Glycerol promoted the anaerobic production of rhamnolipids by different Pseudomonas aeruginosa strains

Background: Rhamnolipids is the most widely studied and applied biosurfactants. The anaerobic biosynthesis of rhamnolipids has important research and practical signicance, such as meeting the in situ production of biosurfactant in anoxic environments and the foamless fermentation of biosurfactants. A few studies have reported the anaerobic biosynthesis of rhamnolipids from rare Pseudomonas aeruginosa strains. What did promote the anaerobic biosynthesis of rhamnolipids, the specicity of the rare strains or the effect of specic substrates? Here, anaerobic production of rhamnolipids by different P. aeruginosa strains was investigated using diverse substrates. The anaerobic biosynthesis mechanism of rhamnolipids were also discussed from the substrate point of view. Results: All P. aeruginosa strains anaerobically grew well using the tested substrates. But all P. aeruginosa strains anaerobically produced rhamnolipids only using substrates containing glycerol and nitrate. Fourier transform infrared (FTIR) spectra analysis conrmed the anaerobic production of rhamnolipids from all P. aeruginosa strains. All the anaerobically produced rhamnolipids decreased air-water surface tension from 72.6 mN/m to below 29.0 mN/m and emulsied crude oil with EI 24 above 65%. Using crude glycerol as low-cost substrate, all P. aeruginosa strains can anaerobically grow and produce rhamnolipids to reduce the culture surface tension below 35 mN/m. The glycerol metabolic intermediate, 1, 2-propylene glycol, can also achieve the anaerobic production of rhamnolipids by all P. aeruginosa strains. Conclusions: Not the specicity of the rare P. aeruginosa strains but the effect of specic substrates promote the anaerobic biosynthesis of rhamnolipids by P. aeruginosa. Glycerol and nitrate are the excellent substrates for anaerobic production of rhamnolipids from all P. aeruginosa strains. Results indicated that glycerol metabolism involveed the anaerobic biosynthesis of rhamnolipids in P. aeruginosa. Results also showed the feasibility of using crude glycerol as low cost substrate to anaerobically biosynthesize rhamnolipids by P. aeruginosa. different P. aeruginosa strains were used. Diverse carbon sources and nitrogen sources were tested for anaerobic production of rhamnolipids by P. aeruginosa strains. Anaerobically produced rhamnolipids was conrmed by Fourier transform infrared (FTIR) spectra analysis. The physicochemical properties of rhamnolipids products were also evaluated. The feasibility of using crude glycerol as low cost substrate to anaerobically biosynthesize rhamnolipids by P. aeruginosa was studied. Prospects and bottlenecks existing in the anaerobic biosynthesis of rhamnolipids were also discussed. Results will guide to enhance anaerobic production of P. aeruginosa rhamnolipids by culture conditions development or strain improvement.

without aeration can avoid the foam problem. These breakthroughs require to further research the anaerobic synthesis of rhamnolipids, including the involved strain and culture medium.
Although the mainly rhamnolipids producers, Pseudomonas aeruginosa, are facultative bacteria [8,13,14], only few studies have reported the anaerobic biosynthesis of rhamnolipids by rare P. aeruginosa strains [9,10,15,16]. P. aeruginosa can grow and metabolize at both aerobic and anaerobic conditions. But studies on production of rhamnolipids by P. aeruginosa were almost performed under aerobic conditions. Few studies reported that rare P. aeruginosa strains can anaerobically produce rhamnolipids [8,17].
What did promote the anaerobic biosynthesis of rhamnolipids, the speci city of the rare strains or the effect of nutritional conditions? In previous studies, using facultative anaerobic and denitrifying bacterial strain P. stutzeri DQ1, the constructed engineering bacterial strain P. stutzeri Rhl achieved heterologous production of P. aeruginosa rhamnolipids under anaerobic conditions [18]. A wild-type P. aeruginosa SG that can anaerobically produce rhamnolipids was subsequently screened from production water of Xinjiang oil reservoir [9]. During the process of medium optimization, it was found that strain P. aeruginosa SG could anaerobically grew well using glucose and glycerol, but strain SG anaerobically produced rhamnolipids using glycerol not glucose [19]. Whether the anaerobic synthesis of rhamnolipids is caused by the strain speci city of P. aeruginosa SG or the promoting effect of glycerol and nitrate? This study aims to analyze this problem by assessing the anaerobic production of rhamnolipids using different P. aeruginosa strains fed with diverse substrates. Results will deepen the biosynthesis theory of rhamnolipids and guide the process for anaerobic production of rhamnolipids.
In the present study, different P. aeruginosa strains were used. Diverse carbon sources and nitrogen sources were tested for anaerobic production of rhamnolipids by P. aeruginosa strains. Anaerobically produced rhamnolipids was con rmed by Fourier transform infrared (FTIR) spectra analysis. The physicochemical properties of rhamnolipids products were also evaluated. The feasibility of using crude glycerol as low cost substrate to anaerobically biosynthesize rhamnolipids by P. aeruginosa was studied. Prospects and bottlenecks existing in the anaerobic biosynthesis of rhamnolipids were also discussed.
Results will guide to enhance anaerobic production of P. aeruginosa rhamnolipids by culture conditions development or strain improvement.

Strains and culture conditions
Four rhamnolipids-producing strains, P. aeruginosa strains (SG, L6-1, WJ1 and P4), were used in this study. Strains P. aeruginosa SG was used as positive control for anaerobic production of rhamnolipids [9]. Strain P. aeruginosa WJ1 was isolated from production uid of Huabei oil reservoirs, China [20]. Strains P. aeruginosa SG, L6-1 and P4 were isolated from production uid of Xinjiang oil reservoirs, China [9]. LB medium was used to prepare seed culture at 35°C and 180 rpm. For anaerobic production of rhamnolipids, four P. aeruginosa strains were cultured in 100 ml serum bottles containing 80 ml anaerobic medium with inoculum amount of 3%, respectively. Brie y, the anaerobic medium was prepared as follows. The medium was boiled for 15 min, and then 99.99% purity of N 2 gas was injected into the boiling medium for 5 min. Then medium was sub-packaged in serum bottles when it was hot. Press the rubber plug and press the aluminum cap under N 2 gas protection. After sterilization and cooling to 30°C, deoxidizer Na 2 S·9H 2 O was added into the medium in serum bottles to a nal concentration of 0.02% (w/v) [18]. The anaerobic fermentation experiments were performed at 35°C and 50 rpm for 10 days. Three parallel experiments were set for each bacterial strain. The non-inoculated medium was used as the negative control. The medium inoculated with strains P. aeruginosa SG was used as positive control.

Analytical methods
Culture samples were taken from serum bottles using sterile syringes. Bacterial strains biomass was represented by OD 600 values of the culture samples. Culture samples were centrifuged at 10,000g for 10 minutes. The surface tension of supernatant was measured by surface tensiometer (BZY-1, Shanghai Hengping Instrument and Meter Factory, Shanghai, China). The diameter of oil spreading circle formed by culture supernatant was determined as previous reported [21]. The crude oil (7-Middle area, Xinjiang oil eld, China) was used. Emulsi cation index (EI 24 ) was measured to evaluate the emulsifying activity of produced rhamnolipids products as previous study described [18]. EI 24 values were calculated as the height of the emulsi ed layer (mm) divided by the total height of the liquid column (mm) and multiplied by 100.

P. aeruginosa strains fed with different carbon sources
The common substrates, glucose, sucrose, molasses, soybean oil, palmitic acid, glycerol and starch, were tested as carbon sources for the anaerobic production of rhamnolipids by four P. aeruginosa strains. Strains P. aeruginosa SG was used as positive control for anaerobic production of rhamnolipids. The concentrations of carbon sources in the medium were all 40 g/l. The medium except carbon source After the anaerobic fermentation process, the OD 600 , surface tension and oil spreading activity of culture samples were analyzed. Anaerobic production of rhamnolipids by strains was de ned as that the surface tension of anaerobic culture was reduced to lower than 40 mN/m and the oil spreading circles diameters formed by anaerobic culture were greater than 10 mm. P. aeruginosa strains fed with possible metabolic intermediates and analogues of glycerol The possible metabolic intermediates and analogues of glycerol (hydroxyacetone, 1, 2-propylene glycol and 1, 3-propylene glycol) were also evaluated for the anaerobic production of rhamnolipids by four P. aeruginosa strains. After the anaerobic fermentation process, the OD 600 , surface tension and oil spreading activity of culture samples were analyzed.

P. aeruginosa strains fed with different nitrogen sources
The common nitrogen nutrients, NH 4 Cl, NaNO 3 , yeast extract and peptone, were tested as nitrogen sources for the anaerobic production of rhamnolipids by four P. aeruginosa strains. Strains P. aeruginosa SG was used as positive control for anaerobic production of rhamnolipids. The concentrations of nitrogen sources in the medium were all 4 g/L. The medium except nitrogen source contained 40 g/l of glycerol, 4 g/l of K 2 HPO 4 ·3H 2 O, 3 g/l of KH 2 PO 4 , 1.0 g/l of MgSO 4 ·7H 2 O, 0.5 g/l of KCl, 0.5 g/l of NaCl, 0.2 g/l of CaCl 2 ·2H 2 O and 5 ml of trace elements solution. After the anaerobic fermentation process, the OD 600 , surface tension and oil spreading activity of culture samples were analyzed. Anaerobic production of rhamnolipids by strains was de ned as that the surface tension of anaerobic culture was reduced to lower than 40 mN/m and the oil spreading circles diameters formed by anaerobic culture were greater than 10 mm.

P. aeruginosa strains fed with different the complex nitrogen sources
The complex nitrogen sources of NaNO 3 and yeast extract (3:7, 5:5, 7:3) were also tested for the anaerobic production of rhamnolipids by four P. aeruginosa strains. The amounts of complex nitrogen sources of NaNO 3 and yeast extract was 4 g/l. After the anaerobic fermentation process, the OD 600 , surface tension and oil spreading activity of culture samples were analyzed.

Rhamnolipids extraction from the anaerobic culture
The anaerobiacally produced rhamnolipids were extracted as previous described [10]. Brie y, the cell free anaerobic culture broth was heated at 80 °C for 30 min to denature the extracellular proteins. Samples were centrifuged at 10,000g for 10 minutes. Using 2 mol/l of HCl-water solution, the pH value was adjusted to 2.0. Chloroform and methanol organic solvent (v/v, 2:1) was used for rhamnolipids extraction. The rhamnolipids products were extracted from the organic phase using a rotary evaporator (50 rpm, 45 °C).

FTIR spectra analysis
Anaerobically produced rhamnolipids was con rmed by FTIR spectra analysis using rhamnolipids produced by strain P. aeruginosa SG as positive control. Brie y, 10 mg of rhamnolipids extract and 90 mg of KBr was mixed to make the translucent pellet with pressure of 25 Mpa for 25 s. A NICOLET 380 FTIR spectrometer was used to record the FTIR spectra of the translucent pellet with the wave number from 400 cm −1 to 4000 cm −1 [10,18]. P. aeruginosa strains fed with crude glycerol and nitrate To further reduce the medium cost, crude glycerol instead of glycerol was assessed for the anaerobic production of rhamnolipids by four P. aeruginosa strains. The concentration of crude glycerol in the medium was 40 g/L. The medium except crude glycerol contained 4 g/l of NaNO 3 , 4 g/l of K 2 HPO 4 ·3H 2 O, 3 g/l of KH 2 PO 4 , 1.0 g/l of MgSO 4 ·7H 2 O, 0.5 g/l of KCl, 0.5 g/l of NaCl, 0.2 g/l of CaCl 2 ·2H 2 O and 5 ml of trace elements solution. After the anaerobic fermentation process, the OD 600 , surface tension and oil spreading activity were analyzed.

Results And Discussion
Anaerobic production of rhamnolipids by P. aeruginosa strains using different carbon sources Using the tested substrates as carbon sources, the results of biomass (OD 600 ), surface activity and oil spreading activity were shown in Fig. 1. At anaerobic conditions, four P. aeruginosa strains obtained the biomass with OD 600 values between 1.80 to 4.00 using the tested substrates (Fig. 1a). Results indicated that four P. aeruginosa strains can grow well under anaerobic conditions using all the tested substrates. Results also con rmed that P. aeruginosa are facultative bacteria [8,13,14]. From the point of view of biomass, molasses, glucose and glycerol were more favorable to the anaerobic growth and reproduction of P. aeruginosa strains. Molasses, glucose and glycerol as water-soluble substrates may be more conducive to the rapid absorption and anaerobic metabolize by bacterial cells of P. aeruginosa.
As shown in Fig. 1b, the surface tension values of anaerobic culture were reduced to lower than 40 mN/m using soybean oil (from 45 mN/m to about 37 mN/m), palmitic acid (from 49 mN/m to about 37 mN/m) and glycerol (from 63 mN/m to about 32 mN/m). Using glycerol, four P. aeruginosa strains reduced the surface tension of anaerobic culture with the greatest decrease (49.5%) in the surface tension and the lowest surface tension values. As shown in Fig. 1c, the oil spreading circles diameters formed by anaerobic culture of four strains were greater than 10 mm using soybean oil (about 11 mm), palmitic acid (about 11 mm) and glycerol (about 20 mm). The oil spreading circles diameters formed by bacterial culture can be used to indirectly characterize the biosurfactants yield of bacterial strain [21]. Results demonstrated that four P. aeruginosa strains can e ciently produce biosurfactants under anaerobic conditions using glycerol as carbon sources.
Though all the tested substrates can be used as carbon sources for production of rhamnolipids by P. aeruginosa under aerobic conditions [22][23][24][25]. In this study, only glycerol can be used for anaerobic production of rhamnolipids by P. aeruginosa strains among the tested substrates. As previous studies described, strain P. aeruginosa SG anaerobically produced rhamnolipids using glycerol [9,19]. In this study, P. aeruginosa strains except strain SG also anaerobically produced rhamnolipids using glycerol as carbon source. Here, anaerobic production of rhamnolipids is not induced by the speci city of the rare P. aeruginosa (strain SG) but the nutrient factor of glycerol.
Anaerobic production of rhamnolipids by P. aeruginosa strains using different nitrogen sources Using NH 4 Cl, NaNO 3 , yeast extract and peptone as nitrogen sources, anaerobic cell growth and rhamnolipids production by P. aeruginosa strains were comparatively studied. The results of biomass (OD 600 ), surface activity and oil spreading activity were shown in Fig. 2. Anaerobic culture of four P. aeruginosa strains obtained the biomass with OD 600 values between 2.50 to 4.50 using the inorganic nitrogen sources, organic nitrogen sources and compound nitrogen sources (Fig. 2a). Four P. aeruginosa strains can grow well under anaerobic conditions using different nitrogen sources. Results also con rmed that P. aeruginosa are facultative bacteria [13,14]. As shown in Fig. 2a, the organic nitrogen sources were more favorable to the anaerobic growth and reproduction of P. aeruginosa strains. The small molecular peptides contained in yeast extract and peptone may provide abundant and available nutrition for cell growth of P. aeruginosa [26].
As shown in Fig. 2b, the surface tension values of anaerobic culture were all reduced from 63 mN/m to lower than 40 mN/m using all tested nitrogen sources. Using NaNO 3 as nitrogen sources, four P. aeruginosa strains reduced the surface tension of anaerobic culture with the greatest decrease (51.3%) in the surface tension and the lowest surface tension values of about 31 mN/m. As shown in Fig. 2c, the oil spreading circles diameters formed by anaerobic culture of four strains were greater than 10 mm using all tested nitrogen sources. Using NaNO 3 as nitrogen sources, anaerobic culture of four P. aeruginosa strains formed oil spreading circles with diameters about 20 mm. Although four P. aeruginosa strains also obtained higher biomass using the complex nitrogen sources of NaNO 3 and yeast extract (Fig. 2a), the anaerobic production of rhamnolipids were relatively less (Fig. 2c). Results demonstrated that NaNO 3 was more favorable to the anaerobic production of rhamnolipids by P. aeruginosa. Studies also reported that organic nitrogen sources were not conducive to the synthesis of rhamnolipids [27]. Nitrate was considered to be the best nitrogen source for rhamnolipid production under aerobic conditions [28.29]. At anaerobic conditions, nitrate was not only the excellent nitrogen source for rhamnolipids production but also the good electron acceptor for anaerobic metabolism of P. aeruginosa. P. aeruginosa, as facultative bacteria, can use other electron acceptors except oxygen, such as nitrate [8,15], so P. aeruginosa can grow and metabolize at both aerobic and anaerobic conditions [13,14]. FTIR spectra analysis of anaerobically produced rhamnolipids using glycerol and nitrate The anaerobically produced rhamnolipids from four P. aeruginosa strains using glycerol and nitrate were con rmed by FT-IR spectra, respectively. Rhamnolipids produced by strain P. aeruginosa SG was used as the positive control. As shown in Fig. 3, the FTIR spectra of the anaerobically produced rhamnolipids from P. aeruginosa strains WJ1, L6-1 and P4 were similar to that of rhamnolipids produced from strain P. aeruginosa SG. The FTIR spectra of the anaerobically produced rhamnolipids from four P. aeruginosa strains were also similar to the spectra of reported rhamnolipids [30,31]. All the FTIR spectra had the characteristic absorption bands around 2927 cm −1 , 2858 cm −1 and 1465 cm −1 causing by the C-H stretching vibrations of aliphatic groups and the absorption bands around 1735 cm −1 causing by the ester groups. All the FTIR spectra also had the absorption area between 1452 cm −1 and 1045 cm −1 causing by the C-H and O-H vibrations that are typical vibrations for carbohydrates. Results showed that four P. aeruginosa strains did produce rhamnolipids under anaerobic conditions using glycerol and nitrate.

Activity of anaerobically produced rhamnolipids using glycerol and nitrate
The rhamnolipids-water solutions (200 mg/L) were prepared using the extracted rhamnolipids products.
The air-water surface tension and the emulsi cation index (EI 24 ) to crude oil was measured to evaluate their surface activity and emulsifying activity. All the anaerobically produced rhamnolipids using glycerol and nitrate decreased the air-water surface tension from 72.6 mN/m to lower than 29 mN/m. Studies reported the aerobically produced rhamnolipids can decrease the air-water surface tension to lower than 27 mN/m [32]. In this study, the anaerobically produced rhamnolipids from P. aeruginosa exhibited excellent surface activity as well. Good surface activity helps to change contact angle, increase wetting activity and even facilitate wetting reversal. The excellent surface activity can also improve the capillary effect and facilitate the ow of groundwater or oil in porous media. These are of great signi cance for enhanced oil recovery and environmental pollution remediation [33]. Anaerobically produced rhamnolipids all showed better emulsifying activity to crude oil with EI 24 values higher than 65%. Studies reported the aerobically produced rhamnolipids can emulsi ed crude oil with EI 24 values ranging from 53% to 90% [32,34]. In this study, the anaerobically produced rhamnolipids from P. aeruginosa also exhibited good emulsifying activity, which would be signi cant to enhanced oil recovery and pollution remediation [5,35,36]. The good emulsifying activity of biosurfactants can assist oil dispersion in oil reservoir to reduce oil viscosity and oil ow [37,38]. Besides, the excellent emulsi cation effect can increase the solubility of hydrophobic pollutants in the environment and increase the bioavailability of hydrophobic pollutants [35,39]. Anaerobic production of rhamnolipids by P. aeruginosa strains using crude glycerol Using crude glycerol as low-cost substrate, all P. aeruginosa strains can grow under anaerobic conditions and obtained biomass with OD 600 values between 2.10 to 2.50. As shown in Fig. 4, four P. aeruginosa strains anaerobically produced rhamnolipids to reduce the culture surface tension below 35 mN/m. Compared to using glycerol as carbon source, the diameters of oil spreading circles formed by four P. aeruginosa strains anaerobic culture were smaller using crude glycerol. Results showed that using crude glycerol for anaerobic production of rhamnolipids by P. aeruginosa is feasible. Previous studies reported aerobic production of rhamnolipids by P. aeruginosa using crude glycerol [40,41]. Using glycerol or crude glycerol, the rhamnolipids yields from P. aeruginosa were both lower under anaerobic conditions. Enhancing the anaerobic production of rhamnolipids from P. aeruginosa is in demand. In future, it is a good choice to develop high-yielding strains of rhamnolipids through biosynthesis pathways regulation and functional genes manipulation. Identifying the biosynthesis pathways and key genes involved in anaerobic production of rhamnoipids are of great importance Research perspectives to the anaerobic biosynthesis mechanism of rhamnolipids Glycerol is water soluble, easy to be absorbed and used by microorganisms, and is an excellent metabolic substrate for microorganisms. In this study, only glycerol promoted the anaerobic production of rhamnolipids by P. aeruginosa. But the mechanism of anaerobic biosynthesis of rhamnolipids by P. aeruginosa using glycerol is still unclear. In this study, from the point of substrate, the possible metabolic intermediates and analogues of glycerol (hydroxyacetone, 1, 2-propylene glycol and 1, 3-propylene glycol) were also evaluated for the anaerobic production of rhamnolipids by P. aeruginosa strains. The results of OD 600 , surface activity and oil spreading activity were shown in Fig. 5. P. aeruginosa strains obtained the biomass with OD 600 values between 2.00 to 3.00 using the possible metabolic intermediates and analogues and analogues of glycerol (Fig. 5a). Four P. aeruginosa strains can anaerobically grow well using the possible metabolic intermediates and analogues of glycerol. These substrates are water-soluble and easily enter into the glycolysis and gluconeogenesis pathways, which may be conducive to the rapid absorption and anaerobic metabolize by P. aeruginosa [42]. Among the possible metabolic intermediates and analogues of glycerol, only 1, 2-propylene glycol promoted four P. aeruginosa strains to produce rhamnolipids under anaerobic conditions, reducing the surface tension of anaerobic culture from 63.2 mN/m to about 31 mN/m (Fig. 5b). Using 1, 2-propylene glycol, the oil spreading circles diameters formed by anaerobic culture of four strains were about 20 mm (Fig. 5c). Results demonstrated that 1, 2-propylene glycol, similar to glycerol, can promote P. aeruginosa to anaerobically biosynthesize rhamnolipids.
Here, results again con rmed that anaerobic production of rhamnolipids is not induced by the speci city of the rare P. aeruginosa (strain SG) but the speci c substrates. Glycerol and 1, 2-propylene glycol can promote P. aeruginosa to anaerobically biosynthesize rhamnolipids. What is the mechanism of anaerobic rhamnolipids biosynthesis by P. aeruginosa metabolizing glycerol and 1, 2-propylene glycol? Hauser and Karnovsky cultured P. aeruginosa strain using 14 C-labeled glycerol-α-14 C and glycerol-β-14 C under aerobic conditions. And they found that the C 6 unit of rhamnose group in rhamnolipids product was directly condensed from the two molecules glycerol (C 3 unit) without carbon chain rearrangement [44]. The polyol can be oxidized into dihydroxyacetone (DHA), then DHA can be converted into dihydroxyacetone phosphate (DHAP) which enters glycolysis and gluconeogenesis pathways [42,43]. This may be why P. aeruginosa can metabolize glycerol and 1, 2-propylene glycol for biosynthesis the precursors of rhamnolipids, rhamnose and fatty acids. But the biosynthesis pathways and key genes involved in anaerobic biosynthesis of rhamnoipids using glycerol is still unclear. Elucidating the mechanism of anaerobic biosynthesis of rhamnolipids is helpful to the development and application of strains high producing rhamnolipids.
The anaerobic biosynthesis of rhamnolipids has important research and practical signi cance. Anaerobic production of rhamnolipids can meet the in situ production of biosurfactant in anoxic environments, such as deep soil, oil reservoirs, sediments [8][9][10]. Based on anaerobic production of rhamnolipids, fermentation without aeration can avoid the foam problem in production of rhamnolipids. However, enhancing the anaerobic production yield of rhamnolipids is critical. Future research will be concentrate on elucidating the mechanism of anaerobic biosynthesis of rhamnolipids through revealing the pathways and key genes involved using transcriptome technology.

Conclusions
Tested P. aeruginosa strains e ciently produced rhamnolipids under anaerobic conditions. Not the speci city of the rare P. aeruginosa strains but the effect of speci c substrates promote the anaerobic biosynthesis of rhamnolipids by P. aeruginosa. Glycerol and nitrate are the excellent substrates for anaerobic production of rhamnolipids from all P. aeruginosa strains. Glycerol metabolism promoted the anaerobic biosynthesis of rhamnolipids in P. aeruginosa. Crude glycerol can be used as low-cost substrate for anaerobically producing rhamnolipids by P. aeruginosa. Transcriptomic analysis would facilitate to reveal the metabolic pathways and key genes involved in anaerobic production of rhamnolipids in future.

Declarations
Authors' contributions FZ conceived and designed the study, carried out the experiments and drafted the manuscript. CG participated in analyzing the data. QFC assisted in revising the manuscript. All authors read and approved the nal manuscript. Anaerobic growth and production of rhamnolipids by four P. aeruginosa strains using different carbon sources: a) biomass (OD600), b) surface activity and c) oil spreading activity. Strain P. aeruginosa SG was used as positive control. Anaerobic growth and production of rhamnolipids by four P. aeruginosa strains using different nitrogen sources: a) biomass (OD600), b) surface activity and c) oil spreading activity. Strain P. aeruginosa SG was used as positive control. Fourier Transform infrared (FTIR) spectra analysis of anaerobically produced rhamnolipids from four P. aeruginosa strains using glycerol and nitrate. Anaerobically produced rhamnolipids by strain SG was used as control.
Page 18/19 Figure 4 Anaerobic production of rhamnolipids by four P. aeruginosa strains using crude glycerol.