Browsing by Subject "Bacillus subtilis"

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Adaptation of model organisms and environmental bacilli to glyphosate gives insight to species-specific peculiarities of the shikimate pathway
(2024) Schwedt, Inge; Commichau, Fabian M.
Glyphosate (GS), the active ingredient of the popular herbicide Roundup, inhibits the 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase of the shikimate pathway, which is present in archaea, bacteria, Apicomplexa, algae, fungi, and plants. In these organisms, the shikimate pathway is essential for de novo synthesis of aromatic amino acids, folates, quinones and other metabolites. Therefore, the GS-dependent inhibition of the EPSP synthase results in cell death. Previously, it has been observed that isolates of the soil bacteria Burkholderia anthina and Burkholderia cenocepacia are resistant to high amounts of GS. In the framework of this PhD thesis, it could be demonstrated that B. anthina isolates are not intrinsically resistant to GS. However, B. anthina rapidly adapts to the herbicide at the genome level and the characterization of GS-resistant suppressor mutants led to the discovery of a novel GS resistance mechanism. In B. anthina, the acquisition of loss-of-function mutations in the ppsR gene increases GS resistance. The ppsR gene encodes a regulator of the phosphoenolpyruvate (PEP) synthetase PpsA. In the absence of a functional PpsR protein, the bacteria synthesize more PEP, which competes with GS for binding in the active site of the EPSP synthase, increasing GS resistance. The EPSP synthase in B. anthina probably does not allow changes in the amino acid sequence as it is the case in other organisms. Indeed, the Gram-negative model organism Escherichia coli evolves GS resistance by the acquisition of mutations that either reduce the sensitivity of the EPSP synthase or increase the cellular concentration of the enzyme. Unlike E. coli, the EPSP synthase is also critical for the viability of Gram-positive model bacterium Bacillus subtilis. This observation is surprising because the enzyme belongs to the class of GS-insensitive EPSP synthases. In fact, the EPSP synthase is essential for growth of B. subtilis. The determination of the nutritional requirements allowing the growth of B. subtilis and E. coli mutants lacking EPSP synthase activity revealed that the demand for shikimate pathway intermediates is higher in the former organism. This finding explains why laboratory as well as environmental Bacilli exclusively adapt to GS by the mutational inactivation of glutamate transporter genes. Here, it was also shown that a B. subtilis mutant lacking EPSP synthase activity grows in minimal medium only when additional mutations accumulate in genes involved in the regulation of aerobic/anaerobic metabolism and central carbon metabolism. The characterization of these additional mutants will help to elucidate the peculiarities of the shikimate pathway in B. subtilis. Moreover, the mutants could be useful to identify the aromatic amino acid transporters that still await their discovery.
Bacillus subtilis high cell density fermentation using a sporulation-deficient strain for the production of surfactin
(2021) Klausmann, Peter; Hennemann, Katja; Hoffmann, Mareen; Treinen, Chantal; Aschern, Moritz; Lilge, Lars; Morabbi Heravi, Kambiz; Henkel, Marius; Hausmann, Rudolf
Bacillus subtilis 3NA is a strain capable of reaching high cell densities. A surfactin producing sfp+ variant of this strain, named JABs32, was utilized in fed-batch cultivation processes. Both a glucose and an ammonia solution were fed to set a steady growth rate μ of 0.1 h-1. In this process, a cell dry weight of up to 88 g L-1 was reached after 38 h of cultivation, and surfactin titers of up to 26.5 g L-1 were detected in this high cell density fermentation process, achieving a YP/X value of 0.23 g g-1 as well as a qP/X of 0.007 g g-1 h-1. In sum, a 21-fold increase in surfactin titer was obtained compared with cultivations in shake flasks. In contrast to fed-batch operations using Bacillus subtilis JABs24, an sfp+ variant derived from B. subtilis 168, JABs32, reached an up to fourfold increase in surfactin titers using the same fed-batch protocol. Additionally, a two-stage feed process was established utilizing strain JABs32. Using an optimized mineral salt medium in this high cell density fermentation approach, after 31 h of cultivation, surfactin titers of 23.7 g L-1 were reached with a biomass concentration of 41.3 g L-1, thus achieving an enhanced YP/X value of 0.57 g g-1 as well as a qP/X of 0.018 g g-1 h-1. The mutation of spo0A locus and an elongation of AbrB in the strain utilized in combination with a high cell density fed-batch process represents a promising new route for future enhancements on surfactin production.
Decontamination of black peppercorn (Piper nigrum L.) using microwave-generated low pressure air plasma
(2011) Müller, Joachim; Stroth, U.; Aurich, S.; Argyropoulos, Dimitrios; Janzen, O.; Krause, N.; Romano, Guiseppe; Heindl, A.; Heberle, B.; Leins, M.; Schulz, A.; Voesgen, W.
The preliminary results show that microwave-generated low pressure air plasmas could be a very efficient method for the decontamination of spices since the population of a microorganism (Bacillus subtilis) commonly found in black pepper (Piper nigrum L.) was significantly reduced on test substrates in a very short period of time. Based on the experimental results, a laboratory apparatus was developed for the sterilisation of spices.
Evaluation of an oxygen‐dependent self‐inducible surfactin synthesis in B. subtilis by substitution of native promoter PsrfA by anaerobically active PnarG and PnasD
(2021) Hoffmann, Mareen; Braig, Alina; Fernandez Cano Luna, Diana Stephanie; Rief, Katharina; Becker, Philipp; Treinen, Chantal; Klausmann, Peter; Morabbi Heravi, Kambiz; Henkel, Marius; Lilge, Lars; Hausmann, Rudolf
A novel approach targeting self-inducible surfactin synthesis under oxygen-limited conditions is presented. Because both the nitrate (NarGHI) and nitrite (NasDE) reductase are highly expressed during anaerobic growth of B. subtilis, the native promoter PsrfA of the surfactin operon in strain B. subtilis JABs24 was replaced by promoters PnarG and PnasD to induce surfactin synthesis anaerobically. Shake flask cultivations with varying oxygen availabilities indicated no significant differences in native PsrfA expression. As hypothesized, activity of PnarG and PnasD increased with lower oxygen levels and surfactin was not produced by PsrfA::PnarG as well as PsrfA::PnasD mutant strains under conditions with highest oxygen availability. PnarG showed expressions similar to PsrfA at lowest oxygen availability, while maximum value of PnasD was more than 5.5-fold higher. Although the promoter exchange PsrfA::PnarG resulted in a decreased surfactin titer at lowest oxygen availability, the strain carrying PsrfA::PnasD reached a 1.4-fold increased surfactin concentration with 696 mg/L and revealed an exceptional high overall YP/X of 1.007 g/g. This value also surpassed the YP/X of the reference strain JABs24 at highest and moderate oxygen availability. Bioreactor cultivations illustrated that significant cell lysis occurred when the process of “anaerobization” was performed too fast. However, processes with a constantly low agitation and aeration rate showed promising potential for process improvement, especially by employing the strain carrying PsrfA::PnasD promoter exchange. Additionally, replacement of other native promoters by nitrite reductase promoter PnasD represents a promising tool for anaerobic-inducible bioprocesses in Bacillus.
Exploiting novel strategies for the production of surfactin in Bacillus subtilis cultures
(2021) Hoffmann, Mareen; Hausmann, Rudolf
Biosurfactants are synthesized by various microorganisms. These surface-active molecules are a promising alternative to petrochemically and oleochemically produced surfactants. Advantageously, biosurfactants are reported to be better biodegradable and less toxic. The cyclic lipopeptide surfactin synthesized by Bacillus subtilis displays one interesting biosurfactant. Many studies report on the outstanding physico-chemical characteristics and add on benefits such as antimicrobial properties. Hence, surfactin has the potential to be used in a variety of industrial sectors. Nevertheless, processes ensuring both robustness and high titers are rare, especially as conventional aerobic bioreactor cultivations share one major disadvantage, namely excessive foaming. To approach industrial processes, different methods are applied, which can be categorized in three practices. These are (1) media and process parameter optimization, (2) strain engineering, and (3) investigating novel process strategies. For the latter category, the anaerobic growth by nitrate respiration poses an interesting foam-free alternative. In this sense, the anaerobic cultivation of B. subtilis to produce surfactin coupled with the three afore mentioned practices was addressed in this thesis targeting at a foam-free surfactin production process. In the 1st publication, the genome reduced strain B. subtilis IIG-Bs20-5-1, a derivative of the laboratory strain 168 able to synthesize surfactin, was evaluated with respect to its suitability as surfactin producer at various temperatures under both aerobic and anaerobic conditions. It was hypothesized that a deletion of 10% of the genome, e.g., non-essential genes synthesizing prophages or the antibiotic bacilysin, saves metabolic resources and hence results in increased surfactin titers. Strains B. subtilis JABs24, a 168 derivative able to synthesize surfactin but without genome reduction, and the surfactin producer B. subtilis DSM 10T served for comparison. Although strain IIG-Bs20-5-1 was superior regarding specific growth rate µ and biomass yield YX/S, the strain was inferior with respect to surfactin titers, product related yields YP/S and YP/X, and specific productivity q. Indeed, compared to others in literature described strains, B. subtilis JABs24 was emphasized as promising target strain for further process development, reaching high surfactin titers of 1147 mg/L aerobically and 296 mg/L anaerobically as well as exceptionally high product yields YP/X under anaerobic conditions. Accordingly, iterative process optimization was hypothesized to improve anaerobically achieved surfactin titers. However, several aspects to consider of anaerobic growth of B. subtilis by nitrate respiration were described in the 2nd publication. Amongst others, increasing ammonium concentrations, resulting from nitrate reduction to ammonium via nitrite, were shown to have no impact on growth of strain JABs24, but surfactin titers and expression of nitrate reductase promoter PnarG were reduced. Nitrite was shown to peak within the first hours of cultivation and concentrations up to 10 mmol/L resulted in prolonged lag-phases. Moreover, acetate accumulated drastically during the time course of cultivation independent of glucose availability, thus decreasing the glucose flux into biomass. Acetate additionally influenced both specific growth rate µ and PnarG expression negatively. Concluding, the general feasibility of anaerobic fed-batch cultivations to synthesize surfactin was shown, but several aspects must be addressed in future works to make this strategy an equated process with aerobic cultivations. In the 3rd publication, a self-inducible surfactin synthesis process was presented where expression of the surfactin operon in B. subtilis JABs24 was induced under oxygen limited conditions. The native promoter of the srfA operon PsrfA was replaced by anaerobically inducible nitrate reductase promoter PnarG and nitrite reductase promoter PnasD. Shake flask cultivations with varying oxygen availabilities demonstrated that both PnarG and PnasD can serve as auto-inducible promoters. At high oxygen availability, surfactin was not produced in the promoter exchange strains. At lowest oxygen availability, the strain carrying PnarG reached lower surfactin titers than the native JABs24 strain, although expression levels of PnarG and PsrfA were similar. However, strain B. subtilis MG14 with PsrfA::PnasD reached 1.4-fold higher surfactin titers with 696 mg/L and an exceptionally high YP/X of 1.007 g/g with overall lower foam levels. Though, bioreactor cultivations have illustrated that the anaerobic induction must be performed slowly as to avoid cell lysis, resulting in so-defined aerobic-anaerobic switch processes. With further appropriate process optimization, a simple and robust surfactin production process with highly reduced or even no foam formation can be achieved employing strain B. subtilis MG14.
Influence of B. subtilis 3NA mutations in spo0A and abrB on surfactin production in B. subtilis 168
(2021) Klausmann, Peter; Lilge, Lars; Aschern, Moritz; Hennemann, Katja; Henkel, Marius; Hausmann, Rudolf; Morabbi Heravi, Kambiz
Background: Bacillus subtilis is a well-established host for a variety of bioproduction processes, with much interest focused on the production of biosurfactants such as the cyclic lipopeptide surfactin. Surfactin production is tightly intertwined with quorum sensing and regulatory cell differentiation processes. As previous studies have shown, a non-sporulating B. subtilis strain 3NA encoding a functional sfp locus but mutations in the spo0A and abrB loci, called JABs32, exhibits noticeably increased surfactin production capabilities. In this work, the impacts of introducing JABs32 mutations in the genes spo0A, abrB and abh from 3NA into strain KM1016, a surfactin-forming derivative of B. subtilis 168, was investigated. This study aims to show these mutations are responsible for the surfactin producing performance of strain JABs32 in fed-batch bioreactor cultivations. Results: Single and double mutant strains of B. subtilis KM1016 were constructed encoding gene deletions of spo0A, abrB and homologous abh. Furthermore, an elongated abrB version, called abrB*, as described for JABs32 was integrated. Single and combinatory mutant strains were analysed in respect of growth behaviour, native PsrfA promoter expression and surfactin production. Deletion of spo0A led to increased growth rates with lowered surfactin titers, while deletion or elongation of abrB resulted in lowered growth rates and high surfactin yields, compared to KM1016. The double mutant strains B. subtilis KM1036 and KM1020 encoding Δspo0A abrB* and Δspo0A ΔabrB were compared to reference strain JABs32, with KM1036 exhibiting similar production parameters and impeded cell growth and surfactin production for KM1020. Bioreactor fed-batch cultivations comparing a Δspo0A abrB* mutant of KM1016, KM681, with JABs32 showed a decrease of 32% in surfactin concentration. Conclusions: The genetic differences of B. subtilis KM1016 and JABs32 give rise to new and improved fermentation methods through high cell density processes. Deletion of the spo0A locus was shown to be the reason for higher biomass concentrations. Only in combination with an elongation of abrB was this strain able to reach high surfactin titers of 18.27 g L−1 in fed-batch cultivations. This work shows, that a B. subtilis strain can be turned into a high cell density surfactin production strain by introduction of two mutations.
The influence of growth rate-controlling feeding strategy on the surfactin production in Bacillus subtilis bioreactor processes
(2024) Hiller, Eric; Off, Manuel; Hermann, Alexander; Vahidinasab, Maliheh; Benatto Perino, Elvio Henrique; Lilge, Lars; Hausmann, Rudolf
Background The production of surfactin, an extracellular accumulating lipopeptide produced by various Bacillus species, is a well-known representative of microbial biosurfactant. However, only limited information is available on the correlation between the growth rate of the production strain, such as B. subtilis BMV9, and surfactin production. To understand the correlation between biomass formation over time and surfactin production, the availability of glucose as carbon source was considered as main point. In fed-batch bioreactor processes, the B. subtilis BMV9 was used, a strain well-suited for high cell density fermentation. By adjusting the exponential feeding rates, the growth rate of the surfactin-producing strain, was controlled. Results Using different growth rates in the range of 0.075 and 0.4 h-1, highest surfactin titres of 36 g/L were reached at 0.25 h-1 with production yields YP/S of 0.21 g/g and YP/X of 0.7 g/g, while growth rates lower than 0.2 h-1 resulted in insufficient and slowed biomass formation as well as surfactin production (YP/S of 0.11 g/g and YP/X of 0.47 g/g for 0.075 h-1). In contrast, feeding rates higher than 0.25 h-1 led to a stimulation of overflow metabolism, resulting in increased acetate formation of up to 3 g/L and an accumulation of glucose due to insufficient conversion, leading to production yields YP/S of 0.15 g/g and YP/X of 0.46 g/g for 0.4 h-1. Conclusions Overall, the parameter of adjusting exponential feeding rates have an important impact on the B. subtilis productivity in terms of surfactin production in fed-batch bioreactor processes. A growth rate of 0.25 h-1 allowed the highest surfactin production yield, while the total conversion of substrate to biomass remained constant at the different growth rates.
Investigations on the mechanisms of sterilization by non-thermal low-pressure nitrogen-oxygen plasmas
(2011) Roth, Stefan; Hertel, Christian
Plastic based materials are increasingly used for packaging of pharmaceuticals (especially biologicals), food or beverages and production of medical devices. Their heat sensitivity requires safe and efficient non-thermal methods for decontamination. Plasma technology has the potential to provide a suitable means since it works at low temperatures and ? in contrast to conventional methods like application of ionizing radiation or ethylene oxide exposure ? is safe to operate, is free of residues and does not alter the bulk properties of the materials. Plasmas can generate various agents potentially active in decontamination like ultra-violet (UV) radiation, radicals and other reactive particles. To acquire an approval for plasma technology as a novel sterilization method, its process safety has to be proven. The research community has proposed hypotheses and models on its mechanisms of action, which are at least partially speculative. Still little is known about the details of the biologic effects of the combination of the various plasma agents on the components of microbial cells or spores. Especially, the question remains open which components of a cell or spore are the primary targets, and which of the agents are most effective in the inactivation process. The acquisition of such knowledge is necessary to identify parameters suitable to control, monitor, and assess the safety of plasma sterilization processes. The aims of the presented work are to elucidate which components of a cell or spore are the primary targets in low-pressure plasma sterilization, and which of the putative agents contained in the plasma are most effective in the inactivation process. To accomplish this, in the presented work suitable microbiological methods were established and the inactivation of bacterial spores and cells and fungal conidia by microwave induced low-pressure low-temperature nitrogen-oxygen plasmas was investigated. Moreover, two strategies were pursued that have hitherto not been applied in published plasma sterilization studies: (i) Using spores of Bacillus subtilis mutants to identify structural components serving as targets for sterilization with plasma and (ii) characterizing the response of Deinococcus radiodurans R1 cells to plasma treatment and identify repair processes during recovery from plasma induced damages in viable cells. Plasmas producing a maximum of UV emission were most effective in inactivating bacterial cells and spores. The inactivation followed a biphasic kinetics consisting of a log-linear phase with rapid inactivation followed by a slow inactivation phase. A continuous model fit was applied to the experimental data allowing reliable calculation of decimal reduction values for both phases. Cells of D. radiodurans were found to be more resistant than spores of B. subtilis. For B. subtilis spores, in the course of plasma treatment damage to DNA, proteins and spore membranes were observed by monitoring the occurrence of auxotrophic mutants, inactivation of catalase (KatX) activity and the leakage of dipicolinic acid, respectively. Spores of the wild-type strain showed highest resistance to plasma treatment. Spores of mutants defective in nucleotide excision repair (uvrA) and small acid-soluble proteins (ΔsspA ΔsspB) were more sensitive than those defective in the coat protein CotE or spore photoproduct repair (splB). Exclusion of reactive particles and spectral fractions of UV radiation from access to the spores revealed that UV-C radiation is the most effective inactivation agent in the plasma, whereby the splB and ΔcotE mutant spores were equally and slightly less sensitive, respectively, than the wild-type spores. The extent of damages in the spore DNA as determined by quantitative PCR correlated with the spore inactivation. Spore inactivation was effectively mediated by a combination of DNA damage and protein inactivation. DNA was identified to be the primary target for spore inactivation by UV radiation emitted by the plasma. Coat proteins were found to constitute a protective layer against the action of the plasma. For the investigation of the recovery from plasma-induced damages, cells of D. radiodurans R1 were subjected to short plasma treatments with various plasmas. A part of the survivors was sublethally injured as determined by their ability to form colonies on standard medium but not on stress medium and by the observation of a prolonged lag phase. Incubation of the cells in a recovery medium after plasma treatment allowed a part of the survivors to recover their ability to grow on stress medium. This recovery strongly depended on transcriptional and translational processes and cell wall synthesis, as revealed by addition of specific inhibitors to the recovery medium. Genes involved in DNA repair, oxidative stress response and cell wall synthesis were induced during recovery, as determined by quantitative RT-PCR. Damage to chromosomal DNA caused by plasma agents and in-vivo repair during recovery was directly shown by quantitative PCR. Plasmas with less UV radiation emission were also effective in killing D. radiodurans cells but resulted in less DNA damage and lower induction of the investigated genes. The response of D. radiodurans to plasma indicated that DNA, proteins and cell wall are primary targets of plasma, whose damage initially leads to the cells' death. Protein oxidation was more important for the killing of D. radiodurans cells than of B. subtilis spores. Thus, the plasma process parameters must regard the expected contaminating biological material in order to obtain a high-level sterilization. The results provide new insight into the interaction of non-thermal low-pressure plasmas with microorganisms. This knowledge supports the definition of suitable parameters for novel plasma sterilization equipment to control process safety. For example, monitoring the UV intensity below 280 nm and spectrometric online measurement of bands related to excited reactive gas particle species during the process is recommended.
The low mutational flexibility of the EPSP synthase in Bacillus subtilis is due to a higher demand for shikimate pathway intermediates
(2023) Schwedt, Inge; Schöne, Kerstin; Eckert, Maike; Pizzinato, Manon; Winkler, Laura; Knotkova, Barbora; Richts, Björn; Hau, Jann-Louis; Steuber, Julia; Mireles, Raul; Noda‐Garcia, Lianet; Fritz, Günter; Mittelstädt, Carolin; Hertel, Robert; Commichau, Fabian M.
Glyphosate (GS) inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that is required for aromatic amino acid, folate and quinone biosynthesis in Bacillus subtilis and Escherichia coli. The inhibition of the EPSP synthase by GS depletes the cell of these metabolites, resulting in cell death. Here, we show that like the laboratory B. subtilis strains also environmental and undomesticated isolates adapt to GS by reducing herbicide uptake. Although B. subtilis possesses a GS-insensitive EPSP synthase, the enzyme is strongly inhibited by GS in the native environment. Moreover, the B. subtilis EPSP synthase mutant was only viable in rich medium containing menaquinone, indicating that the bacteria require a catalytically efficient EPSP synthase under nutrient-poor conditions. The dependency of B. subtilis on the EPSP synthase probably limits its evolvability. In contrast, E. coli rapidly acquires GS resistance by target modification. However, the evolution of a GS-resistant EPSP synthase under non-selective growth conditions indicates that GS resistance causes fitness costs. Therefore, in both model organisms, the proper function of the EPSP synthase is critical for the cellular viability. This study also revealed that the uptake systems for folate precursors, phenylalanine and tyrosine need to be identified and characterized in B. subtilis.
Metabolic rewiring enables ammonium assimilation via a non‐canonical fumarate‐based pathway
(2024) Mardoukhi, Mohammad Saba Yousef; Rapp, Johanna; Irisarri, Iker; Gunka, Katrin; Link, Hannes; Marienhagen, Jan; de Vries, Jan; Stülke, Jörg; Commichau, Fabian M.
Glutamate serves as the major cellular amino group donor. In Bacillus subtilis, glutamate is synthesized by the combined action of the glutamine synthetase and the glutamate synthase (GOGAT). The glutamate dehydrogenases are devoted to glutamate degradation in vivo. To keep the cellular glutamate concentration high, the genes and the encoded enzymes involved in glutamate biosynthesis and degradation need to be tightly regulated depending on the available carbon and nitrogen sources. Serendipitously, we found that the inactivation of the ansR and citG genes encoding the repressor of the ansAB genes and the fumarase, respectively, enables the GOGAT-deficient B. subtilis mutant to synthesize glutamate via a non-canonical fumarate-based ammonium assimilation pathway. We also show that the de-repression of the ansAB genes is sufficient to restore aspartate prototrophy of an aspB aspartate transaminase mutant. Moreover, in the presence of arginine, B. subtilis mutants lacking fumarase activity show a growth defect that can be relieved by aspB overexpression, by reducing arginine uptake and by decreasing the metabolic flux through the TCA cycle.
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.
Toward effects of hydrophobicity on biosurfactant production by Bacillus subtilis isolates from crude-oil-exposed environments
(2024) Hashemi, Seyedeh Zahra; Fooladi, Jamshid; Vahidinasab, Maliheh; Hubel, Philipp; Pfannstiel, Jens; Pillai, Evelina; Hrenn, Holger; Hausmann, Rudolf; Lilge, Lars
Background: Due to their structural features, biosurfactants reveal promising physicochemical properties, making them interesting for various applications in different fields, such as the food, cosmetics, agriculture, and bioremediation sectors. In particular, the bioproduction of surfactin, one of the most potent microbially synthesized biosurfactant molecules, is of great interest. However, since the wild-type productivities are comparably low, stimulatory environmental conditions have to be identified for improved bioproduction This study aims to find a correlation between the hydrophobicity and production of the biosurfactant surfactin by B. subtilis isolates from crude-oil-contaminated soil and water. Methods: The surfactin production yield was characterized in adapted batch cultivations using high-performance thin-layer liquid chromatography (HPTLC). Defined hydrophobic environmental conditions were achieved by supplementation with hexadecane or polystyrene beads, and the effects on biosurfactant production were measured. Adaptations at the protein level were analyzed using mass spectrometry measurements. Results: The correlation between hydrophobicity and surfactin production was characterized using Bacillus subtilis strains ZH1 and P7 isolated from crude-oil-contaminated soil and water. Since these isolates show the biodegradation of crude oil and hexadecane as hydrophobic substrates, respectively, a first-time approach, using polystyrene beads, was applied to provide a hydrophobic environment. Interestingly, contrary to popular opinion, reduced biosurfactant production was determined. Using mass spectrometric approaches, the physiological effects of co-cultivation and the cellular response at the protein level were investigated, resulting in altered quantities of stress proteins and proteins involved in the carbon metabolism counter to polystyrene beads. Conclusions: Contrary to common opinion, increasing hydrophobicity does not have a stimulating effect, and even reduces the effect on the bioproduction of surfactin as the main biosurfactant using selected B. subtilis strains.
Understanding the role of plant growth promoting bacteria on sorghum growth and biotic suppression of striga infestation
(2014) Mounde, Lenard Gichana; Sauerborn, Joachim
Witchweeds (Striga sp.) are parasitic weeds of great agricultural significance, parasitizing the roots of their hosts. Striga, like all other root parasitic weeds, drain essential organic and inorganic resources from their hosts leading to poor crop development and low yield. In Africa, about 50 million ha in over 30 countries are infested by Striga spp. causing grain loss of cereals. Estimated yield losses of maize, sorghum, millets and upland rice are between 30 and 90%. The parasite, therefore, is ranked as the leading biotic constraint to cereal production in the continent. Plant growth promoting rhizobacteria (PGPR) are promising components for integrated solutions to agro-environmental problems because inoculants possess the capacity to promote crop growth and reduce the population of deleterious microbes in the rhizosphere. Although there are numerous studies on crop growth promotion and biological control of diseases, weeds, nematodes and parasitic weeds using PGPR, little is known about the potential of some Bacillus subtilis, B. amyloliquefaciens and Burkholderia phytofirmans strains in sorghum growth promotion and resistance against Striga infection. The main objective of the study was to assess the effect of B. subtilis Bsn5, B. subtilis GBO3, B. amyloliquefaciens FZB42 and Burkholderia phytofirmans PsJN on growth promotion of sorghum crop and suppression of Striga development, thus providing a basic understanding on the sorghum-PGPR-Striga interaction. This study opens with an elaborate review of the state-of-the-art knowledge on the tripartite interactions between Striga, sorghum and different species of PGPR. Prior to this, bipartite relationship between sorghum and Striga, PGPR-sorghum and PGPR-Striga are reviewed with a focus on understanding Striga impact on sorghum, sorghum defence responses to infection, plant growth and disease suppression benefits by PGPR on sorghum, and the effect of PGPR on Striga development. Knowledge gaps in both bipartite and tripartite relationships are described, and future research recommendations given. A key recommendation from the review is to conduct experiments under controlled environmental conditions using Bacillus subtilis, B. amyloliquefaciens and Burkhoderia phytofirmans strains in order to understand their relationship with sorghum and Striga at bipartite and tripartite levels. Petri dish bioassays and root chamber experiments under controlled conditions were conducted at the Institute of Plant Production and Agroecology in the Tropics and Subtropics, University of Hohenheim between 2012 and 2014. B. subtilis Bsn5, B. subtilis GBO3, B. amyloliquefaciens FZB42 and Burkholderia phytofirmans strain PsJN inocula and their corresponding cell culture supernatants were evaluated for their growth promotion potential on sorghum and suppressiveness on Striga development. Sorghum root exudates and synthetic stimulant GR24 were used to induce Striga seed germination. Bacillus subtilis Bsn5 supernatant, which showed the greatest inhibitory activity on Striga germination and radicle elongation, was separated by ethyl acetate into lipophilic and hydrophilic phases. The purpose of this extraction was to try and identify the polarity of the inhibitor. Protein composition by mass spectrometry (MS) was also done on the supernatant with a view of establishing the presence of peptides because peptides have been associated with Orobancheceae germination and radicle inhibition in previous studies. In addition, determination of plant growth hormones in bacteria supernatants was also conducted using Radio-Immuno-Assay (RIA) in order to relate PGPR hormone production and sorghum growth enhancement. Burkholderia phytofirmans PsJN significantly (<0.05) induced a higher vigor index (VI) on sorghum seedlings (>18,000) compared to other PGPR and control treatments. The lowest VI (7626) was recorded in seeds inoculated with Bacillus amyloliquefaciens FZB42. Complete Striga germination inhibition (0% germination) occurred in seeds exposed to all PGPR inocula suspended while the highest germination (>60%) occurred in control treatments (10% Luria Bertani (LB) + GR24 and sterile distilled water (SDW) + GR24). The effect of bacterial supernatants on the germination percentage and radicle length of Striga seeds was also significantly (<0.05) different among treatments. The least germination (7.4 %) was observed in Bacillus subtilis Bsn5 + GR24 while the highest (66 %) was observed in SDW + GR24 control. Bacillus subtilis Bsn5 supernatant produced the lowest mean radicle lengths (0.1 mm) while the highest radicle lengths were observed in SDW + GR24 (2.2 mm). Therefore, Bacillus subtilis Bsn5 supernatant was selected for further investigation of compounds causing inhibition of Striga germination and preventing radicle elongation. The supernatant was separated into hydrophilic and hydrophobic fractions using ethyl acetate. Each fraction was then prepared in 1%, 25%, 50%, 75% and 100% concentrations before being evaluated for their inhibitory activity in Striga germination and radicle elongation. The highest germination percentage (63%) and radical length (2.9 mm) was observed in SDW + GR24 control treatment. The ethyl acetate (lipophilic) fraction at both 100% and 1% concentration + GR24 produced a germination percentage of >40% which was similar to 10% LB + GR24 and ethyl acetate + GR24 controls. There was complete inhibition of Striga seed germination after exposure to either Bacillus subtilis Bsn5 supernatant + GR24 or 100% hydrophilic fraction of the supernatant + GR24. However, at 25% and 1% concentration + GR24, Striga germination percentage increased to 34% and 49%, respectively. Light microscopy examination of Striga radicles exposed to Bacillus subtilis Bsn5 supernatant + GR24 revealed that stunting of the radicles was due to reduction in cell sizes at the radicle elongation zone. Extended agar gel assays (EAGA) experiments showed a similar trend of results with B. subtilis Bsn5 showing the highest inhibitory activity on Striga germination and radicle elongation compared to other PGPR and control treatments. Results from root chamber experiments demonstrated significant (p<0.05) differences in biomass production between Striga-free and Striga-infected sorghum. Total biomass yield in uninoculated Striga-free plants was 40% higher than uninoculated Striga-infected sorghum plants. Bacillus amyloliquefaciens FZB42, B. subtilis GBO3 and Burkholderia phytofirmans PsJN inoculated Striga-free sorghum showed a 75%; 142% and 158% increase in biomass yield, respectively, compared to uninoculated Striga-free sorghum. There were no significant differences in biomass yield observed between inoculated and uninoculated Striga-infected plants. All PGPR supernatants and 10% LB media showed production of phytohormones cytokinin, IAA, GAs and ABA. Cytokinin content in PGPR supernatants was significantly (>0.05) higher than blank 10% LB control media. There was a significant negative correlation (r= -0.96) between IAA and cytokinins. However, there was no significant positive correlation between any phytohormone and sorghum plant height, SPAD values, biomass production, Striga germination, attachment and tubercle death. Finally, this study shows that Bacillus subtilis Bsn5, B. subtilis GBO3, B. amyloliquefaciens FZB42 and Burkholderia phytofirmans PsJN might accelerate sorghum growth and suppress key stages of Striga development under laboratory conditions. Greenhouse and field experiments are recommended to better understand these interactions under natural conditions where other biotic and abiotic factors come into play. These findings could contribute to a better understanding of sorghum and beneficial bacteria interactions and provide novel information of the long-term effects of a PGPR on sorghum development, opening new avenues for Striga control and sustainable, ecofriendly sorghum production.