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 film rupture [32]. In response to gravity the liquid flows out of the foam and this process is called drainage [33]. Drainage reduces the liquid fraction in the foam and increases film 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 films 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 film 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 field 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 firstly 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 flask 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 overflowed. Given that overflowed 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 table 1, compared with control strain 0, all the mutant strains showed higher values of BC. Strain 3 showed the highest value of BC with 16 mm blue circle diameters and 7 mm colony diameters. Strain 8 and strain 15 also showed significant enhanced production capacity with the high values of BC. Therefore, strain 3, strain 8 and strain 15 were isolated for the EMS mutagenesis. For EMS mutagenesis, strain 3, strain 8 and strain 15 were mutated separately according to the method abovementioned in “Mutagenesis” and 20 colonies with large blue circles were screened and the values of BC were calculated. As shown in table 1, strain 7 showed the highest BC value followed by strain 18, strain 15, strain 19 and strain 8, which indicated that the rhamnolipids production may have a significant increase by mutagenesis in these strains. Therefore, strain 7, strain 8, strain 15, strain 18 and strain19 were isolated for further studies.
Table 1
BC values of strains in culture plate after UV and EMS composite mutagenesis
| UV | | | EMS | |
Code | R1(mm) | R2 (mm) | BC | Code | R1(mm) | R2 (mm) | BC |
0 | 6.0 | 4.0 | 1.5 | | | | |
1 | 10.0 | 6.5 | 1.5 | 1 | 14.5 | 7.0 | 2.1 |
2 | 9.5 | 5.0 | 1.9 | 2 | 19.0 | 7.5 | 2.5 |
3 | 16.0 | 7.0 | 2.3 | 3 | 15.0 | 6.0 | 2.5 |
4 | 7.5 | 4.0 | 1.9 | 4 | 19.0 | 9.0 | 2.1 |
5 | 9.0 | 5.0 | 1.8 | 5 | 15.5 | 7.0 | 2.2 |
6 | 16.5 | 9.0 | 1.8 | 6 | 14.0 | 6.5 | 2.2 |
7 | 9.5 | 5.5 | 1.7 | 7 | 21.0 | 7.0 | 3.0 |
8 | 13.0 | 6.0 | 2.2 | 8 | 16.0 | 6.0 | 2.7 |
9 | 8.5 | 5.0 | 1.7 | 9 | 17.0 | 7.0 | 2.4 |
10 | 8.0 | 4.5 | 1.8 | 10 | 15.0 | 6.0 | 2.5 |
11 | 9.0 | 5.5 | 1.6 | 11 | 18.0 | 7.5 | 2.4 |
12 | 11.5 | 6.5 | 1.8 | 12 | 17.0 | 6.5 | 2.6 |
13 | 8.0 | 5.0 | 1.6 | 13 | 19.0 | 7.5 | 2.5 |
14 | 12.5 | 6.0 | 2.1 | 14 | 18.0 | 8.0 | 2.3 |
15 | 14.5 | 6.5 | 2.2 | 15 | 18 | 6.5 | 2.8 |
16 | 14.5 | 8.5 | 1.7 | 16 | 19.0 | 7.5 | 2.5 |
17 | 9.5 | 5.0 | 1.9 | 17 | 17.0 | 6.5 | 2.6 |
18 | 13.0 | 7.5 | 1.7 | 18 | 20.0 | 7.0 | 2.9 |
19 | 9.0 | 5.0 | 1.8 | 19 | 17.0 | 6.0 | 2.8 |
20 | 13.0 | 7.5 | 1.7 | 20 | 18.0 | 7.0 | 2.6 |
R1: Blue circle diameters; R2: Colony diameters; BC: R1/ R2. Code “0” is the control strain without mutation induction treatment. |
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 fixed at 300 rpm/min and aeration flux 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 film viscosity [35]. The formation of bubbles is the process of increasing film surface area, and the increase in film 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 film of bubbles formed by low viscosity solution have low viscosity and strength [40, 41], which are also conducive to accelerating the film 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 significant 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 significantly suppressed the foaming ability of rhamnolipids.
In condition, according to the previous findings, 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, emulsifiers, oil recovery and other fields should be adjusted to neutral or alkaline.