Enhancing rhamnolipids production and exploring the mechanism of low-foaming fermentation under weakly acidic conditions

The commercial applications of rhamnolipids have seriously been impeded by the high cost of production caused by severe foaming. Effective low-foaming fermentation and mechanism study have become the most urgent requirement for the larger-scale rhamnolipids production. In this study, the low-foaming fermentation of rhamnolipids is realized by controlling fermentation at pH 5.5. Further, the rhamnolipids production is enhanced 0.9-fold by ultraviolet and ethyl methanesulfonate composite mutagenesis. To the best of our knowledge, this is the rst report to enhance production of rhamnolipids by strain improvement under weakly acidic conditions. The mechanism exploration tests indicated that increasing surface tension and decreasing viscosity are conducive to reducing foaming ability of rhamnolipids fermentation. The decrease of negatively charged ions may weaken the electrostatic repulsion between charged substances adsorbed on membranes of bubbles such as rhamnolipids and cells, making liquid membranes prone to rupture. In addition, the vesicle or lamellar structures of rhamnolipids are formed under pH 5.0-6.0, weakening the foaming ability of rhamnolipids. The work reveals promising potentiality by genetic modication to enhance rhamnolipids production and is conducive to understanding the pH-associated foaming behavior as well as developing the more effective pH-associated control strategies for large-scale rhamnolipids production under weakly acidic conditions.

However, according to the previous studies, the most of rhamnolipids fermentation by controlling pH at pH 5.0-6.0 are unsatisfactory, because the cells growth and rhamnolipids synthesis are conspicuously inhibited [25,26]. Therefore, in this study, we rstly prove the potentiality of low-foaming rhamnolipids fermentation of Pseudomonas aeruginosa SW1 by evaluating foaming behavior and productivity at weakly acidic conditions. Further, the production ability of P. aeruginosa SW1 is enhanced by ultraviolet (UV) and ethyl methanesulfonate (EMS) composite mutagenesis under pH 5.5. Finally, the mechanism of low-foaming rhamnolipids fermentation caused by weakly acidic conditions are systematically explored by characterizing the effects of fermentation pH on viscosity, surface tension, zeta potential, foaming behavior and morphological change of rhamnolipids.

Materials And Methods
Bacterial strain and medium A Pseudomonas aeruginosa SW1 stored in our laboratory was grown in nutrient broth at 37℃ and 200 rpm in an orbital shaker until the optical density at 600 nm reached approximately 2.0. The broth culture then was used as the fermentation inoculum. The medium of fermentation contained: 40 g/L soybean oil; 5 g/L NaNO 3 ; 1.1 g/L KCl; 1.1 g/L NaCl; 4 g/L KH 2 PO 4 ; 4.4 g/L K 2 HPO 4 ; and 0.5 g/L MgSO 4 ·7H 2 O. The medium was sterilized at 121 ℃ for 20 min for fermentation. Chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. The bean oil was purchased from Shandong Luhua group Industry Co., Ltd (Laiyang, China).

Fermentation
The seed culture was inoculated at 1:10 into medium of fermentation. In 250 ml Erlenmeyer asks, fermentation was conducted at 37 °C with orbital shaking at 200 rpm for 96 h. In the fermenter, a 7.5 L fermenter produced by Shanghai Baoxing Bio-Engineering Co, Ltd. (Shanghai, China) with 4 L working volume was employed to the fermentation. Four different fermentation strategies were used as described in Fig 6. The temperature of fermentation was maintained at 37 °C. The pH was controlled by addition of 3M HCl or ammonia. Agitation was xed at 300 rpm/min and aeration ux was set to 3 L/min.

Rhamnolipids concentration and biomass
The production of rhamnolipids in the fermentation broth were quanti ed by the colorimetric determination of sugars moiety with orcinol [14]. The culture broth of rhamnolipids was initially centrifuged (10 000 g, 10 min) to separate the cells from the supernatant. 0.5 ml of supernatant was extracted thrice using 1 ml of chloroform/ethanol (2:1, v/v). The organic phase was collected and evaporated to dryness, the rhamnolipids residue was then dissolved in deionized water. Approximately 2.7 ml of a solution containing 0.19% orcinol (in 53% H 2 SO 4 ) was then added to 0.3 ml of each sample.
After heating for 30 min at 80°C, the samples were cooled in an ice-water bath for 2 min, and the samples' absorbance at 421 nm was measured. Rhamnolipids were quanti ed in triplicate and rhamnolipids concentration was calculated by multiplying the amount of rhamnose by 3.4 [27]. The dry weight method was employed to measure biomass. Error bars in the gure were standard deviations from triplicate experiments.

Rhamnolipids puri cation
The rhamnolipids puri cation was performed according to wang's method with minor modi cation [28].
Brie y, the culture medium was centrifuged at 10000 rpm at 4°C for 10 min. The supernatant was precipitated by acidifying culture supernatant to pH 2.0 with 6 M hydrochloric solution and kept at 4°C overnight, then recovered by centrifugation at 10,000 rpm at 4°C for 1 h and dissolved in deionized water. Two volumes of chloroform:ethanol (2:1, v/v) were added to rhamnolipids solution and shaken 1 h for extraction at room temperature. The organic phase was evaporated to dryness to remove the solvent at 40 °C. After solvent evaporation, the rhamnolipids residue was obtained.

Foaming properties
The fermentation broth of rhamnolipids (22 g/L, 30 ml) from 250 ml Elenmeyer ask were controlled at pH 4, pH 5, pH 6, pH 7 and pH 8 by 3M HCl and ammonia. Among them, the pH 7 and pH 8 were control groups for neutral pH and weak alkaline pH, respectively. Then they were whipped with a rotor-stator disperser (AM400W-H, Angni Instruments Ltd, Shanghai, China) at 1000 rpm for 2 min at 37 °C, after whipped, the foam was poured into a 100 mL graduated cylinder immediately. Foaming ability (FA) was determined by comparing the foam volume at 1 min to the initial liquid volume of samples. The foam volume of fermentation broth of rhamnolipids at 5 min was measured to evaluate the accumulation ability of foam. FA (%) = V 0 /30 × 100 (1) where V 0 is the foam volume at 1 min.

Foaming microstructure observation by optical microscopy
According to the method of Xiong's report [29] with slight modi cation, the microstructures of foams from fermentation broth of rhamnolipids at pH 4-8 were visualized using a microscope (CX43, Olympus). The samples (10 μL) were placed on microscopy glass slides. Foams were observed after being stored at 37°C for 1 and 5 min. The magni cation was 10 (objective) × 10 (eyepiece).
Composite mutagenesis by UV and EMS.
For UV mutagenesis, the collected cells (3000 r/min for 10 min) were resuspended in nutrient broth at approximately 1 x 10 4 cells m/L. Then the cells were exposed to UV for 5, 10, 20, 30, 40, and 50 s using a 30 W lamp (245 nm, Shengxing ZW30S19W) at a distance of 30 cm. The irradiated cells suspension was diluted 100-fold with sterilized saline, and then 0.1 mL of dilution was spread on the blue agar plates [30] (the nutrient broth agar plates added 0.2 g cetyltrimethylammonium bromide (CTAB) and 0.005 g methylene blue, the value of pH was adjusted to 5.5 with 3 M HCl and 1M NaOH ). The plates were incubated at 37 °C for 4 days. All these manipulations were conducted in darkness to avoid possible photoreactivation. Strains with large blue circle were isolated for the further EMS mutagenesis. For EMS mutagenesis, the collected cells (10000 r/min for 5 min) were resuspended in sterilized saline at approximately 1 x 10 6 cells ml −1 . Cells were subjected to 5 ml 2% (v/v) EMS over 0.5 h in a dark room and then EMS was inactivated by addition of sterile sodium thiosulfate to a nal concentration of 5%. Cells were pelleted at 10000 r/min and washed three times with sterilized saline and resuspended in nutrient broth at approximately 1 x 10 6 cells ml −1 . The cells suspension of 0.1 ml was spread on the blue agar plates and were incubated at 37 °C for 4 days.

Surface tension, viscosity and zeta potential
The fermentation broth of rhamnolipids from 7.5 L fermenter was adjusted to pH 4-7 by utilizing 3M HCl and ammonia. The surface tension was measured by the Wilhelmy plate method [31], using a tensiometer (SFZL-E, Innuo Pecision Instruments Ltd, Shanghai, China). The Wilhelmy plate was immersed in 20 mL of aqueous phase at 37 °C. The viscosity was measured with a viscometer (NDJ-9S, Pingxuan Scienti c Instrument CO., Ltd. Shanghai, China) operating in 60 rpm. The zeta potential was measured using a Zetasizer Nano ZSP (Malvern Instruments Ltd, Malvern, UK).

Rhamnolipids morphology
The morphologies of rhamnolipids residue were observed with transmission electron microscopy (TEM).
A drop of the test rhamnolipids solution sample was placed onto a copper grid and dried naturally then was stained 2 min with a 1% phosphomolybdic acid aqueous solution. The excess phosphomolybdic acid aqueous solution was removed by absorbing the drop with a piece of lter paper and the grid was dried naturally. The samples were imaged under a transmission electron microscope (JEM-2100Plus, JEOL).

Results And Discussion
Evaluation of foaming properties in pH 4-8 As shown in Fig. 1A, the FAs of fermentation broth at pH 4, pH 5, pH 6, pH 7 and pH 8 were 32%, 41%, 42%, 146% and 174%, respectively (the corresponding visual foam volume image at Fig. 1C). After being stored for 5 min, there are less accumulation of foam at pH 4, pH 5 and pH 6 (Fig. 1B). These indicated that the foaming behavior of fermentation broth of rhamnolipids could be obviously suppressed at pH 4, pH 5 and pH 6. The time evolutions of bubbles formed by fermentation broth of rhamnolipids were shown in Fig. 2. The fermentation broth of rhamnolipids at pH 4-6 exhibited lesser multiple bubbles layers and smaller size of bubble at 1 min, which indicated that the fermentation broth of rhamnolipids at pH 4-6 had a poor foaming ability. A main property of aqueous foams is that there is out-of-equilibrium systems, and they evolve in time by gravitational drainage, coarsening and lm rupture [32]. In response to gravity the liquid ows out of the foam and this process is called drainage [33]. Drainage reduces the liquid fraction in the foam and increases lm surface area of the bubbles, causing an easier gas diffusion between bubbles. In foam, bubbles grow because of the gas diffusion from one bubble to the next and the coalescence of bubbles when lms are breaking. Foams are coarsen because of pressure differences between bubbles of different sizes [29]. These result in an increase in the mean bubble size. The bubbles growth eventually causes lm rupture. From the Fig. 2, after being stored for 5 min, a lot of bubbles have ruptured and only a few small sizes bubbles existed in the eld of vision at pH 4-6, but the fermentation broth of rhamnolipids at pH 7-8 still had more multiple bubbles layers and bigger size of bubble. These suggested that the ability of bubbles to prevent drainage or coarsening at pH 7-8 was stronger than these at pH 4-6 and the foam was easy to be accumulated. The results of time evolution of air bubbles also provided a better understanding for high foaming behavior of fermentation of rhamnolipids.
Based on the above results, the foaming behavior of fermentation broth of rhamnolipids was typically pH dependency and could be conspicuously suppressed by weakly acidic conditions.
Fermentation of rhamnolipids at pH 5.5 and pH 6.0 Owing to the metabolic activities of the microorganism, the pH value of fermentation of rhamnolipids by Pseudomonas aeruginosa would be increased from weak acid to weak alkaline (in this study, the value pH of was increased from 6.6 to 8.2, Fig. S1) [2,34]. Base on above results, the foaming behavior could be enhanced at alkaline environments caused by metabolic of microorganism. Therefore, in order to reduce the foaming behavior of fermentation of rhamnolipids, the fermentation was rstly conducted at pH 5.5. As shown in Fig. 3A, the fermentation exhibited a low-foaming behavior and could be conducted for 120 h or longer, which was conspicuously longer than the fermentation time of no control pH in Fig.  S1. In addition, although the concentration of rhamnolipids and biomass increased through the entire fermentation, the maximum rhamnolipids concentration (8.1 g/L) and biomass (6.5 g/L) were obviously lower than that of Erlenmeyer ask fermentation (22 g/L and 12.9 g/L, Fig. S1). These results suggested that the foaming behavior, rhamnolipids synthesis and cells growth were simultaneously inhibited at pH 5.5.
To improve the production of rhamnolipids, the pH of fermentation was adjusted to 6.0. Anti-foam agent was added at 1 ml/L broth at 60, 72 and 86 h of the fermentation. The production of rhamnolipids and biomass were plotted in Fig. 3B. It was observed that the increase of pH could promote cells growth and rhamnolipids synthesis. The maximum production of rhamnolipids (12.5 g/L) and biomass (8.7 g/L) were 1.5-fold and 1.3-fold higher than that of pH 5.5, respectively. However, severe foam occurred during fermentation (Fig. 3B) and a small amount of foam was over owed. Given that over owed cultivation was unacceptable in fermentation [35]. Therefore, the fermentation of rhamnolipids should be conducted below pH 6.0. In addition, the fermentation results of pH 5.5 and pH 6.0 further indicated that the foaming behavior of rhamnolipids fermentation was typical pH-sensitive and 0.5 change of pH value will cause a conspicuous decline of foaming ability.
Improved the production of rhamnolipids by mutagenesis at pH 5.5 In order to improve the production of rhamnolipids, the P. aeruginosa SW1 was mutated by UV and EMS, and then screened by the blue agar plates. The rhamnolipids produced from strain colony can form blue circle around the colony in the blue agar plates [30]. The blue circle diameter/colony diameter (BC) is proportional to the rhamnolipids concentration produced by strain. For UV mutagenesis, to obtain the mutant strains with increased rhamnolipids production, 20 colonies with large blue circles were screened and the values of BC were calculated. As shown in  The high production performance of strains obtained by mutagenesis may be instability because of the mutation reversion often occurring during subculture [36]. To evaluate the stability of production, strain 7, strain 8, strain 15, strain 18 and strain19 were inoculated at a 7.5 L fermenter with 2 L initial fermentation medium respectively. After that, 1 L of fermentation liquid was removed and simultaneously 1 L of fresh fermentation medium was added by timer control digital pump every 48 h for continuous subculture. The pH 5.5 was controlled by addition of 3M HCl or ammonia. Agitation was xed at 300 rpm/min and aeration ux was set to 2 L/min. The productions of rhamnolipids were detected at 240, 288 and 336 h, respectively. As shown in Fig. S2, after 336 h of continuous subculture, all the 5 strains showed excellent production stability and rhamnolipids production exceed 11 g/L. The strain 7 still showed the highest productions of rhamnolipids (13.6 g/L) followed by strain 15 (12.9 g/L) and strain 18 (12 g/L).
Further, the strain 7 and strain15 were conducted to the fermentation according to the method of fermentation involved in "materrials and method". As shown in Fig. 2, after 120 h of fermentation, the production of rhamnolipids and biomass of strain 7 reached 15.4 g/L and 10.3 g/L, which were 1.9-fold and 1.6-fold of the original strain, respectively. Corresponding, the production of rhamnolipids (14.7 g/L) and biomass (10.1 g/L) of strain 15 were about 1.8-fold and 1.6-fold higher than that of the original strain. The fermentation still exhibited low-foaming behavior through whole process of fermentation.
These results suggested that enhancing rhamnolipids production by mutagenesis was a promising strategy to realize low-foaming rhamnolipids fermentation under weakly acid conditions. In addition, other methods for strain improvement like metabolic engineering and synthetic biology techniques [37,38] should be included in future studies for improving rhamnolipids productivity under weakly acid conditions.
Characterizing the effects of pH 5-6 on foaming of rhamnolipids fermentation According to these results abovementioned that fermentation at pH 5.5 was a suitable strategy to realize low-foaming production of rhamnolipids by P. aeruginosa SW1. In addition, according to the previous reports, the pH 5-6 were also considered to be suitable range to carry out the low-foaming fermentation of rhamnolipids [25,26,35]. Therefore, in order to explore the mechanism of low-foaming caused by weak acid pH, we used a weak-alkaline condition (pH 8) as the control group to systematically evaluate the effects of rhamnolipids fermentation broths and residues on foaming behavior at pH 5.0, pH 5.5, and pH 6.0.

Effects of pH 5-6 on viscosity and surface tension
Generally speaking, the foaming ability of solution is related to its surface tension and lm viscosity [35]. The formation of bubbles is the process of increasing lm surface area, and the increase in lm surface area means that the energy of the system of bubbles also is increased correspondingly [39]. Therefore, from a thermodynamic point of view, the high surface tension of fermentation broth is obviously not conducive to foaming, because high surface tension will increase the surface energy of bubbles and producing the same volume of foam do more work. In addition, the liquid lm of bubbles formed by low viscosity solution have low viscosity and strength [40,41], which are also conducive to accelerating the lm rupture of bubbles. Therefore, according to the Fig. 5A and Fig. 5B, in comparison to that at pH 8, the fermentation broth of rhamnolipids at pH 5-6 exhibited lower viscosity and higher surface tension, which were conducive to suppressing the severe foaming in rhamnolipids fermentation.
Effects of pH 5-6 on zeta potential and foaming behavior of rhamnolipids The foaming ability (Fig. 5C) and zeta potential (Fig. 5D) of solution of rhamnolipids residue were simultaneously reduced with the decline of pH. Specially, there were obvious molecules aggregation of rhamnolipids and less foam at pH 5.0 (Fig. S3). Given that the trend of the change of foaming behavior was accordance with the zeta potential, it was considered that the decrease of foaming could be also partly attributed to the decline in charge potential of rhamnolipids. Because rhamnolipids containing carboxyl group has negatively charge and smaller fractions of rhamnolipids will be present as negatively charged ions in aqueous solution [42,43]. The decrease of negatively charged ions can weaken the electrostatic repulsion between rhamnolipids and make the rhamnolipids molecules easy to aggregate and further impede the foaming ability [11,44]. On the other hand, the decrease of negatively charged ions can also weaken the electrostatic repulsion of the charged cells adsorbed inner and outer membranes of bubbles and make the liquid membrane prone to drain, thin and rupture [45].
Effects of pH 5-6 on morphology of rhamnolipids According to previous studies, the reported pKa of rhamnolipids are about 4.3-5.6, so rhamnolipids molecules may present vesicles and/or lamellar structures while at pH 5.5 [46,47]. To verify the rhamnolipids structure at pH 5-6, TEM was used to examine the morphology of rhamnolipids microstructure. Fig. 6 shown the conversion of rhamnolipids molecular aggregates is typical pH-sensitive. The vesicles structure predominates at pH 5.0, while the larger lamellar were presented at pH 5.5. This was consistent with obvious molecules aggregation of rhamnolipids shown in pH 5.0 (Fig. S3). The size of lamellar signi cant became small at pH 6.0, but no lamellar or vesicles was observed at pH 8.0. Compared with the foaming behavior of rhamnolipids presented in Fig. 5C, it may be that formation of such vesicles or lamellar structures signi cantly suppressed the foaming ability of rhamnolipids.
In condition, according to the previous ndings, there are a paradox that rhamnolipids has been previously characterized as having low foam ability, but its fermentation presented severely foaming [48,49]. The reason may be explained by this study that the pH of rhamnolipids fermentation by P. aeruginosa is generally carried out in weakly alkaline conditions caused by the alkaline substances of microbial metabolism (Fig. S1), based on the foaming properties of rhamnolipids in 3.4.2 and 3.4.3, which are conducive to enhancing the foaming ability of rhamnolipids during fermentation. However, aqueous solution of rhamnolipids is a weak acid (the value of pH was about 4.5 in our study) due to the existence of the carboxylic acid moiety [11,47], which make rhamnolipids molecules low foaming.
Therefore, it was advised that rhamnolipids solution used in foaming agents, emulsi ers, oil recovery and other elds should be adjusted to neutral or alkaline.

Conclusions
The foaming behavior of fermentation broth of rhamnolipids produced by P. aeruginosa SW1 was con rmed to be typical pH dependency and the low-foaming fermentation of rhamnolipids could be realized at pH 5.5. Effective low-foaming fermentation of rhamnolipids was realized by UV and EMS composite mutagenesis at pH 5.5 and the mechanism of low-foaming fermentation of rhamnolipids caused by weakly acid pH were also systematically characterized. The work provided a guidance for further strain improvement of P. aeruginosa and developing a more effective pH-associated control strategies for large-scale low-foaming production of rhamnolipids under weakly acidic conditions. Abbreviations UV, ultraviolet; EMS, ethyl methanesulfonate; FA, Foaming ability; TEM, transmission electron microscopy; BC, Blue circle diameters/Colony diameters; CTAB, cetyltrimethylammonium bromide.

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