Layered Modeling and Simulation of Complex Biotechnological Processes - Optimizing Rhamnolipid Production by Pseudomonas aeruginosa during Cultivation in a Bioreactor

In this thesis, a model for the regulation of rhamnolipid production and data obtained from metabolic balancing were combined with a process model on a bioreactor scale. The model was used to derive an optimized process control stategy for enhanced product formation. This thesis provides a missing piece in a puzzle for knowledge-based strategies for enhanced rhamnolipid formation

Substitution of the native <i>srfA</i> promoter by constitutive P<i>veg</i> in two <i>B. subtilis </i>strains and evaluation of the effect on surfactin production

The genetic enhancement of Surfactin production increasingly gained attention in the last years, since relatively low product yields limit the industrial application of this biosurfactant. The natural quorum sensing regulation of the srfA operon (coding for the Surfactin synthetase) can reasonably be assumed to be the bottleneck of Surfactin synthesis. Therefore, the replacement of the naturally quorum sensing regulated, and herewith cell density dependent, promoter PsrfA against the Bacillus subtilis endogenous and constitutive promoter Pveg was hypothesized to generally enhance Surfactin yields. The markerless promoter replacement was conducted in the two B. subtilis Surfactin producer strains 3A38 and DSM 10T. The promoter substitution led to an enhancement of Surfactin concentrations in the producer strain 3A38, initially producing only minor amounts of Surfactin (0.07 g/L increased to 0.26 g/L). In contrast, promoter exchange in B. subtilis DSM 10T (wild-type strain producing 0.62 g/L Surfactin) did not achieve an enhancement of Surfactin concentrations (detrimental reduction to 0.04 g/L). These findings implicate that Surfactin synthesis is differently regulated in minor and strong Surfactin producer strains. The hypothesized general enhancement of Surfactin yields after substitution of the native promoter was therefore not confirmed

PRESERVATION OF MICROBIAL SPOILAGE OF FOOD BY BIOSURFACTANT-BASED COATING

Objectives: Microbial spoilage of food is one of the leading causes of food scarcity worldwide, which could have devastating effect on the socioeconomic of any country. Along with classical food preservation methods, various innovative approaches can significantly increase the food safety. Biosurfactants are the amphiphilic microbial metabolites, while many of them have potential antimicrobial properties and therefore can be used for food preservation. Methods: During this study, three biosurfactants obtained from Bacillus subtilis (two of them) and Pseudomonas sp. (rhamnolipid) were screened for their antifungal activity against Aspergillus oryzae (MTCC 1846), Fusarium solani (MTCC 350), and Curvularia sp. by various in vitro and in vivo methods. Results: During this study, among three surfactants only Pseudomonas sp. biosurfactant (rhamnolipid) exhibited significant antifungal activity against A. oryzae, F. solani, and Curvularia sp. Further, the rhamnolipid coating (1 mg/ml) on lemon, potato and tomato protected them from fungal spoilage up to 15 days at room temperature in contrast to untreated samples which started spoiling in 6–7 days. Discussion: Above findings emphasis on the potential use of biosurfactants for the preservation of food items, however, a detailed study to ensure the safety of biosurfactant is of prerequisite.Â

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 Y P/X and specific surfactin productivity q surfactin 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, Y P/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.</p

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

Potential of biotechnological conversion of lignocellulose hydrolyzates by Pseudomonas putida KT2440 as a model organism for a bio‐based economy

Lignocellulose‐derived hydrolyzates typically display a high degree of variation depending on applied biomass source material as well as process conditions. Consequently, this typically results in variable composition such as different sugar concentrations as well as degree and the presence of inhibitors formed during hydrolysis. These key obstacles commonly limit its efficient use as a carbon source for biotechnological conversion. The gram‐negative soil bacterium Pseudomonas putida KT2440 is a promising candidate for a future lignocellulose‐based biotechnology process due to its robustness and versatile metabolism. Recently, P. putida KT2440_xylAB which was able to metabolize the hemicellulose (HC) sugars, xylose and arabinose, was developed and characterized. Building on this, the intent of the study was to evaluate different lignocellulose hydrolyzates as platform substrates for P. putida KT2440 as a model organism for a bio‐based economy. Firstly, hydrolyzates of different origins were evaluated as potential carbon sources by cultivation experiments and determination of cell growth and sugar consumption. Secondly, the content of major toxic substances in cellulose and HC hydrolyzates was determined and their inhibitory effect on bacterial growth was characterized. Thirdly, fed‐batch bioreactor cultivations with hydrolyzate as the carbon source were characterized and a diauxic‐like growth behavior with regard to different sugars was revealed. In this context, a feeding strategy to overcome the diauxic‐like growth behavior preventing accumulation of sugars is proposed and presented. Results obtained in this study represent a first step and proof‐of‐concept toward establishing lignocellulose hydrolyzates as platform substrates for a bio‐based economy

Novel insights into biosynthesis and uptake of rhamnolipids and their precursors

The human pathogenic bacterium Pseudomonasaeruginosa produces rhamnolipids, glycolipids with functionsfor bacterial motility, biofilm formation, and uptake of hydrophobicsubstrates. Rhamnolipids represent a chemically heterogeneousgroup of secondary metabolites composed of one ortwo rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosyntheticpathway involves rhamnosyltransferase I encoded by the rhlABoperon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoicacids (HAAs) followed by their coupling to one rhamnose moiety.The resulting mono-rhamnolipids are converted to dirhamnolipidsin a third reaction catalyzed by therhamnosyltransferase II RhlC. However, the mechanism behindthe biosynthesis of rhamnolipids containing only a singlefatty acid is still unknown. To understand the role of proteinsinvolved in rhamnolipid biosynthesis the heterologous expressionof rhl-genes in non-pathogenic Pseudomonas putidaKT2440 strains was used in this study to circumvent the complexquorum sensing regulation in P. aeruginosa. Our resultsreveal that RhlA and RhlB are independently involved inrhamnolipid biosynthesis and not in the form of a RhlAB heterodimercomplex as it has been previously postulated.Furthermore, we demonstrate that mono-rhamnolipids providedextracellularly as well as HAAs as their precursors are generallytaken up into the cell and are subsequently converted todi-rhamnolipids by P. putida and the native host P. aeruginosa.Finally, our results throw light on the biosynthesis ofrhamnolipids containing one fatty acid,which occurs by hydrolyzationof typical rhamnolipids containing two fatty acids,valuable for the production of designer rhamnolipids with desiredphysicochemical properties