Isolation and characterization of a novel rhamnolipid producer Pseudomonas sp. LGMS7 from a highly contaminated site in Ain El Arbaa region of Ain Temouchent, Algeria

Abstract

This study aims to isolate and characterize a novel rhamnolipid producer within the recent bioremediation approaches for treating hydrocarbon-contaminated soils in Algeria. In this context, from a hydrocarbon-contaminated soil, a newly bacterium designated LGMS7 was screened and identified, belonged to the Pseudomonas genus, and was closely related to Pseudomonas mucidolens, with a 16S rRNA sequence similarity of 99.05%. This strain was found to use different hydrocarbons and oils as a sole carbon and energy source for growth. It showed a stable emulsification index E₂₄ (%) of 66.66% ± 3.46 when growing in mineral salts medium (MSM) supplemented with 2% (v/v) glycerol after incubation for 6 days at 30 °C. Interestingly, it was also able to reduce the surface tension of the cell-free supernatant to around 30 ± 0.65 mN m⁻¹ with a critical micelle concentration (CMC) of 800 mg l⁻¹. It was found to be able to produce around 1260 ± 0.57 mg l⁻¹ as the yield of rhamnolipid production. Its biosurfactant has demonstrated excellent stability against pH (pH 2.0–12.0), salinity (0–150 g l⁻¹), and temperature (−20 to 121 °C). Based on various chromatographic and spectroscopic techniques (i.e., TLC, FTIR, ¹H-NMR), it was found to belong to the glycolipid class (i.e., rhamnolipids). Taken altogether, the strain LGMS7 and its biosurfactant display interesting biotechnological capabilities for the bioremediation of hydrocarbon-contaminated sites. To the best of our knowledge, this is the first study that described the production of biosurfactants by Pseudomonas mucidolens species.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-021-02751-6.

Introduction

Algeria is a leading African oil producer, and its economy relies principally on fossil fuels, such as natural gas and oil (Harrouz et al. 2017). In 2015, the production rate was estimated at 1.7 barrels per day of total petroleum products. This sector causes massive terrestrial and marine pollution by the hydrocarbon leakages accompanying the production, refining, and export activities (Djahnit et al. 2019). For several decades, the local contaminations continue to cause harmful effects on human and animal health and reach the groundwater level, which constitutes a significant source for Algeria’s fresh water supply (Bouderbala et al. 2016). The local authorities have recently started to request researchers and companies to move towards novel bio-based approaches to tackle these urgent environment-menacing issues by isolating indigenous biosurfactant-producing microorganisms with interesting features in the hydrocarbons bioremediation approaches (Sekkour et al. 2019).

Environmental contamination caused by industrial activity is generally due to accidental or deliberate releases of organic and/or inorganic compounds into the environment. Those compounds pose several problems for remediation, as they become easily bound to soil particles. The hydrocarbon-contaminated soils generally contain at least six phases: bacteria, soil particles, water, air, immiscible liquid and solid hydrocarbon. The hydrocarbons can be partitioned among different states: solubilized in the water phase, or/absorbed to soil particle, sorbed to cell surfaces, and as a free/insoluble phase (Banat et al. 2010). By definition, a surfactant consists of hydrophobic and hydrophilic components and can merge two immiscible liquids, such as water and oil through mainly surface tension reduction (Almansoory et al. 2019; Mulligan 2005).

Biosurfactants (BS) are diverse groups of natural amphiphilic compounds, which could also be synthesized by several microorganisms (Chebbi et al. 2017a, 2021). Most of the studied biosurfactants are low molecular weight molecules (e.g., glycolipids, lipopeptides), reducing the surface tension of water to 25–35 mN m⁻¹ (Franzetti et al. 2010). Depending on the carbohydrate nature of the hydrophilic moiety, glycolipids can be subdivided into rhamnolipids, trehalolipids, sophorolipids, cellobiolipids, mannosylerythritol lipids, lipomannosyl-mannitols, lipomannans and lipoarabinomannanes, diglycosyl diglycerides, monoacylglycerol and galactosyl-diglyceride (Mnif and Ghribi 2016). However, rhamnolipids were firstly described by Jarvis and Johnson (1949) (Abdel-Mawgoud et al. 2010). They are among the best-known and studied BS classes, which are mainly produced by Pseudomonas aeruginosa. They have diverse biological activities like hemolytic, anticancer, antifungal, antibacterial, and antiviral and are used as bio-control agents. Furthermore, rhamnolipids are also well recognized by diverse properties like their higher emulsifying and surface activities permitting the ability to disperse hydrophobic substances in water and the enhancement of hydrocarbons solubilization and biodegradation (Mnif et al. 2018).

Last two decades, BS have gained both academic and industrial interests due to various advantages such as structural diversity, low toxicity, higher biodegradability, ability to act in wide ranges of pH, temperature, and salinity, higher selectivity, lower critical micelle concentration (CMC) and production on renewable sources (i.e., industrial and agricultural wastes) (Da Rosa et al. 2015; Makkar et al. 2011). Biosurfactants have proved a wide range of biotechnological applications in various industries, e.g., petroleum, food, medicine, pharmaceutical, chemical, paper and pulp, textile, and cosmetics (Mujumdar et al. 2019). Among several BS classes, rhamnolipids are considered promising candidates for large-scale industrial production and applications in numerous fields such as remediation of contaminated sites (Moussa et al. 2014).

Numerous remediation methods of petroleum hydrocarbons contamination have been described, such as physical, chemical, and biological approaches (Franzetti et al. 2010). Among these methods, the biological processes mediated by microbes (or their fermentation products) are considered economical and eco-friendly and generally result in total degradation of petroleum hydrocarbons (Logeshwaran et al. 2018). The BS application into the organic compounds’ remediation aims to increase their bioavailability (biosurfactant-enhanced bioremediation) or mobilize and remove the contaminants by pseudosolubilisation and emulsification (Banat et al. 2010). In fact, the increase of hydrocarbon solubility can enhance their availability in aqueous environment, promoting, therefore, their elimination by hydrocarbon degrading bacteria. Many reports have described the enhancement of hydrocarbon assimilation in the presence of rhamnolipid biosurfactants. Nie et al. (2010) and Chandran and Das (2010) have described that in-situ rhamnolipid and sophorolipid biosurfactants synthesis can also enhance hydrocarbon solubility and thus their biodegradation, respectively. In addition, a patent of Yin et al. (2011) described the beneficial effect of rhamnolipid derived from P. aeruginosa strain NY3 on the treatment of water and soil contaminated with hydrocarbons or pesticides.

Currently, the isolation and characterization of novel BS producers, with promising abilities, from highly contaminated sites are still gaining particular attention (Cheffi et al. 2020; He et al. 2020).

In this context, our study highlighted the promising potential of a newly bacterial strain Pseudomonas sp. LGMS7, isolated from a hydrocarbon-contaminated soil in western Algeria, after 16S rRNA sequencing analysis of numerous bacterial strains from two contaminated particular sites. Its BS was also characterized for further application strategies compared to other related biosurfactant-producing strains of Pseudomonas spp.

Materials and methods

Sampling

In this study, we were interested in petroleum hydrocarbon-contaminated soil near to well-known Company (SOPRETA) at the Ain El Arbaa region of Ain Temouchent (35°24′49.5″ N 0°53′55.9″ W), due to their prolonged contamination incidents. The second site was to collect effluents from the Terga thermal power plant near the sea (35°27′43.2″ N 1°13′40.5″ W). For the first site, soil sampling was performed at various points (0–10 cm depth) using pre-sterilized materials, while for the second site, the samples were collected at 60 m before the discharge area into the sea. All the samples were collected in sterile flasks and transferred directly to the laboratory facilities, and placed in a cold room (+ 4 °C) for further analysis.

Chemicals

All the chemicals used in our experiments were purchased from Sigma-Aldrich Company, including glycerol, ethyl acetate, sodium hydroxide, hydrogen chloride, sodium chloride, sodium Dodecyl Sulfate (SDS), toluene, diethyl ether, chloroform, acetic acid, methanol, and hexadecane. The crude oil and the industrial waste oil were obtained from Naftec Company (oil refinery) of Arzew, Algeria, while the diesel fuel was purchased from a filling station in Oran, Algeria. The crude rhamnolipid was purchased from the IDRABEL Italia Company. Sugarcane molasses and soft wheat bran were obtained from a sugar refinery and a soft wheat flour production plant, respectively, in Algeria.

Media composition

The nutrient broth (NB) employed for the enrichment and isolation of microorganisms is composed of (g/l): peptone (10), yeast extract (5), NaCl (5) per liter of distilled water. The mineral salt medium (MSM) consisted of (g/l): NaNO₃ (2), Na₂HPO₄ (0.9), KH₂PO₄ (0.7), MgSO₄. 7H₂O (0.4), CaCl₂. 2H₂O (0.1), FeSO₄. 7H₂O (0.001) and 1 ml of a trace element solution containing (g/l): ZnSO₄. 7H₂O (0.7), CuSO₄. 5H₂O (0.5), MnSO_4. H₂O (0.5), H₃BO₄ (0.26), Na₂MoO₄. 2H₂O (0.06) per liter of distilled water (Chebbi et al. 2017a). The pH was adjusted to 7.2–7.4 using (5 M) NaOH and (6 N) HCl solutions. All the media were sterilized by autoclaving at 121 °C for 20 min. The sugarcane molasses was diluted and clarified according to the protocol described by Raza et al. (2007). The soft wheat bran was sieved and crushed. The olive oil was obtained from the olive oil mill (Ain El Arbaa, Algeria), and the coconut oil was obtained from commercial sources (manufactured by Hemani International KEPZ Karachi, Pakistan). Olive oil, coconut oil, and molasses were sterilized by filtration (Sterile syringe filters, pore size 0.45 µm).

Enrichment, isolation, and screening of biosurfactant-producing bacteria

One gram of hydrocarbon-contaminated soil and one milliliter of the effluent sample were aseptically transferred to 9 ml of NB medium, individually. After incubation at 30 °C for 1–2 days, decimal dilutions series were performed according to the method described by Nandhini and Josephine (2013). For The isolation, volumes of the bacterial cultures were diluted with 0.85% sodium chloride pre-sterilized solutions. Decimal dilution series were performed from 10^–1 to 10^–7, while only the last three dilutions (i.e., 10^–5, 10^–6, and 10^–7) were plated onto NB agar plates. The Petri dishes were incubated at 30 °C under aerobic conditions for 2 days. After incubation, colonies of different morphologies were isolated and purified. The pure isolates were stored at (− 20 °C) on a NB medium supplemented with glycerol (20%, v/v) for further studies. Therefore, the ability of the pure isolates to produce BS was studied by the addition of 3% (v/v) of olive oil as the sole source of carbon in MSM medium adjusted to pH 7.2 using sodium hydroxide solution (5 M) and sterilized by autoclaving at 121 °C for 20 min. Bacterial growth was studied using 3% (v/v) of inoculum size. Therefore, the bacterial cultures were incubated at 28 °C at 150 rpm for 4 days of incubation (Chebbi et al. 2017a). The emulsification E₂₄ (%) index, oil spreading technique, and surface tension measurement were used as preliminary tests to screen biosurfactant-producing bacteria. Results are expressed as the mean of three replicates tests ± standard deviation.

Biochemical and molecular studies

The isolated bacterial strains were identified based on their phenotypic and biochemical characteristics using the gallery API 20 E and Bergey’s Manual of Determinative Bacteriology (Levine et al. 1975). From the strains, the 16S rRNA gene was amplified by colony PCR and sequenced for phylogenetic analysis. The reaction mixture of the PCR was composed of: 6 μl of (27F) forward primer (1 μM) 5′ AGAGTTTGATCMTGGCTCAG 3′, 6 μl of (1492R) reverse primer (1 μM): 5′-GGTTACCTTGTTACGACTT-3′ (Weisburg et al. 1991), 30 μl of Green Taq Master Mix, 18 μl of the bacterial cells. The reaction was made in a BIOER thermocycler. The 16S rRNA gene was visualized after migration on an electrophoresis gel composed of 0.8 g of agarose per 80 ml of TBE buffer containing 2 μl of gel star (DNA marker). Purification of PCR products was performed using a special cleaning kit (WizardR SV Gel and PCR Clean-Up System) containing two reagents (Membrane Binding Solution and Membrane Wash Solution). Quantification of pure 16S rRNA gene was performed also using a kit containing two reagents (QubitTM ds DNA BR Buffer and Reagent Buffer) by Thermo Fisher Scientific (Invitrogen) using a mini spectrophotometer (Qubit fluorometer), Invitrogen. Sequencing was carried out according to the Sanger method in the Interministerial Service Center for Biotechnology of Agricultural, Chemical, and Industrial Interest (C.I.B.I.A.C.I.) by taking 150 ng of the purified 16S rRNA gene. The obtained sequences were imported into the editor BioEdit ver. 5.0.9 (Hall 1999). The full sequences were aligned using the RDP Sequence Aligner program (Maidak et al. 2000). Then, they were aligned with other sequences via BLAST (Basic Local Alignment Search Tool) using NCBI (National Center for Biotechnology Information) database, and the construction of the phylogenetic tree was performed using MEGA X: Molecular Evolutionary Genetics Analysis (Kumar et al. 2018). The evolutionary history was inferred by the Maximum Likelihood method and Jukes-Cantor model (Jukes and Cantor 1969), and the tree was generated at 1000 repetitions via the bootstrap method (Felsenstein 1985). Their 16S rRNA sequences were compared with those closest type strains in the NCBI database. All the sequences were deposited in the NCBI database collection (Accession numbers: MT071345…MT071350).

Growth and biosurfactant production studies by strain LGMS7 using various substrates

Growth studies of the LGMS7 strain were carried out on various carbon sources (coconut oil, olive oil, molasses, soft wheat bran, glycerol, toluene, diesel oil, diethyl-ether at 2% (v/v); crude oil, industrial waste oil at 1% (v/v), and Hexadecane (0.5%, v/v). Experiments were carried out with an inoculum size proportion of 3% (v/v) in 500 ml Erlenmeyer flasks containing 100 ml of the MSM medium for 27 days of incubation at 28 °C and 150 rpm. The cell growths were assessed by optical density (OD) measurement at 600 nm and Colonies Forming Units (CFU) counting on plates (when it is needed). Glycerol, sugarcane molasses, and soft wheat bran were selected for the BS production towards a further biotechnological valorization. The potential of the selected strain LGMS7 to secrete BS(s) was then studied in 100 ml of MSM medium (pH 7.2) (500 ml Erlenmeyer flasks), containing 2% (v/v) of glycerol, 2% (v/v) of sugarcane molasses, and 2% (v/v) of soft wheat bran. The three flasks were incubated at 30 °C and 150 rpm for 5–6 days. Biotic control (MSM medium without substrate addition) was also monitored under the same conditions. During the incubation period, the cell growth monitoring was assessed by determining the optical density (Absorbance 600 nm) using a spectrophotometer (Ultrospec 3000, UV–Visible) while the BS activity was examined out on different incubation times (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h) by determining both E₂₄ (%) and the oil spreading test. The bacterial purity was evaluated by regular microscopic observations from liquid and solid cultures. Results are expressed as the mean of three independent tests ± standard deviation.

Emulsification stability (E₂₄) test, oil spreading technique, measurement of surface tension (ST), the critical micelle dilution (CMD)

To measure the emulsification index (E₂₄), the culture broth was centrifuged at 6000 rpm for 20 min, and the obtained cell-free supernatant was filtered through a filter paper (Whatman paper). A volume of 2 ml of filtered supernatant was mixed with 2 ml of diesel and vortexed vigorously for 2 min at room temperature (Chebbi et al. 2017a). After 24 h of incubation, the emulsion layer's height was measured as the percentage of the height of the emulsified layer divided by the total height of the liquid column × 100 (Chebbi et al. 2017a). For this test, sodium dodecyl sulfate (SDS) (Chemical surfactant) and water were used as positive and negative controls, respectively. For oil spreading tests, crude oil (20 µl) was dropped to form a thin oil layer on the surface of the water (20 ml) into a Petri dish (diameter of 90 mm), and therefore, 10 μl of a test solution has been dropped onto the surface of the oil as described elsewhere (Chebbi et al. 2017a). The maximum diameter of the clear zone was measured. Sodium Dodecyl Sulfate (SDS) and water were used as positive and negative controls, respectively. All values presented are the mean of three replicates ± standard deviation.

The surface tension measurements were repeatedly determined using a tensiometer (Model: Manual KRUSS Tensiometer k6, Germany) by taking 10 ml of the cell-free supernatant of the bacterial culture after centrifugation at 6000 rpm for 20 min according to the Du Noüy Ring method (Kim et al. 2000). Distilled water was used as a negative control. Critical micelle dilution (CMD) was determined by measuring the cell-free supernatant surface tension at different concentrations and during the incubation times (72 h, 96 h, and 120 h) at 150 rpm. Milli-Q water was used for the preparation of the dilutions. The dilution is a critical factor in the dilution of micronutrients (CMD), which is the factor by which the effective BS concentration exceeds the critical micelle concentration (Marchesi et al. 1991). Results are expressed as the mean of three replicates ± standard deviation.

Extraction of biosurfactant and study of the effect of pH, temperature, and salinity on biosurfactant stability

The BS extraction was carried out according to the method described by Smyth et al. (2010). The LGMS7 bacterial culture's cell-free supernatant was obtained after centrifugation at 8000 rpm for 15 min and acidification to pH 3 with HCl solution (4.8 N). Then, the extraction was carried out with ethyl acetate (v/v) repeated three times. The organic phase was dried in the presence of magnesium sulfate (MgSO₄) (0.1 g per 100 ml of the solvent) and obtained by evaporation of the extraction solvent using a rotavapor (BṺCHI R-114) at 45 °C. A partially purified BS was recovered, weighed, and expressed in (g l⁻¹). The yield of BS production is represented by the mean of two independent experiments ± standard deviation. The critical micelle concentration (CMC) of the crude BS was determined by measuring the surface tension at different concentrations. The crude BS was diluted in milli-Q water at concentrations ranging from (0–1600 mg l⁻¹), and the CMC was thus determined by measuring the surface tension until a constant value was obtained. To evaluate the BS stability, the surface tension was determined on the cell-free supernatant (at 120 h of incubation), which was exposed to different pH (pH 2–pH 12); various concentrations of NaCl (from 0 to 250 g/l), and different temperatures (from −80 °C to 121 °C). For temperature tests, the measurements were determined after overnight incubation at 4 to 70 °C (after one hour for at 100 °C and after autoclaving (20 min) at a temperature of 121 °C) (Cheffi et al. 2020; Hentati et al. 2019).

Characterization of biosurfactant: TLC, FTIR, and ¹H-NMR analyses

The characterization of the BS was carried out using thin-layer chromatography (TLC), FTIR, and ¹H-NMR analyses. For the TLC, the separation was performed on a TLC Silica gel 60 F₂₅₄, MERCK, Germany by the solvent system (chloroform: methanol: 20% aqueous acetic acid) (65:15:2) as a mobile phase, in the presence of a standard of rhamnolipid (Dos Santos et al. 2016). To identify the chemical structure of the BS, the Fourier Transform Infrared (FTIR) analyses were carried out using a Bruker Alpha spectrometer, Germany. The ATR (Attenuated Transmission Resonance) was also used for analyzing the BS with a wavenumber range of 500 to 4000 cm⁻¹. The ¹H-NMR spectrum was then determined using an NMR spectrometer (Bruker Avance III 300 MHz NMR), Germany (deuterated chloroform (CDCl₃) as a solvent). Chemical shifts were expressed in parts per million (ppm), which were shifted downfield from (Internal standard) tetramethylsilane TMS.

Statistical analysis

Emulsification and oil spreading activities, ST and CMD determinations were performed at three replicates tests, and the production yield of BS was conducted at two independent tests. Means and standard deviations were calculated using GraphPad Prism 6 (Trial version).

Results

Isolation and screening of biosurfactant-producing bacteria

After microbial enrichments from the two contaminated sites (i.e., hydrocarbon-contaminated soil and of the liquid effluents), several strains were isolated and tested separately for their emulsifying potential E₂₄ (%), oil displacement tests, and their potential for surface tension reduction (Table 1). Four isolates (from 10 tested) showed BS activities from the hydrocarbon-contaminated soil, including LGMS7, LGMS9, LGMS2, and LGMS12. For the liquid effluents, only three isolates were selected for the same activities, comprising LGMT16, LGMT11, and LGMT14, from 12 tested isolates (Table 1). Based on those results, the strain LGMS7 showed higher emulsifying potential and significant oil displacement abilities. Moreover, it showed the best surface tension reduction. Accordingly, the strain LGMS7 was selected for further studies.

Table 1.

Preliminary screening tests of BS producing isolates, growing in the presence of olive oil (3%, v/v) as the sole carbon source, at 96 h of incubation and 150 rpm and 30 °C

Bacteria code	Origin	E24 Emulsification index (%)	Oil spreading test (mm)	Surface tension (ST)
LGMS7	Hydrocarbon- contaminated soil	15.27 ± 1.9	15.33 ± 1.2	35.5 ± 0.70
LGMS9		9.72 ± 1.9	8 ± 0.8	44 ± 1.41
LGMS2		8.33 ± 2.0	7.33 ± 0.4	47 ± 1.41
LGMS12		11.11 ± 1.9	6.33 ± 0.4	43 ± 2.82
LGMT16	Effluents of the thermal power plant	9.72 ± 1.9	7.33 ± 0.9	40.66 ± 1.15
LGMT11		7.16 ± 2.0	6.66 ± 0.4	42.33 ± 1.52
LGMT14		9.72 ± 1.9	8.33 ± 0.4	42.33 ± 0.57
Abiotic control		0.66 ± 0.57	1.66 ± 0.57	63.33 ± 0.57

Characterization of bacteria

Strain LGMS7 is a short rod Gram-negative, catalase- and oxidase-positive, which forms small round colonies with a diameter of 1 mm on the surface of the nutrient agar medium after 24 h of incubation at 30 °C. It can hydrolyze skimmed milk casein and Tween 80, while it is unable to hydrolyze starch. Growth was observed between 4 and 40 °C of temperature, 0 and 60 g l⁻¹ of NaCl concentration, while the pH range required for growth was between pH 5.0 and pH 9.0. Phenotypic and biochemical analyses were conducted based on the Bergey’s Manual of Determinative Bacteriology.

Phylogenetic analysis showed that strain LGMS7 was most closely related to genus Pseudomonas members, especially to the species Pseudomonas mucidolens IAM 12406^T (D84017), displaying a sequence 16S rRNA similarity of 99.05% (Fig. 1). The corresponding sequence was deposited in the GenBank nucleotide database as Pseudomonas sp. strain LGMS7 under the accession number (MT071345). Table 2 shows the characteristics that differentiate the LGMS7 strain and type strains of closely related species, while (Table S1: Supplementary material) displays the percentage of 16S rRNA similarities of all isolates, including LGMS7, LGMS2, LGMS9, LGMT16, LGMT11, and LGMT14 strains, each with the closest type strain.

Fig. 1.

Maximum-likelihood phylogenetic tree based on 16S rRNA sequence of strain LGMS7 and related type strains species. The tree was generated with 1000 repetitions Bootstrap method, and the evolutionary history was inferred using the Maximum Likelihood method and Jukes-Cantor model. The percentages (%) at the node represent the probability values of similarity robustness. Bar = 0.05 nucleotide substitution per site. Micrococcus luteus DSM20030^T was used as an out-group

Table 2.

Differential phenotypic characteristics of the strain LGMS7 and other related type strains of Pseudomonas species

Characteristics	Strain LGMS7	Pseudomonas mucidolens IAM 12406 T (D84017) (Levine and Anderson 1932)	Pseudomonas paralactis WS 4672 T (KP756921) (Von Neubeck et al. 2017)	Pseudomonas aeroginosa DSM 50071 T (HE978271) (Colwell 1965)
Similarity of 16S rRNA with strain LGMS7 (%)	100	99.05	99.05	88.15
Gram	−	−	−	−
Oxydase	+	+	+	+
Catalase	+	+	+	+
Mobility	+	+	+	+
NaCl growth range (%, w/v)	0–6	nd	0–6	0–5
Temperature growth range (°C)	4–40	10–33	4–35	7–44
pH growth range	5–9	nd	5–8	5–9
B-galactosidase	−	−	−	−
Arginine dihydrolase	+	−	+	+
Lysine decarboxylase	+	nd	−	nd
Ornithine decarboxylase	+	nd	−	nd
Utilization of citrate	+	nd	nd	+
H2S production	−	nd	−	−
Urease	+	−	−	−
Tryptophan deaminase	+	nd	−	nd
Indole production	−	−	−	−
Acetoin Production	+	nd	−	−
Gelatinase	+	−	+	+
Nitrate reduction (NO2 production)	+	+	−	+
Utilization of
Glucose	+	+	+	−
Mannitol	−	±	+	+
Inositol	+		+	−
Sorbitol	−	+	−	−
Rhamnose	+	+	−	−
Saccharose	+	−	−	−
Melibiose	+	nd	−	−
Amygdaline	−	−	−	nd
Arabinose	+	+	+	−
Voges–Proskauer test	+	−	−	−
Methyl red test	−	nd	nd	−
Hydrlolysis of skimmed milk	+	nd	+	+
Hydrolysis of starch	−	−	−	−
Hydrolysis of tween 80	+	nd	nd	+

 +  Positive, − Negative, ± Weak, nd not determined

Growth and biosurfactant production studies: effect of pH, temperature and salinity on biosurfactant stability

The strain LGMS7 was found to grow on several hydrocarbons and oils, as sole carbon and energy sources, including coconut oil, olive oil, molasses, soft wheat bran, glycerol, toluene, diesel oil, diethyl-ether, crude oil, industrial waste oil, and Hexadecane (Table 3). Among the three tested substrates (i.e., molasses, soft wheat bran, and glycerol), the BS production has been significantly detected in MSM medium supplemented with 2% (v/v) glycerol, showing an emulsifying capacity E₂₄ (%) index = 66.66% ± 3.46 (at 120 h and 144 h of incubation) and a significant oil spreading potential (OST = 4 cm halos at 96 h and 120 h of incubation) (Fig. 2a, d). While in the same medium supplemented with 2% (v/v) of molasses, the E₂₄ (%) test showed an average percentage of 45.45% at 144 h of incubation (Fig. 2b), and no halo formation was recorded for the oil spreading tests. When the wheat bran was used as a substrate, both the E₂₄ (%) index and the oil spreading technique showed negative results. The glycerol was, therefore, selected as a substrate for BS production to peruse further studies.

Table 3.

Growth pattern of the strain LGMS7 on various oils and hydrocarbons in MSM medium for 27 days of incubation at 28 °C at 150 rpm

Carbone source	OD max (600 nm)	Growth
Coconut oil (2%, v/v)	15.3 (14 109 CFU ml−1)	++
Olive oil (2%, v/v)	9.14 (82 108 CFU ml−1)	++
Molasses (2%, v/v)	1.79	+
Soft wheat bran (2%, v/v)	0.6	+
Glycerol (2%, v/v)	0.9	+
Crude oil (1%, v/v)	0.49 (30 108 CFU ml−1)	+
Industrial waste oil (1%, v/v)	0.23	±
Toluene (2%, v/v)	0.39	+
Hexadecane(0.5%, v/v)	0.26 (58 107 CFU ml−1)	+
Diesel oil (2%, v/v)	0.89 (20 108 CFU ml−1)	+
Diethyl-ether (2%, v/v)	0.39	+
Biotic control	0.14	–

 +  Good growth, ±  weak growth, − No growth.

Fig. 2.

Growth monitoring (OD 600 nm) (Black circle); measurement of surface tension (Black square) and E₂₄ (%) index (Black up pointing triangle) of Pseudomonas sp. strain LGMS7 in MSM medium supplemented with glycerol (2%,v/v) at 30 °C and 150 rpm (a); Growth monitoring (OD 600 nm) (Black circle); E₂₄ (%) Emulsification index (Black square) of Pseudomonas sp. strain LGMS7 in MSM medium supplemented with molasses (2%,v/v) (b); Critical Micelle Dilution (CMD) of the cell-free supernatant of strain LGMS7 in MSM medium supplemented with glycerol at (2%, v/v) at different times of incubation: 3-day old culture (Black square); 4-day old culture (Black circle); 5-day old culture (Black up pointing triangle) (c); Emulsification index E24 (%) of strain LGMS7 on MSM medium + glycerol (a): control T_{0 h}, (b): T_120 h (d)

The strain LGMS7 was able to reduce the surface tension of the cell-free supernatant from 66 mN m⁻¹ to 30 ± 0.65 mN m⁻¹ after 48 h (Fig. 2a). The surface tension remained stable up to 144 h of incubation and even during the stationary phase (Fig. 2a). The greatest CMD was recorded after 3 days and 4 days of incubation with a percentage of 34% (v/v), by measuring the surface tension of the different concentrations of the cell-free supernatant of strain LGMS7 (Fig. 2c). After extraction with ethyl acetate (v/v), a yield of BS production of 1.26 ± 0.57 g l⁻¹ was noted, while the critical micelle concentration (CMC) of the crude BS was recorded at 800 mg l⁻¹ (Fig. 3a). At this concentration and more, the surface tension was almost stable. Below the concentration of 800 mg l⁻¹ (700 mg l⁻¹ and less) an increase of surface tension was detected to around 55 mN m⁻¹, and surface tension continues to increase to around 72 mN m⁻¹ (Fig. 3a). The effect of pH, temperature, and salinity on BS stability was studied (Fig. 3b, c, d). Interestingly, the BS of strain LGMS7 showed excellent stability against pH (pH 2 to pH 12), salt concentrations of NaCl (0 to150 g l⁻¹), and temperature (−20 to 121 °C). However, lower stability was noted at higher concentrations of NaCl (200 to 250 g l⁻¹) and temperature at −80 °C (Fig. 3b, c, d).

Fig. 3.

Determination of critical micelle concentration (CMC) of the crude BS (a); pH stability (b); Sodium chloride concentration stability (c); Temperature stability (d) of the BS produced by Pseudomonas sp. strain LGMS7

Characterization of biosurfactant (TLC, FTIR, and ¹H-NMR)

The Thin-layer chromatography of the BS of strain LGMS7, in the presence of a rhamnolipid control, showed two bands corresponding to a mono-rhamnolipid and a di-rhamnolipid with a retention factor (Rf), respectively, Rf = 0.9 and 0.6 (Fig. 4a). An ATR-FTIR spectrum of its BS was also determined (Fig. 4b). The FTIR spectra indicated the functional groups of the glycolipid biosurfactant, containing rhamnolipid. The elongation appearing at 3368.08 cm⁻¹ indicated the presence of a hydroxyl group (–OH stretching). The triple bands at 2949.83, 2917.71, and 2849.02 cm⁻¹ are derived from symmetric C-H stretching vibrations of aliphatic groups, while the –C=O stretching band at 1740 cm⁻¹ was attributed to ester bonds characteristic and carboxylic acid groups. The absorption peaks between 1235 to 1455 cm⁻¹ were associated with C–H and O–H deformation vibrations, typical of carbohydrates (rhamnose units of the molecule). Therefore, the structure of rhamnolipid was confirmed by ¹H-NMR and the results are shown in Fig. 5. The chemical shifts at 0.87, 1.26, 2.49, 5.01, 5.19, and 8.19 ppm recorded on ¹H-NMR spectra likely match L-rhamnose's characteristics peaks moiety and an aliphatic moiety of rhamnolipid. TLC, FTIR, and ¹H-NMR could affiliate our produced BS to rhamnolipids, a class of glycolipid biosurfactants.

Fig. 4.

TLC of rhamnolipids shows the presence of mono-rhamnolipid: MRL and di-rhamnolipid: DRL, with a retention factor (Rf), respectively, of 0.9 and 0.6 (1: Standard rhamnolipids; 2: Crude BS of Pseudomonas sp. strain LGMS7) (a); ATR-FTIR spectrum of crude rhamnolipids produced by Pseudomonas sp. strain LGMS7 (b)

Fig. 5.

¹H-NMR spectrum of the crude BS produced by Pseudomonas sp. strain LGMS7

Discussion

Various Algerian local and even international organisms are currently focusing on developing bio-based methodologies to tackle the soil-hydrocarbon contamination in the nearby oil-producing companies (Sekkour et al. 2019). In this context, our study highlighted the isolation, screening of biosurfactant-producing bacterium; strain LGMS7, after microbial enrichment from a contaminated site in the western region, Ain Temouchent, Algeria. For that, six biosurfactant-producing bacteria were screened based on their emulsifying potential E₂₄ (%), surface tension reduction, and oil displacement capacities, belonging to Pseudomonas sp., Bacillus sp., Cronobacter sp., and Enterobacter sp. Among them, the strain LGMS7 isolated from hydrocarbon-contaminated soil was selected for growth and BS production studies on various substrates, e.g., glycerol, molasses, and soft wheat bran chosen in this context. In this region, some studies have addressed the hydrocarbon-degrading capacities of some bacterial species. However, little is known about their capability to produce BS, which are currently requested for their local hydrocarbon bioremediation approaches. For instance, Ferhat et al. (2017) have indicated the production of BS by a bacterial strain 1C of the species Ochrobactrum intermedium. The authors showed that the cell-free culture broth of the strain 1C recorded a surface tension below 31.5 mN m⁻¹ on olive oil as a substrate. Its BS, identified as a glycolipid, was also found to solubilize naphthalene and phenanthrene effectively, which are widely detected at several petroleum-contaminated soils (Ferhat et al. 2017). Our results are in agreement with other studies on the BS producing capacities by those species, including, Cronobacter sakazakii studied by Jain et al. (2012), Enterobacter cloacae by Karmakar et al. (2019), Bacillus subtilus by Veshareh et al. (2019), and Pseudomonas aeruginosa by Sun et al. (2019). Based on the literature, we have presented some strains that were described in the microbial surface-active agents’ production, with particular interests in rhamnolipid production among Pseudomonas spp. (Table 4).

Table 4.

Comparative study among some Pseudomonas strains on the production of BS

Strains	BS Type	Substrate for BS production	BS yield (g l−1)	BS proprieties	BS Stability	Study
Pseudomonas sp. strain LGMS7	Rhamnolipids	Glycerol	1.26 ± 0.57	CMC = 800 mg l−1 ST = 30 ± 0.65 mN m−1	Salinity (0–150 g l−1) Temperature (− 20 to 121 °C); pH (2–12)	Our study
Pseudomonas aeruginosa strain W10	Rhamnolipids	Olive oil Glycerola	2 10a	CMC = 400 mg l−1 ST = 32 mN m−1	Salinity (0–150 g l−1) Temperature (0–100 °C) pH (2–12)	Chebbi et al. (2017a) Chebbi et al. (2017b)
Pseudomonas aeruginosa strain UCP0992	Rhamnolipids	Glycerol	8	CMC = 700 mg l−1 ST = 27.4 mN m−1	Salinity (0–100 g l−1) Temperature (4–120 °C) pH (4–12)	Silva et al. (2010)
Pseudomonas aeruginosa strain LBI	Rhamnolipids	Soap stock	15.9	CMC = 120 mg l−1 ST = 24 mN m−1	nd	Benincasa et al. (2004)
Pseudomonas nitroreducens strain TSB.MJ10	Lipopeptide bioemulsifier	Sodium benzoate	2.9	ST = 72 mN m−1	Salinity (0–250 g l−1) Temperature (20–90 °C) pH (5–11)	De Sousa and Bhosle (2012)
Pseudomonas alcaligenes strain PCL	Rhamnolipids	Palm oil	2.3	ST = 28 mN m−1	nd	Oliveira et al. (2009)

nd not determined

^aAnother study using the same strain

The data available in the literature regarding biosurfactant-producing bacteria show that the emulsification index E₂₄ (%) differs among strains, depending on several factors including the type of BS, the abundances of congeners, the genus and species of the producer, the used substrate, and other factors. Our study has demonstrated an excellent value of E₂₄ (%) index of 66.66% ± 3.46 after 120–144 h compared to other reports on glycerol as a substrate and diesel oil as the hydrocarbon model for this test. For example, Pseudomonas aeruginosa strain 181 was found to record the greater BS production ability after 120 h of incubation (Govindammal and Parthasarathi 2013). It was also demonstrated that the maximum emulsifying potentials by Pseudomonas aeruginosa strain F23 was observed during its stationary phase at 96 h of incubation, with an E₂₄ (%) value of 54% (Patil et al. 2014). Moreover, Saikia et al. (2012) have demonstrated that the E₂₄ (%) reached a maximum of (76%) at 68–72 h of incubation by the Pseudomonas fluorescens strain RS29 in MSM medium supplemented with glycerol as a substrate. Similarly, Suryanti et al. (2015) demonstrated that the value of E₂₄ (%) obtained by the growth of Pseudomonas fluorescens on molasses, as a substrate was about 50%. Makkar and Camerotra (1997) also used molasses to produce BS by two strains of Bacillus under thermophilic conditions. The BS obtained by both strains showed E₂₄ (%) values of 80% and 95% for B. subtilis MTCC 1427 and MTCC 2423, respectively. However, it is important to mention that several studies in the literature are showing only the emulsification index values of biosurfactant-producing microorganisms on various hydrophobic substrates e.g., olive oil, crude oil (Abouseoud et al. 2008; Bento et al. 2005), which is less precise in comparison to yields of biosurfactants obtained after extraction and purification (Chebbi et al. 2017a). Further studies must emphasize re-evaluating this key parameter between strains for this activity by determining the BS yields for specific classes on specific substrates at the same conditions e.g., among rhamnolipid producers. Currently, the available biosurfactant-producing microorganisms in various bacterial collections are being revaluated by determining the yield of production on specific substrates, especially for the non-pathogenic strains, which could be very advantageous for novel bio-based approaches and sustainability (Chebbi et al. 2021).

On the other hand, it was observed that a good surfactant could lower the surface tension of water from 72 to around 35 mN m⁻¹ (Mulligan 2005). The strain LGMS7 has presented an interesting ability to reduce the surface tension of the cell-free supernatant compared to its closest strains, such as Pseudomonas aeruginosa strain W10, showing a surface tension reduction of the cell-free supernatant to around 32 mN m⁻¹, in the presence of 1% of olive oil as a substrate (Chebbi et al. 2017a). Varadharajan and Subramaniyan (2014) also demonstrated that Pseudomonas aeruginosa strain PB3A could reduce surface tension to 41 mN m⁻¹ and 42 mN m⁻¹, when used corn oil and cassava flour waste amended medium, respectively, after 48 h of incubation. Almost the same results have been noted by Sidkey et al. (2016), who showed that Pseudomonas aeruginosa PAO1 was able to reach the surface tension of 28.23 mN m⁻¹ in MSM medium supplemented with 1% olive oil wastes.

Throughout the literature, the yields of BSs and their CMCs vary between studies, which mainly depend on the class of BS, the biosurfactant-producing microorganism, carbon sources, and fermentation conditions and others (Chebbi et al. 2017a) (Table 4). In our study, when the glycerol was used as a substrate with some specified experimental conditions (not optimized), we have recorded an average in the production yield of 1.26 ± 0.57 g l⁻¹, which may be increased with the optimization of production conditions. Generally, the yield of rhamnolipids produced by Pseudomonas species is greater than 2 g l⁻¹ and can go up to 20 g l⁻¹ (even more in some studies) (Benincasa et al. 2004; Chebbi et al. 2017a, b; De Sousa and Bhosle 2012).

The CMC is defined as the minimum concentration of BS required to give maximum surface tension reduction of water and initiate micelle formation. Efficient surfactant generally assumed to have very low CMC values, i.e., a low surfactant concentration is required to decrease surface tension (Silva et al. 2010). In our study, the non-purity of extracted rhamnolipids (partially purified) might be responsible for the higher value of CMC obtained compared to CMCs described in the literature. In fact, the rhamnolipids’ compositions and the relative abundances of congeners may influence the CMC value, due to the intrinsic variability of the accumulated rhamnolipids and the variability of its composition, the number of homologues, the presence of unsaturated bonds, the branching and length of the aliphatic chains of rhamnolipids (Asfora et al. 2006). For instance, the production yield and the CMC recorded by the Pseudomonas aeruginosa UCP0992 strain are 8 g l⁻¹ and 700 mg l⁻¹, respectively (Silva et al. 2010). Another study showed a production yield of 2 g l⁻¹ and a CMC of 400 mg l⁻¹ using Pseudomonas aeruginosa strain W10 (Chebbi et al. 2017a). Furthermore, Benincasa et al. (2004) noted a production yield and a CMC of 15.9 g l⁻¹ and 120 mg l⁻¹, respectively, by Pseudomonas aeruginosa LBI.

The industrial applications of BS require multiple characters comprising mainly their stability at extreme operational conditions. For instance, within the Microbial Enhanced Oil Recovery processes, the BS use as coadjutants is promising under harsh reservoir conditions i.e., high/hardness temperatures and salinities and others (Haloi et al. 2020; Joshi et al. 2008). For that, BSs were found to be promising candidates for green applications due to the stability proprieties into bioremediation processes of contaminated terrestrial and marine saline sites (Eddouaouda et al. 2012; Jamal and Pugazhendi 2018). Given their interesting characteristics of stability in wide ranges of salinity, temperature, and pH, the researchers are still concentrated their studies on isolation and screening of biosurfactant-producing microorganisms from particular sites with extreme contamination conditions (Eddouaouda et al. 2012). The effects of NaCl concentration on the BS stability (i.e., reduction of surface tension) up to specific limits was previously described in literature. Helvaci et al. (2004) have explained this phenomenon by the presence of electrolytes that directly affect rhamnolipids’ carboxylate groups. The solution/air interface has a net negative charge due to the ionized carboxylic acid groups at alkaline pH with strong repulsive electrostatic forces between the rhamnolipid molecules. This negative charge is shielded by the Na + ions in the electrical double layer in the presence of NaCl, causing the formation of a close-packed monolayer and consequently a decrease in surface tension values (Silva et al. 2010). Other more in-depth studies should be carried out within this framework for the collection of indigenous microorganisms isolated from particular sites not yet explored. The great majority of studies have shown the excellent stability of the BS at different pH, salinity, and temperature in agreement with our results (Chebbi et al. 2017a; De Sousa and Bhosle 2012; Silva et al. 2010). For that, the BSs produced by Pseudomonas species appear to be good candidates for environmental applications compared to other bacterial genera.

The rhamnolipids production is mainly described by the opportunistic pathogenic bacterium Pseudomonas aeruginosa (Wittgens et al. 2011). Recently, other studies have revealed these capacities among Pseudomonas genus and few other genera, which are described as novel alternatives for safe rhamnolipid production, e.g., Pseudomonas putida (Wittgens et al. 2011), Pseudomonas alcaligenes (Oliveira et al. 2009), and Burkholderia thailandensis E264 (Elshikh et al. 2017). In this context, our study highlighted the rhamnolipids production by the strain LGMS7, a non-pathogenic Pseudomonas mucidolens, which could be a novel alternative toward replacing the well-known pathogenic Pseudomonas aeruginosa (Chebbi et al. 2017b). Though, these conclusions are preliminary and should be supported with further phylogenetic and ecotoxicological studies.

Based on data from the literature, the FTIR spectrum indicated the functional groups of the glycolipid biosurfactant, including rhamnolipid. These results are in agreement with the structure reported by other studies (Gogoi et al. 2016; Leitermann et al. 2008; Moussa et al. 2014; Sabturani et al. 2016). The obtained ¹H-NMR spectrum corresponded to characteristics peaks of L-rhamnose moiety and an aliphatic moiety of rhamnolipid, as previously noted by previous studies (Gogoi et al. 2016; Moussa et al. 2014). However, liquid chromatography-mass spectrometry (LC–MS) will be performed in the future to analyse the abundance of rhamnolipids congeners in this BS. Based on those promising results, we are currently studying the gene expression involved in rhamnolipids production, which might be highly required to design further large-scale production strategies. Besides, the whole genome sequencing is also currently being studied to an improved affiliation of this novel Pseudomonas sp. strain LGMS7. In addition, further studies are also needed to reveal the key genes involved in both hydrocarbons’ degradations and BS production.

Conclusion

In this work, several bacterial isolates were screened and evaluated for their capacities to produce BS from two contaminated sites in north Algeria. A rhamnolipid producer, Pseudomonas sp. strain LGMS7, was isolated from hydrocarbon-contaminated soil after enrichment in NB medium. This strain could be a new alternative for rhamnolipid safe production, which presents a novel pathway towards sustainable industrial applications. It showed remarkable capacities for BS production and thus reducing the surface tension of the cell-free supernatants using glycerol as a substrate at 2% (v/v) in MSM medium after incubation for 6 days at 150 rpm and 30 °C. Moreover, it revealed interesting abilities to use several hydrocarbons and oils, including crude oil, hexadecane, industrial waste oil, toluene, diesel oil, diethyl-ether, as sole carbon and energy sources. Interestingly, its BS has also shown excellent stabilities against wide ranges of pH, NaCl, and temperature. The current data highlight the importance to keep isolating novel microorganisms from highly contaminated sites in developing countries with contamination incidents in history. With increasing environmental awareness, those efforts will be very advantageous for those countries to get involved in the bio-based approaches currently taking place by leading fuel-producing countries. Our next challenge will be to study the ability of this strain (and the others selected) to degrade recalcitrant hydrocarbons using more sensitive techniques, such as gas chromatography-mass spectrometry (GC–MS), gas chromatography with flame-ionization detection (GC-FID), and the key genes responsible for those traits. Further research is also ongoing to produce this BS at a larger scale for further applications on contaminated soils in Algeria.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

AC: conceptualization, writing–reviewing and editing. Dr. AC: supervision, methodology, software, writing–reviewing and editing. Pr. FB and Pr. AF: visualization, writing–reviewing, investigation, and supervision.

Declarations

Conflict of interest

The authors declare that there is no conflict of interest.