**2. Materials and methods**

#### **2.1 Production and purification of biosurfactants**

*Pseudomonas sp.* PDD-14b-2 (GenBank accession number of the 16S rRNA gene sequence: DQ512788), *Xanthomonas campestris* PDD-32b-52 (HQ256850), and *Pseudomonas syringae* PDD-32b-74 (HQ256872) were isolated from cloud water sampled at the Puy de Dôme summit (1465 m) [5]. The isolates obtained in pure cultures (R2A, 17°C) were stored in 10% (v/v) glycerol at −80°C.

For each strain, preculture was performed from the glycerol stocks in 100 mL of a R2A growth medium [36] at 17°C. After 3 days, the inoculum was grown in 10 Erlenmeyer flasks containing 200 mL of R2A medium (2% v/v plating). Cultures were incubated at 17°C at 200 rpm. The growth was monitored through measurement of optical density and pH, the production of biosurfactants by measurement of the surface tension of the supernatant. After 3–5 days, cultures were centrifuged (8000 rpm) at 4°C for 15 min. Supernatants were combined (1.8 L) and pH adjusted to 6.1.

Concentration of biosurfactants was achieved using a chromatographic method initially described by Reiling [37] for rhamnolipids, through adsorption chromatography on an Amberlite XAD2 (Sigma-Aldrich) column [38]. After equilibration to pH 6.1 using 0.1 M phosphate buffer, supernatant was passed through the resin (300 mL.h<sup>−</sup><sup>1</sup> outflow) until saturation of the resin occurred (monitored by measuring the surface tension of the column outlet). The column was then washed by three volumes of distilled water before eluting the biosurfactants with three volumes of methanol. The process is repeated until complete treatment of the supernatant. Methanol fractions are collected and evaporated to dryness using a rotary evaporator.

Purification was then undergone by flash chromatography (puriFlash®) on a Chromabond® C18 column. Gradient elution was applied (water (A), acetonitrile (B), 5–95% B in 30 min.). Fractions (25 mL) were collected at a flow rate of 12.5 mL per minute over a period of 40 min. UV-detection of biosurfactants was made simultaneously at 219 and 237 nm.

## **2.2 Structure identification by LC-MS/MS**

Identification of biosurfactants was performed using an ultra-high-resolution mass spectrometer (LTQ-Orbitrap™, Thermo Scientific) coupled to an electrospray ionization (ESI) source.

Samples were directly infused into the ESI source. The mobile phases consisted in (A) 0.1% formic acid in water (Fluka, 98%) and (B) acetonitrile (CAN; Optima LC-MS, Fischer). The gradient elution was performed at a flow rate of 5 μL min<sup>−</sup><sup>1</sup> using 5–95% of B within 11 min. The sample injection volume was 10 μL. Each sample was measured in the negative and positive ionization modes, with the following optimized settings: source voltage 4 kV and capillary temperature 350°C, in the positive mode. Transient acquisition time was set to 1 s, which corresponds to a nominal resolution of 3 ppm, and to observe individual peaks resolution (FWHM) typically better than 70,000 (m/z 200). Identification was performed using MS/MS fragmentation to confirm the structure of the products. MS/MS experiments were carried out with a collision energy of 5 eV.

Data were collected from m/z 50 to 1200 in the positive and negative ionization modes. Elemental compositions from exact mass measurement were assigned using Xcalibur® software (Thermo Scientific). The data processing was done through the following steps: (1) the assignment of m/z for each spectrum signal, (2) internal calibration of spectrum by homologs biosurfactant using the most intense class, (3) assignment of molecular formula for each signal by comparing experimental m/z with a theoretical m/z database for possible biosurfactant, and (5) solving of dubieties on molecular formula assignments by confirming the isotopic ratio.

#### **2.3 Surface tension measurements**

Samples were centrifuged (10,480 g/3 min) just prior to surface tension measurements. All surface tension measurements were performed using the pendant drop method with an OCA 15 Pro tensiometer (Data Physics, Germany). The camera analyzes the pendant drop profile of the crude extract. A dosing needle with a 1.65-mm outside diameter was used, producing drops of 12 μL. The software fits this latter measurement to the Young-Laplace equation and averages out surface tension from all measurements [39]. The measurements were obtained at 295 K every second. The tensiometer was calibrated using Milli-Q water. The uncertainty of the instrument was ±0.01 mN.m<sup>−</sup><sup>1</sup> . Each dynamic surface tension curve was measured three times for the most efficient biosurfactant-producing microorganisms, and the measurements displayed ±10% variation. These dynamic surface tension measurements lasted until the equilibrium region was reached (maximum 30 min [27]). Along with the surface tension, each measurement also provided real-time monitoring of the droplet volume, facilitating an assessment of evaporation. No significant evaporation (<5%) was observed during the experiments [27].

### **2.4 Modeling**

Molecular dynamics based on all-atoms simulation was performed using NAMD programs with CUDA gpu acceleration designed especially for large

**77**

*Cloud Microorganisms, an Interesting Source of Biosurfactants*

biomolecular systems. The force field used is CHARMM with CHARMM22 parameter files. All air/water interface models were constructed using VMD as molecular visualization program. The water used in our solvent boxes is TIP3 water. Each model undergoes the same treatment: a first phase of minimization, followed by a canonical dynamics for reaching the set temperature of 298 K, and a second phase of pressure equilibration at 1 atm in isothermal-isobaric ensemble followed by a third phase of production in the same isothermal-isobaric

The coarse-grain (CG) molecular dynamics were performed by using the MARTINI force field [1, 2] at 300 K in the NVT statistical ensemble. The liquidvapor interfaces were modeled using a parallelepipedic box of dimensions Lx = 60 Å, Ly = 60 Å, and Lz = 570 Å. The total number of CG water molecules was fixed to 6000 and the number of surfactants was varied from 4 to 32 for each interface. A CG water molecule corresponds to four water molecules. The equilibration time was set to 110 ns whereas the thermodynamics and structural properties

The 3 strains *Pseudomonas sp.* PDD-14b-2, *Xanthomonas campestris* PDD-32b-52,

and *Pseudomonas syringae* PDD-32b-74 were selected from the screening of 480 strains isolated from cloud waters for their effectiveness in reducing surface tension [27]. They all belong to the class of Gammaproteobacteria and are representative of

For the production of biosurfactants, the bacterial cultures were carried out in R2A medium, a relatively poor but diversified medium in carbon and nitrogen sources, initially developed to isolate microorganisms from tap water. We choose this medium, without supplementing with compounds known to favor the production of biosurfactants, as it is representative of the cloud environment in its

On each culture, the simultaneous kinetic monitoring of the bacterial growth and the decrease of the surface tension, using as reference value that of the medium

Extraction of the lipopeptides was carried out by adapting an adsorption column chromatography method (using Amberlite) initially described by Reiling [37] for the concentration of rhamnolipids. The surface tension of the crude

monitored by the measurement of the surface tension of the aqueous fractions at the column outlet, and the saturation of the column was detected when the

were then eluted with methanol. One advantage of the technique is that if the entire supernatant cannot be processed at one time, the operation is repeated until it is fully treated. We have thus shown that the use of adsorption column chromatography is particularly suitable for the extraction of lipopeptides. In addition, compared to conventional methods of extraction using organic solvents (preceded or not by acid precipitation) [40], this method effectively eliminates the culture medium nutrients present in the supernatant and leads to a preconcentration of biosurfactants which then allows easier purification by reversed

surface tension reached a value greater than 40 mN.m<sup>−</sup><sup>1</sup>

), allowed us to stop the cultures for an optimal production of

. The adsorption of the biosurfactants was easily

. The biosurfactants

a genus very commonly encountered in cloud water samples [5].

*DOI: http://dx.doi.org/10.5772/intechopen.85621*

ensemble for a duration of 20 nanoseconds.

**3. Results and discussion**

composition.

biosurfactants.

R2A (62–64 mN.m<sup>−</sup><sup>1</sup>

supernatants was 25 mN.m<sup>−</sup><sup>1</sup>

phase chromatography**.**

were averaged during the acquisition phase over 200 ns.

**3.1 Production and purification of biosurfactants**

*Cloud Microorganisms, an Interesting Source of Biosurfactants DOI: http://dx.doi.org/10.5772/intechopen.85621*

biomolecular systems. The force field used is CHARMM with CHARMM22 parameter files. All air/water interface models were constructed using VMD as molecular visualization program. The water used in our solvent boxes is TIP3 water. Each model undergoes the same treatment: a first phase of minimization, followed by a canonical dynamics for reaching the set temperature of 298 K, and a second phase of pressure equilibration at 1 atm in isothermal-isobaric ensemble followed by a third phase of production in the same isothermal-isobaric ensemble for a duration of 20 nanoseconds.

The coarse-grain (CG) molecular dynamics were performed by using the MARTINI force field [1, 2] at 300 K in the NVT statistical ensemble. The liquidvapor interfaces were modeled using a parallelepipedic box of dimensions Lx = 60 Å, Ly = 60 Å, and Lz = 570 Å. The total number of CG water molecules was fixed to 6000 and the number of surfactants was varied from 4 to 32 for each interface. A CG water molecule corresponds to four water molecules. The equilibration time was set to 110 ns whereas the thermodynamics and structural properties were averaged during the acquisition phase over 200 ns.
