Assessing the feasibility and sustainability of a surfactin production process: a techno-economic and environmental analysis.

Biosurfactants have been profiled as a sustainable replacement for chemical-based surfactants since these bio-based molecules have higher biodegradability. Few research papers have focused on assessing biosurfactant production to elucidate potential bottlenecks. This research aims to assess the techno-economic and environmental performance of surfactin production in a potential scale of 65m3, considering different product yields and involving the European energy crisis of 2021-2022. The conceptual design, simulation, techno-economic, and environmental assessments were done by applying process engineering concepts and software tools such as Aspen Plus v.9.0 and SimaPro v.8.3.3. The results demonstrated the high economic potential of surfactin production since the higher values in the market offset the low fermentation yields, low recovery efficiency, and high capital investment. The sensitivity analysis of the economic assessment elucidated a minimum surfactin selling price between 29 and 31 USD/kg of surfactin, while a minimum processing scale for economic feasibility between 4 and 5 kg/h is needed to reach an equilibrium point. The environmental performance must be improved since the carbon footprint was 43 kg CO2eq/kg of surfactin. The downstream processing and energy demand are the main bottlenecks since these aspects contribute to 63 and 25% of the total emissions. The fermentation process and downstream process are key factors for future optimization and research.

Recombinant Production of Pseudomonas aeruginosa Rhamnolipids in P. putida KT2440 on Acetobacterium woodii Cultures Grown Chemo-Autotrophically with Carbon Dioxide and Hydrogen.

The establishment of sustainable processes for the production of commodity chemicals is one of today's central challenges for biotechnological industries. The chemo-autotrophic fixation of CO2 and the subsequent production of acetate by acetogenic bacteria via anaerobic gas fermentation represents a promising platform for the ecologically sustainable production of high-value biocommodities via sequential fermentation processes. In this study, the applicability of acetate-containing cell-free spent medium of the gas-fermenting acetogenic bacterium A. woodii WP1 as the feeder strain for growth and the recombinant production of P. aeruginosa PAO1 mono-rhamnolipids in the well-established nonpathogenic producer strain P. putida KT2440 were investigated. Additionally, the potential possibility of a simplified production process without the necessary separation of feeder strain cells was elucidated via the cultivation of P. putida in cell-containing A. woodii culture broth. For these cultures, the content of both strains was investigated by examining the relative quantification of strain-exclusive genes via qPCR. The recombinant production of mono-rhamnolipids was successfully achieved with maximum titers of approximately 360-400 mg/L for both cell-free and cell-containing A. woodii spent medium. The reported processes therefore represent a successful proof of principle for gas fermentation-derived acetate as a potential sustainable carbon source for future recombinant rhamnolipid production processes by P. putida KT2440.

Evaluation of an external foam column for in situ product removal in aerated surfactin production processes.

In Bacillus fermentation processes, severe foam formation may occur in aerated bioreactor systems caused by surface-active lipopeptides. Although they represent interesting compounds for industrial biotechnology, their property of foaming excessively during aeration may pose challenges for bioproduction. One option to turn this obstacle into an advantage is to apply foam fractionation and thus realize in situ product removal as an initial downstream step. Here we present and evaluate a method for integrated foam fractionation. A special feature of this setup is the external foam column that operates separately in terms of, e.g., aeration rates from the bioreactor system and allows recycling of cells and media. This provides additional control points in contrast to an internal foam column or a foam trap. To demonstrate the applicability of this method, the foam column was exemplarily operated during an aerated batch process using the surfactin-producing Bacillus subtilis strain JABs24. It was also investigated how the presence of lipopeptides and bacterial cells affected functionality. As expected, the major foam formation resulted in fermentation difficulties during aerated processes, partially resulting in reactor overflow. However, an overall robust performance of the foam fractionation could be demonstrated. A maximum surfactin concentration of 7.7 g/L in the foamate and enrichments of up to 4 were achieved. It was further observed that high lipopeptide enrichments were associated with low sampling flow rates of the foamate. This relation could be influenced by changing the operating parameters of the foam column. With the methodology presented here, an enrichment of biosurfactants with simultaneous retention of the production cells was possible. Since both process aeration and foam fractionation can be individually controlled and designed, this method offers the prospect of being transferred beyond aerated batch processes.

Lysinibacillus irui sp. nov., isolated from Iru, fermented African locust beans.

A Gram-positive, motile, aerobic, rod-shaped, endospore-forming strain designated IRB4-01T was isolated from fermented African locust beans (Iru) obtained from Bodija market in the city of Ibadan, southwestern Nigeria, during a screening process from food-related sources. IRB4-01T grew at 10-50 °C (optimum, 35-37 °C), pH 6-10 (optimum, pH 7) and in 0-6 % NaCl (optimum, 1-3 %). Phylogenetic analyses based on 16S rRNA and combined short- and long-read genome sequencing revealed that IRB4-01T is closely related to Lysinibacillus cavernae SYSU K30005T and Lysinibacillus boronitolerans 10aT. The cell-wall peptidoglycan type was A4α (Lys-Asp), containing the diagnostic diamino acid lysine. The major polar lipids in strain IRB4-01T were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and an unidentified phospholipid, while the predominant menaquinone was MK-7. The major cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0. Genomic DNA G+C content was 37.4  mol%, while the digital DNA-DNA hybridization revealed 33.6 and 32.3 % relatedness to L. cavernae SYSU K30005T and L. boronitolerans 10aT, respectively. Based on phenotypic, physiological and chemotaxonomic characteristics, as well as genome comparisons, strain IRB4-01T represents a novel species of the genus Lysinibacillus, for which the name Lysinibacillus irui sp. nov. is proposed. The type strain is IRB4-01T (NCIMB 15452T=LMG 32887T). Hybrid genome data are provided on the NCBI database using the Bioproject number PRJNA906010 and accession numbers CP113527 and CP113528. Additionally, a representative 16S rRNA sequence is available with the GenBank accession number OQ566940.

Characterization ofantifungal properties of lipopeptide-producing Bacillus velezensis strains and their proteome-based response to the phytopathogens, Diaporthe spp.

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.

Design and evaluation of a 3D-printed, lab-scale perfusion bioreactor for novel biotechnological applications.

3D-printing increased in significance for biotechnological research as new applications like lab-on-a-chip systems, cell culture devices or 3D-printed foods were uncovered. Besides mammalian cell culture, only few of those applications focus on the cultivation of microorganisms and none of these make use of the advantages of perfusion systems. One example for applying 3D-printing for bioreactor development is the microbial utilization of alternative substrates derived from lignocellulose, where dilute carbon concentrations and harmful substances present a major challenge. Furthermore, quickly manufactured and affordable 3D-printed bioreactors can accelerate early development phases through parallelization. In this work, a novel perfusion bioreactor system consisting of parts manufactured by fused filament fabrication (FFF) is presented and evaluated. Hydrophilic membranes are used for cell retention to allow the application of dilute substrates. Oxygen supply is provided by membrane diffusion via hydrophobic polytetrafluoroethylene membranes. An exemplary cultivation of Corynebacterium glutamicum ATCC 13032 supports the theoretical design by achieving competitive biomass concentrations of 18.4 g L-1 after 52 h. As a proof-of-concept for cultivation of microorganisms in perfusion mode, the described bioreactor system has application potential for bioconversion of multi-component substrate-streams in a lignocellulose-based bioeconomy, for in-situ product removal or design considerations of future applications for tissue cultures. Furthermore, this work provides a template-based toolbox with instructions for creating reference systems in different application scenarios or tailor-made bioreactor systems.

Deletion of Rap-phosphatases for quorum sensing control in Bacillus and its effect on surfactin production.

The complex regulatory network in Bacillus, known as quorum sensing, offers many opportunities to modify bacterial gene expression and hence to control bioprocesses. One target regulated by this mechanism is the activity of the PsrfA promoter, which is engaged in the formation of lipopeptide surfactin. It was hypothesised that deletion of rapC, rapF and rapH, encoding for prominent Rap-phosphatases known to affect PsrfA activity, would enhance surfactin production. Therefore, these genes were deleted in a sfp+ derivative of B. subtilis 168 with subsequent evaluation of quantitative data. Up to the maximum product formation of the reference strain B. subtilis KM1016 after 16 h of cultivation, the titers of the rap deletion mutants did not exceed the reference. However, an increase in both product yield per biomass YP/X and specific surfactin productivity qsurfactin was observed, without any considerable effect on the ComX activity. By extending the cultivation time, a 2.7-fold increase in surfactin titer was observed after 24 h for strain CT10 (ΔrapC) and a 2.5-fold increase for CT11 (ΔrapF) compared to the reference strain KM1016. In addition, YP/X was again increased for strains CT10 and CT11, with values of 1.33 g/g and 1.13 g/g, respectively. Interestingly, the effect on surfactin titer in strain CT12 (ΔrapH) was not as distinct, although it achieved the highest promoter activity (PsrfA-lacZ). The data presented support the possibility of involving the quorum sensing system of Bacillus in bioprocess control as shown here on the example of lipopeptide production.

Metabolic engineering of Corynebacterium glutamicum for acetate-based itaconic acid production.

BACKGROUND: Itaconic acid is a promising platform chemical for a bio-based polymer industry. Today, itaconic acid is biotechnologically produced with Aspergillus terreus at industrial scale from sugars. The production of fuels but also of chemicals from food substrates is a dilemma since future processes should rely on carbon sources which do not compete for food or feed. Therefore, the production of chemicals from alternative substrates such as acetate is desirable to develop novel value chains in the bioeconomy. RESULTS: In this study, Corynebacterium glutamicum ATCC 13032 was engineered to efficiently produce itaconic acid from the non-food substrate acetate. Therefore, we rewired the central carbon and nitrogen metabolism by inactivating the transcriptional regulator RamB, reducing the activity of isocitrate dehydrogenase, deletion of the gdh gene encoding glutamate dehydrogenase and overexpression of cis-aconitate decarboxylase (CAD) from A. terreus optimized for expression in C. glutamicum. The final strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt) produced 3.43 ± 0.59 g itaconic acid L-1 with a product yield of 81 ± 9 mmol mol-1 during small-scale cultivations in nitrogen-limited minimal medium containing acetate as sole carbon and energy source. Lowering the cultivation temperature from 30 °C to 25 °C improved CAD activity and further increased the titer and product yield to 5.01 ± 0.67 g L-1 and 116 ± 15 mmol mol-1, respectively. The latter corresponds to 35% of the theoretical maximum and so far represents the highest product yield for acetate-based itaconic acid production. Further, the optimized strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt), produced 3.38 ± 0.28 g itaconic acid L-1 at 25 °C from an acetate-containing aqueous side-stream of fast pyrolysis. CONCLUSION: As shown in this study, acetate represents a suitable non-food carbon source for itaconic acid production with C. glutamicum. Tailoring the central carbon and nitrogen metabolism enabled the efficient production of itaconic acid from acetate and therefore this study offers useful design principles to genetically engineer C. glutamicum for other products from acetate.

Characterization of Bacillus velezensis UTB96, Demonstrating Improved Lipopeptide Production Compared to the Strain B. velezensis FZB42.

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.

High-level recombinant protein production with Corynebacterium glutamicum using acetate as carbon source.

In recent years, biotechnological conversion of the alternative carbon source acetate has attracted much attention. So far, acetate has been mainly used for microbial production of bioproducts with bulk applications. In this study, we aimed to investigate the potential of acetate as carbon source for heterologous protein production using the acetate-utilizing platform organism Corynebacterium glutamicum. For this purpose, expression of model protein eYFP with the promoter systems T7lac and tac was characterized during growth of C. glutamicum on acetate as sole carbon source. The results indicated a 3.3-fold higher fluorescence level for acetate-based eYFP production with T7 expression strain MB001(DE3) pMKEx2-eyfp compared to MB001 pEKEx2-eyfp. Interestingly, comparative eyfp expression studies on acetate or glucose revealed an up to 83% higher biomass-specific production for T7 RNAP-dependent eYFP production using acetate as carbon source. Furthermore, high-level protein accumulation on acetate was demonstrated for the first time in a high cell density cultivation process with pH-coupled online feeding control, resulting in a final protein titer of 2.7 g/L and product yield of 4 g per 100 g cell dry weight. This study presents a first proof of concept for efficient microbial upgrading of potentially low-cost acetate into high-value bioproducts, such as recombinant proteins.

Surfactin Shows Relatively Low Antimicrobial Activity against Bacillus subtilis and Other Bacterial Model Organisms in the Absence of Synergistic Metabolites.

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.

Acetate-based production of itaconic acid with Corynebacterium glutamicum using an integrated pH-coupled feeding control.

To date, most bio-based products of industrial biotechnology stem from sugar-based carbon sources originating from food and feed competing resources. Exemplary for bioproducts converted from glucose, the potential C5 platform chemical itaconic acid is presently produced by the filamentous fungus Aspergillus terreus. Here, an engineered strain of the industrial platform organism Corynebacterium glutamicum ATCC 13032 was used for acetate-based production of itaconic acid to overcome current production difficulties. For this purpose, C. glutamicum ICDR453C (pEKEx2-malEcadopt) with a mutated icd variant for reduced isocitrate dehydrogenase activity was constructed harbouring pEKEx2-malEcadopt, that includes a cis-aconitate dehydrogenase gene originating from A. terreus. Overall, a peak volumetric productivity of 1.01 gL-1h-1 was achieved resulting in an itaconate titer of 29.2 g/L, by using an integrated pH-coupled acetate feeding control in a fed-batch process without base titration. The results support the high potential of acetate as alternative substrate for bioproduction.

Expression of degQ gene and its effect on lipopeptide production as well as formation of secretory proteases in Bacillus subtilis strains.

Bacillus subtilis is described as a promising production strain for lipopeptides. In the case of B. subtilis strains JABs24 and DSM10T , surfactin and plipastatin are produced. Lipopeptide formation is controlled, among others, by the DegU response regulator. The activating phospho-transfer by the DegS sensor kinase is stimulated by the pleiotropic regulator DegQ, resulting in enhanced DegU activation. In B. subtilis 168, a point mutation in the degQ promoter region leads to a reduction in gene expression. Corresponding reporter strains showed a 14-fold reduced expression. This effect on degQ expression and the associated impact on lipopeptide formation was examined for B. subtilis JABs24, a lipopeptide-producing derivative of strain 168, and B. subtilis wild-type strain DSM10T , which has a native degQ expression. Based on the stimulatory effects of the DegU regulator on secretory protease formation, the impact of degQ expression on extracellular protease activity was additionally investigated. To follow the impact of degQ, a deletion mutant was constructed for DSM10T , while a natively expressed degQ version was integrated into strain JABs24. This allowed strain-specific quantification of the stimulatory effect of degQ expression on plipastatin and the negative effect on surfactin production in strains JABs24 and DSM10T . While an unaffected degQ expression reduced surfactin production in JABs24 by about 25%, a sixfold increase in plipastatin was observed. In contrast, degQ deletion in DSM10T increased surfactin titer by threefold but decreased plipastatin production by fivefold. In addition, although significant differences in extracellular protease activity were detected, no decrease in plipastatin and surfactin produced during cultivation was observed.

Modeling the time course of ComX: towards molecular process control for Bacillus wild-type cultivations.

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.

Influence of B. subtilis 3NA mutations in spo0A and abrB on surfactin production in B. subtilis 168.

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.

High cell density cultivation of Corynebacterium glutamicum on bio-based lignocellulosic acetate using pH-coupled online feeding control.

Acetate represents a promising alternative carbon source for future industrial biotechnology. In this study, the high potential of Corynebacterium glutamicum for utilizing acetate as sole carbon source was demonstrated. Batch culture studies revealed that C. glutamicum ATCC 13032 naturally exhibits high acetate tolerance with maximum growth rates (µmax = 0.47 h-1) similar to those on D-glucose. Based on a simple and auto-regulated pH-coupled feeding strategy which utilizes bio-acetic acid in pure form, a novel and high-efficient fed-batch process was developed in a 42 L stirred-tank bioreactor. By optimizing the carbon-to-nitrogen (C/N) feeding ratio, maximum biomass concentrations of 80.2 gCDW/L were achieved with a space-time yield of 66.6 gCDW/L·d. In addition, a process model was implemented describing the time-courses of biomass growth and substrate concentrations. This is the first study in which an industrial platform organism was grown to high cell densities using green, lignocellulosic acetate as an alternative carbon source.

Exploiting RNA thermometer-driven molecular bioprocess control as a concept for heterologous rhamnolipid production.

A key challenge to advance the efficiency of bioprocesses is the uncoupling of biomass from product formation, as biomass represents a by-product that is in most cases difficult to recycle efficiently. Using the example of rhamnolipid biosurfactants, a temperature-sensitive heterologous production system under translation control of a fourU RNA thermometer from Salmonella was established to allow separating phases of preferred growth from product formation. Rhamnolipids as bulk chemicals represent a model system for future processes of industrial biotechnology and are therefore tied to the efficiency requirements in competition with the chemical industry. Experimental data confirms function of the RNA thermometer and suggests a major effect of temperature on specific rhamnolipid production rates with an increase of the average production rate by a factor of 11 between 25 and 38 °C, while the major part of this increase is attributable to the regulatory effect of the RNA thermometer rather than an unspecific overall increase in bacterial metabolism. The production capacity of the developed temperature sensitive-system was evaluated in a simple batch process driven by a temperature switch. Product formation was evaluated by efficiency parameters and yields, confirming increased product formation rates and product-per-biomass yields compared to a high titer heterologous rhamnolipid production process from literature.

Bacillus subtilis High Cell Density Fermentation Using a Sporulation-Deficient Strain for the Production of Surfactin.

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. KEY POINTS: • Utilization of a sporulation deficient strain for fed-batch operations • High cell density process with Bacillus subtilis for lipopeptide production was established • High titer surfactin production capabilities confirm highly promising future platform strain.

Surfactin from Bacillus subtilis displays promising characteristics as O/W-emulsifier for food formulations.

BACKGROUND: Biosurfactants are surface-active molecules produced by different microorganisms and display a promising alternative to synthetically derived food emulsifiers. One of these biosurfactants, synthesized by Bacillus subtilis, is the lipopeptide surfactin, which composes a linear fatty acid and cyclic peptide moiety. This study explores the interfacial and emulsion forming properties of surfactin to further characterize its suitability as an O/W emulsifier in food formulations. RESULTS: Surfactin revealed a high interfacial activity with a reduction of interfacial tension of 83.26 % to 4.21 ± 0.11 mN/m. O/W emulsions (coil = 10 % w/w) were prepared by high-pressure homogenization, which yielded volume-based mean particle sizes below 1 µm already at low emulsifier concentrations of 0.01 % (w/w). Environmental stress experiments revealed that emulsions were stable between pH 6 to pH 9. Furthermore, neither phase separation nor extensive emulsion instability was observed with NaCl addition up to 0.5 M. However, CaCl2 addition (> 3 mM) destabilized surfactin mediated emulsions. Finally, the main emulsion forming and stabilization effect of surfactin was related to its high interfacial activity and the high degree of electrostatic repulsion between the oil droplets (i.e. zeta-potential of up to -100 mV). CONCLUSION: In comparison to other natural and synthetic emulsifiers, the results showed that surfactin is a strong candidate to form and stabilize O/W emulsions under the reported conditions.