs were also correlated with the antioxidant activity using PLS-R. In conclusion, the study showed novelty in the application of hyphenated analytical techniques and chemometrics that assist in quality control of herbal medicine.

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