Foam-free production of Surfactin via anaerobic fermentation of Bacillus subtilis DSM 10T
© Willenbacher et al.; licensee Springer. 2015
Received: 28 January 2015
Accepted: 4 March 2015
Published: 17 March 2015
Surfactin is one of the most popular biosurfactants due to its numerous potential applications. The usually aerobic production via fermentation of Bacillus subtilis is accompanied by vigorous foaming which leads to complex constructions and great expense. Therefore it is reasonable to search for alternative foam-free production processes. The current study introduces a novel approach to produce Surfactin in a foam-free process applying a strictly anaerobic bioreactor cultivation. The process was performed several times with different glucose concentrations in mineral salt medium. The fermentations were analyzed regarding specific (qSurfactin, vol. qSurfactin) and overall product yields (YP/X, YP/S) as well as substrate utilization (YX/S). Fermentations in which 2.5 g/L glucose were employed proofed to be the most effective, reaching product yields of YP/X = 0.278 g/g. Most interesting, the product yields exceeded classical aerobic fermentations, in which foam fractionation was applied. Additionally, values for specific production rate qSurfactin (0.005 g/(g∙h)) and product yield per consumed substrate (YP/S = 0.033 g/g) surpass results of comparable foam-free processes. The current study introduces an alternative to produce a biosurfactant that overcomes the challenges of severe foaming and need for additional constructions.
Biosurfactants become increasingly attractive based on their biodegradability and production on the basis of renewable resources (Banat et al. 2010). Surfactin is one of the most popular biosurfactants and was already discovered in 1968 by Arima et al. (1968). The lipopeptide consists of a seven amino acid peptide ring comprising a β-hydroxy fatty acid. Surfactin is produced by Bacillus subtilis, a gram positive bacterium known for its application in several industrial processes, for instance the production of detergent enzymes and others (Schallmey et al. 2004). The molecule exhibits various different characteristics, which might lead to several applications. For instance, Surfactin was shown to improve plant self-resistance mechanism against soil bacteria (Ongena et al. 2007) or vigorously affects mycoplasma cells (Vollenbroich et al. 1997). Therefore, next to an application in detergents, washing agents or food products, a usage in agriculture or pharmaceutical products is also imaginable.
Naturally, amphiphile molecules produced by bacteria in cultivation processes accumulate at gas–liquid interfaces and lead to massive foam formation. The main challenge in cultivating microorganisms producing biosurfactants is to overcome this severe foam production. In the majority of cases foaming is handled by the addition of antifoam agents. Unfortunately, this strategy harbors several disadvantages, as antifoam agents are expensive and very hard to remove in downstream processes. The second most common method to cope with foam formation is to disrupt the foam by shear stress or pressure using foam breakers. However, this method is often insufficient and increases the overall costs for the production of biosurfactants. Another, more elegant, way to manage foaming in biosurfactant production processes is to apply foam fractionation, which was already shown by Cooper et al. 1981 (Cooper et al. 1981). This technique inverts the disadvantage into an advantage by using the accumulation of biosurfactants in the foam for in situ product enrichment and recovery. The Surfactin producer Bacillus subtilis is especially suited for the employment of foam fractionation, yielding high values in product recovery and enrichment (Willenbacher et al. 2014). Although this is a possible way to handle foam and to improve product yields, a realization in industrial scale is probably unrealistic in the near future.
Another artful approach is to avoid foaming at all instead of dealing with it. Several attempts have been made to establish foam-free fermentation processes. Ohno et al. for instance employed a solid state fermentation of recombinant Bacillus subtilis MI113 (pC112), using soybean curd residue as solid substrate (Ohno et al. 1995), which led to a yield of 2.0 g/kg (Surfactin per wet weight). Another attempt to produce Surfactin in a foam-free fashion implemented a membrane bioreactor (Coutte et al. 2010). A culture of Bacillus subtilis ATCC 21332 obtained a maximal Surfactin concentration of 0.242 g/L. However, a significant amount of Surfactin was adsorbed at the membranes and oxygen transfer was reduced significantly. In contrast, Chtioui et al. focused on a rotating disc bioreactor for the production of Surfactin, allowing Bacillus subtilis ATCC 21332 to grow free and immobilized in a biofilm at the same time (Chtioui et al. 2012). Aeration was realized above the fluid level, when the overgrown discs arose from the liquid. Maximal Surfactin concentrations of 0.212 g/L were obtained, but oxygen supply was limited and Fengycin concentrations surpassed Surfactin concentrations by far. While all these studies implemented innovative ideas to circumvent foaming, those processes are either difficult to scale up or lack high specificity.
Bacillus subtilis was for a long time believed to be a strict aerobic bacterium. Since 1995 research on the anaerobic growth behavior of Bacillus subtilis increased dramatically (Hoffmann et al. 1995; Nakano et al. 1997). By using nitrate as the terminal electron acceptor, Bacillus subtilis is able to perform anaerobic respiration via a nitrate reductase encoded by operon narGHJI (Ramos et al. 1995). In this manner nitrate is reduced to nitrite, which thereafter is transformed to ammonium via a nitrite reductase encoded by nasDEF (Nakano et al. 1998).
The production of biosurfactants under anaerobic conditions was already shown in 1985. The study presents the production of an undefined biosurfactant by Bacillus licheniformis in glucose mineral salt medium (Javaheri et al. 1985). The cultivation was performed in shake flasks, in the course of which the decreasing surface tension (from 70 mN/m to 28 mN/m) was measured. Although the characterization of the biosurfactant was only performed by thin layer chromatography and no high pressure liquid chromatography (HPLC) was applied, Javaheri et al. laid the foundation of anaerobic biosurfactant production. Subsequently, Davis et al. investigated the impact of nitrogen, carbon and oxygen conditions on Surfactin production of Bacillus subtilis ATCC 21332 (Davis et al. 1999). Interestingly, maximal product yields were obtained under nitrate-limited and oxygen-depleted conditions (YP/X = 0.075), which gives a further impulse to examine anaerobic Surfactin production. The proof of concept was provided by Zhang et al., who produced Surfactin with Bacillus subtilis ATCC 21332 strictly anaerobic for the first time (Zhang et al. 2007). The investigation focused on a connected shake flask system, introducing a nitrogen flow to induce vigorous foaming. The foam was channeled through several flasks with distilled water to collect the produced biosurfactant. While these studies demonstrate that anaerobic production of Surfactin is possible, none of them propose a solution to overcome foaming.
Materials and methods
All chemicals applied in the current study were of analytical grade and purchased from Carl Roth GmbH (Karlsruhe, Germany). The Surfactin standards for HPLC analysis were obtained from Sigma-Aldrich Laborchemikalien GmbH (Seelze, Germany).
Microorganism and strain maintenance
The wildtype strain Bacillus subtilis DSM 10T was used for all experiments during this study. The microorganism was obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) and stored as glycerol stocks, prepared from a culture in Lysogeny Broth (Bertani 1951) from the exponential growth phase, at −80°C.
The employed mineral salt medium was based on the fermentation medium of Cooper (Cooper et al. 1981): 8.0 × 10−4 M MgSO4, 7.0 × 10−6 M CaCl2, 4.0 × 10−6 M FeSO4, 4.0 × 10−6 M Na2EDTA, 1 × 10−6 M MnSO4. In contrast to the original medium (40 g/L glucose) the concentration of glucose was altered to 2.5 g/L, 5 g/L, 7.5 g/L and 10 g/L, during various cultivations. Furthermore, the former nitrogen source 0.05 M NH4NO3 was replaced with 0.1 M NH4Cl and 0.1177 M NaNO3. The deployed concentration of the phosphate buffer demanded slight changes depending on its usage for inoculum cultures or fermentation processes. For the cultivation in serum bottles the original 0.07 M phosphate buffer (0.03 M KH2PO4 and 0.04 M Na2HPO4) was used, whereas for the cultivation in benchtop bioreactors a 0.01 M phosphate buffer was employed (4.29 × 10−3 M KH2PO4 and 5.71 × 10−3 M Na2HPO4).
The preparation of medium for the cultivation in serum bottles demanded a different approach compared to the preparation of medium for the cultivation in benchtop bioreactors. Four different stock solutions were prepared for the cultivation in serum bottles. One stock solution contained the salt compounds (NH4Cl, NaNO3, KH2PO4, Na2HPO4) and was later completed to the final volume of 50 or 100 mL, respectively. The second stock solution included a 5.56-fold glucose solution of the final glucose concentration. In comparison, the third and fourth stock solution contained a 50-fold MgSO4 solution and a 1000-fold solution of the trace elements (CaCl2, FeSO4, Na2EDTA, MnSO4). All solutions were filled into separate serum bottles and anaerobic conditions were adjusted by 20 alternating cycles of purging with gas (20 vol.-% CO2 in N2, 45 s) and evacuating (70 mbar, 45 s). Subsequently the bottles were autoclaved and the salt stock solution was completed under anaerobic conditions to receive the final concentrations of glucose, MgSO4 and trace elements.
Preparation of inoculum cultures
For the preparation of the first seed culture a loop of B. subtilis DSM 10T from the glycerol stock solution was inoculated in 20 mL of Lysogeny Broth (inside a 100 mL baffled shake flask) and incubated in a shake incubator chamber (Multitron II, HT Infors, Bottmingen, Switzerland) at 30°C and 120 rpm for 24 h. The second seed culture was inoculated with a resulting OD600 of 0.05 under anaerobic conditions in prepared serum bottles with 50 or 100 mL of mineral salt medium, respectively. The serum bottles were incubated in a horizontal position but otherwise in the same manner as the first seed culture. After 24 h of incubation approximately 200 mL of the second seed culture were used to inoculate the aqueous phase of the bioreactor (Figure 1 A). The initial OD600 inside the bioreactor fluctuated between 0.03 and 0.07, depending on bacterial growth of the second seed culture.
Cultivation in a 2.5 L benchtop bioreactor
All cultivations were carried out in 2.5 L benchtop bioreactors (Minifors, HT Infors, Bottmingen, Switzerland) with 1.0 L mineral salt medium. The bioreactors were equipped with pH (Mettler-Toledo International Inc., Greifensee, Switzerland) and pO2 electrodes (Oxyferm, Hamilton Bonaduz AG, Bonaduz, Switzerland), a temperature sensor and Rushton turbines. The temperature was adjusted to 30°C and the pH was controlled to a value of 7.0 by the addition of 4 M NaOH or 4 M H3PO4 (Figure 2). The stirrer was adjusted to 300 rpm the entire time of cultivation. The medium was not exposed to gas flow throughout the whole fermentation process to guarantee an absolutely foam-free cultivation. However, to avoid reflux of air through the exhaust cooler and to allow the measurement of CO2 through the exhaust gas analysis, a constant N2 gas flow through the headspace of the bioreactor with 0.1 Lpm (1.5 L headspace volume) was adjusted (Figure 2: valve 2 open).
The fermentation process was started with 1.0 L of the described mineral salt medium and the additional volume of the inoculated seed culture (200 mL). Since the bioreactor cultivation was realized as a batch cultivation, no further medium components were added. During the cultivation pH, pO2, CO2 exhaust, temperature, stirrer speed and addition of acid and base were consistently monitored (Figure 2). Samples were taken from the cultivation broth (4 mL) without allowing any air flow inside the bioreactor. All fermentations were performed as duplicates.
Sampling and sample processing
By day samples were taken every three hours, whereas during nights the intervals were between five and seven hours. The sampling was designed to prevent air from entering the bioreactor system to guarantee anaerobic conditions inside. The offline analysis of the cultivation broth samples included the determination of the OD600 and the glucose, nitrate and Surfactin concentration of the supernatant. The concentration of glucose and nitrate was analyzed using a glucose assay kit (Cat. No. 10 716 251 035, R-Biopharm AG, Darmstadt, Germany) and a nitrate assay kit (1.09713.0001, Merck KGaA, Darmstadt, Germany). The concentrations were determined according to the manufacturer instructions, utilizing a spectrophotometric method (Ultrospec 2100 pro, General Electric Deutschland Holding GmbH, Frankfurt, Germany). The concentration of Surfactin was determined by analyzing the sample supernatant using HPLC (Willenbacher et al. 2014).
To enable the evaluation of the fermentation processes, several values were calculated to compare the different experiments. Using the results of CDW, glucose and Surfactin mass the values of YX/S [g/g], YP/X [g/g], YP/S [g/g], μ [h−1], qSurfactin [g/(g · h)], volumetric qSurfactin [g/(L · h)], were determined.
Comparison of process parameters during anaerobic fermentation with different glucose concentrations
Summary of the process parameters during various fermentations
Glucose concentration [g/L]
Cultivation time [h]
Max. CDW [g/L]
Max. cSurfactin [g/L]
vol. qSurfactin [g/(L∙h)]
Although cultivations with 2.5 g/L glucose reached only small amounts of CDW and Surfactin, these fermentations are comparably efficient. The cultivation time is much shorter and values for μmax, YX/S and vol. qSurfactin are comparatively high. Moreover, fermentations with 2.5 g/L glucose reached excellent values for YP/X, YP/S and specific production rate qSurfactin emphasizing an outstanding conversion of substrate into product. Nevertheless, fermentations with 2.5 g/L glucose yielded only small amounts of Surfactin, due to the short cultivation time. As a consequence it would be interesting to test whether higher overall amounts of Surfactin can be reached by applying a repeated fed-batch process. Interestingly, on closer inspections Surfactin concentrations did increase simultaneously to rising initial glucose concentrations possibly due to longer cultivation times. Surprisingly, fermentations employing 10 g/L did also achieve an almost equal value for YP/X in comparison to fermentations with 2.5 g/L glucose. But this positive result is misleading as overflow metabolism (as a result of the high initial glucose concentration) leads to low values of CDW, μmax and YX/S. This means that the bacterial growth is already strongly restricted under the employment of 10 g/L glucose. As a result data for YP/S and qSurfactin are comparably low. These findings support the usage of lower initial glucose concentrations for the anaerobe fermentation of B. subtilis DSM 10T for the production of Surfactin to avoid overflow metabolism.
Comparison with other foam-free cultivation systems and aerobic fermentation with foam fractionation
Summary of the process parameters of different foam-free processes
Chtioui et al. 2012
Coutte et al. 2010
The current study
Willenbacher et al. 2014
Bacillus subtilis ATCC 21332
Bacillus subtilis ATCC 21332
Bacillus subtilis DSM 10T
Bacillus subtilis DSM 10T
Anaerobic, no gas flow
Cultivation time [h]
Max. cSurfactin [g/L]
vol. qSurfactin [g/(L∙h)]
The processes of Chtioui et al. and Coutte et al. each yielded above 0.2 g/L Surfactin. Whereas only 0.087 g/L Surfactin were reached in the current study (with 2.5 g/L glucose in mineral salt medium). However, the fermentations of Chtioui et al. and Coutte et al. lasted comparatively longer (72 h instead of 55 h). Aerobic fermentations with Bacillus subtilis using foam fractionation take much shorter time (30 h) and yield much higher concentrations in foam (3.995 g/L). Values for YX/S differ only slightly between the foam-free processes (0.120 g/g – 0.189 g/g), but are relatively low compared to cultivations applying foam fractionation (0.268 g/g). The results for volumetric production rates are very similar, too, between the foam-free fermentations (0.002 g/(L∙h) – 0.003 g/(L∙h)). The foam fractionation fermentation reached a much higher value for vol. qSurfactin in comparison (0.018 g/(L∙h)). The product yield in contrast to substrate utilization is given by the parameter YP/S. The values for cultivations of Chtioui et al. and Coutte et al. are both 0.013 g/g. The current study reached a much higher value of 0.033 g/g for YP/S. However, fermentations employing foam fractionation still yield higher YP/S values (0.052 g/g). The specific production rate qSurfactin is five-times higher in anaerobic fermentations using 2.5 g/L glucose (0.005 g/(g∙h)) in comparison to other foam-free fermentations (0.001 g/(g∙h)). Aerobic processes applying foam fractionation yield rather similar results for qSurfactin (0.006 g(g∙h)). Most surprising are the results for YP/X. Fermentations of Chtioui et al. and Coutte et al. reached 0.068 g/g and 0.078 g/g, respectively. In contrast, anaerobic fermentations of the current study employing 2.5 g/L glucose yielded 0.278 g/g. These findings surpass even YP/X values of aerobic fermentations employing foam fractionation (0.192 g/g).
Interestingly, the results of Chtioui et al. and Coutte et al. show very similar values for efficiency, product yields and substrate utilization although completely different fermentation approaches were applied. This similarity was revealed only after calculating some additional process parameters from the original data of these publications (Table 2). While rotating disc bioreactors or membrane reactors seem very attractive alternatives to common foam fractionation processes the presented data in Table 2 expose their low yields in comparison to the results of a classic foam fractionation process. The comparison of the results of Chtioui et al. and Coutte et al. with data of the current study displays a much higher effectiveness of the anaerobic fermentation approach. Although overall less Surfactin was produced, much more Surfactin was produced per CDW. This implies that the bacterial growth is probably lower compared to the rotating discs or membrane bioreactors, but single cells produce more Surfactin under completely anaerobic conditions. These findings explain the much higher values for YP/X, YP/S and qSurfactin. In comparison to an aerobic fermentation process with foam fractionation some process parameters are lower (e.g., vol. qSurfactin and YX/S), but values for YP/S and qSurfactin are at the same level. Most important is the much higher value for YP/X under anaerobic conditions. This implies a much better production of Surfactin per CDW not only in comparison to other foam-free processes, but even in comparison to aerobic foam fractionation processes.
The current study demonstrates a new approach to produce Surfactin without any foam formation. Moreover, anaerobic cultivation and foam-free biosurfactant production are combined in one process for the first time. The anaerobic production of Surfactin was shown before, but never analyzed for product yields and substrate utilization. The comparison of different fermentations with various glucose concentrations displayed great efficiency for processes applying low glucose concentrations. Furthermore, the confrontation with other foam-free processes revealed a much higher effectiveness of the anaerobic fermentation process of the current study.
The authors would like to thank Florian Oswald, a colleague at the Karlsruhe institute of technology (KIT), for his helpful tips during anaerobic cultivations and contribution to scientific discussions. We acknowledge the support by Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of Karlsruhe Institute of Technology.
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