**1. Introduction**

The structure and function of microbial communities in clouds have been studied only very recently. Although clouds are hostile environments (with acidic pH, low temperature, UV exposure, and oxidative medium), it was shown that microorganisms are alive and metabolically active [1, 2]. Our team was pioneer in isolating and describing microbial strains in cloud water isolated at the summit of the Puy de Dôme mountain, which is referenced as a European site for cloud studies [3–5]. Long-term survey at this site allowed to evaluate concentrations of 105 bacteria mL<sup>−</sup><sup>1</sup> and 104 fungi and yeasts mL<sup>−</sup><sup>1</sup> of cloud water. The most frequently encountered

genera of cultivable bacteria are *Pseudomonas*, *Sphingomonas*, *Streptomyces*, *Rhodococcus*, and *Bacillus*, while *Dioszegia*, *Udeniomyces*, and *Cryptococcus* are dominant genera for cultivable yeasts [5]. Metagenomics and other DNA-based analyses confirmed the predominance of Proteobacteria, Actinobacteria, and Firmicutes [2, 6]. Recent metatranscriptomics data showed that Proteobacteria are the most active in *clouds* [6].

Microorganisms have long been considered as inert particles traveling in the atmosphere; however, the discovery of their metabolic activity suggested they could play a role in atmospheric chemistry and in the microphysics of clouds [1, 2, 7].

Concerning atmospheric microphysics, one of the most difficult scientific problems today is to improve the fundamental understanding and prediction of cloud formation in the atmosphere. Recent papers have highlighted the role of surfactants in atmospheric particles, a role predicted by theory 80 years ago but denied by the scientific community for decades [8, 9]. The group of Barbara Nozière extracted organic compounds from atmospheric aerosols that were able to lower the surface tension (σ) under 30 mN.m<sup>−</sup><sup>1</sup> for concentrations 5 or 6 orders of magnitude lower than those for organic acids [10–14].

These very low values suggested the presence of biosurfactants, and these surfaceactive agents are of microbial origin and are extremely efficient compared to classical surfactants [15–17]. They are amphiphilic with a lipid tail (hydrophobic) and a sugar or peptide moiety (hydrophilic). Although their chemical composition is extremely diverse, they can be classified in two main categories based on their molecular mass [15, 18, 19]: (1) small biosurfactants (PM < 1000 amu) including glycolipids (rhamnolipids, trehalolipids, sophorolipids etc.) and lipopeptides (viscosin, surfactin, polymyxin, syringomycin etc.) and (2) polymeric structures (PM 106 amu) such as polysaccharides, proteins, liposaccharides, lipoproteins (alasan, emulsan etc.).

Biosurfactants could affect atmospheric microphysics by modifying cloud condensation nuclei (CCN) activation. Owing to their exceptional scope in reducing surface tension, these surface-active compounds are thus likely to enhance the propensity of the aerosols to form clouds, as the activation of particles into cloud droplets depends on surface tension according to Köhler's theory [20].

The discovery of the presence of biosurfactants on aerosols raised a new scientific hypothesis: could these biosurfactants be produced by airborne microorganisms? Traditionally, biosurfactant-producing microorganisms were mainly isolated in environments such as soils, seawaters, and sediments contaminated or not by petroleum products [21–24]. Biosurfactants producers can also be isolated from natural sources including fruits, leaves, honey, sugarcane, insects, marine sponges etc. [24]. More recently, extreme environments were described as sources of biosurfactants, microbial producers were isolated from desert and arid soils or from the cryosphere (polar soils and lakes) [24, 25]. The first report concerning the atmospheric environment was made by Ahern [26]. This team showed that 70 fluorescent *Pseudomonas* strains isolated from cloud and rain waters in Scotland were producing biosurfactants; among them, 43 isolates were high producers. More recently, our group screened 480 microbial strains isolated from cloud water collected at the Puy de Dôme station [27]. This microbial collection was composed mainly of Gammaproteobacteria (23.3%), with a majority of *Pseudomonas*; Alphaproteobacteria (19.8%), with a majority of *Sphingomonas*; Actinobacteria (24.2%); and Basidiomycota (19.6%). Using the pending drop method, we measured the decrease of the surface tension of water droplets (12 μL in volume) induced by the addition of crude culture medium of the different strains. Up to 41% of the tested strains were producing biosurfactants (σ ≤ 55 mN.m<sup>−</sup><sup>1</sup> ), 7% of them (*Pseudomonas* and *Xanthomonas* strains) were very active producers (σ ≤ 30 mN.m<sup>−</sup><sup>1</sup> ).

**75**

*Cloud Microorganisms, an Interesting Source of Biosurfactants*

These results show that the biodiversity present in clouds and rain can be a very interesting, still unexplored, source of biosurfactants. As atmospheric environments are cold habitats, these results confirm that cold-adapted organisms are good

Biosurfactants may constitute very valuable compounds of industrial interest as they are promising substitutes for synthetic surfactants with higher biodegradability and lower toxicity. They reach such low surface tensions, even for trace concentrations. Typical desirable properties include solubility enhancement, surface tension reduction, and low critical micelle concentrations, higher foaming, higher selectivity, and specific gravity at extreme temperature, pH, and salinity. In terms of economics, biosurfactants can be synthetized from a renewable stock; however, large-scale production remains challenging [28, 29]. The enormous diversity of biosurfactants also makes them an interesting group of materials for application in many areas such as agriculture, public health, food, health care, medicine, cleaning, textiles, nanotechnologies, waste utilization, and environmental pollution control such as in degradation of hydrocarbons present in soil or extraction of heavy metals [15, 16, 18, 19, 28, 30–35]. Considering the potential industrial interest of biosurfactants, we decided to go further in investigating our unique collection of microbial strains isolated from clouds as a source of biosurfactants. The objective of this work was thus to isolate biosurfactants produced by some of the best producers as determined from our previous screening in order to study their structure and their critical micelle concentration (CMC) properties. In addition, we were interested in modeling their conformation at the water-air interface to understand better their behavior in making cloud droplets. To reach these goals, we selected three strains isolated from clouds (*Pseudomonas* sp. PDD-14b-2, *Xanthomonas campestris* PDD-32b-52, and *Pseudomonas syringae* PDD-32b-74) that were high biosurfactant producers [27].

*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

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

cultures (R2A, 17°C) were stored in 10% (v/v) glycerol at −80°C.

are collected and evaporated to dryness using a rotary evaporator.

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

candidates to produce biosurfactants [25].

**2. Materials and methods**

**2.1 Production and purification of biosurfactants**

*Surfactants and Detergents*

tension (σ) under 30 mN.m<sup>−</sup><sup>1</sup>

than those for organic acids [10–14].

*clouds* [6].

genera of cultivable bacteria are *Pseudomonas*, *Sphingomonas*, *Streptomyces*,

*Rhodococcus*, and *Bacillus*, while *Dioszegia*, *Udeniomyces*, and *Cryptococcus* are dominant genera for cultivable yeasts [5]. Metagenomics and other DNA-based analyses confirmed the predominance of Proteobacteria, Actinobacteria, and Firmicutes [2, 6]. Recent metatranscriptomics data showed that Proteobacteria are the most active in

Microorganisms have long been considered as inert particles traveling in the atmosphere; however, the discovery of their metabolic activity suggested they could play a role in atmospheric chemistry and in the microphysics of clouds [1, 2, 7].

Concerning atmospheric microphysics, one of the most difficult scientific problems today is to improve the fundamental understanding and prediction of cloud formation in the atmosphere. Recent papers have highlighted the role of surfactants in atmospheric particles, a role predicted by theory 80 years ago but denied by the scientific community for decades [8, 9]. The group of Barbara Nozière extracted organic compounds from atmospheric aerosols that were able to lower the surface

These very low values suggested the presence of biosurfactants, and these surfaceactive agents are of microbial origin and are extremely efficient compared to classical surfactants [15–17]. They are amphiphilic with a lipid tail (hydrophobic) and a sugar or peptide moiety (hydrophilic). Although their chemical composition is extremely diverse, they can be classified in two main categories based on their molecular mass [15, 18, 19]: (1) small biosurfactants (PM < 1000 amu) including glycolipids (rhamnolipids, trehalolipids, sophorolipids etc.) and lipopeptides (viscosin, surfactin,

polymyxin, syringomycin etc.) and (2) polymeric structures (PM 106

droplets depends on surface tension according to Köhler's theory [20].

polysaccharides, proteins, liposaccharides, lipoproteins (alasan, emulsan etc.). Biosurfactants could affect atmospheric microphysics by modifying cloud condensation nuclei (CCN) activation. Owing to their exceptional scope in reducing surface tension, these surface-active compounds are thus likely to enhance the propensity of the aerosols to form clouds, as the activation of particles into cloud

The discovery of the presence of biosurfactants on aerosols raised a new scientific hypothesis: could these biosurfactants be produced by airborne microorganisms? Traditionally, biosurfactant-producing microorganisms were mainly isolated in environments such as soils, seawaters, and sediments contaminated or not by petroleum products [21–24]. Biosurfactants producers can also be isolated from natural sources including fruits, leaves, honey, sugarcane, insects, marine sponges etc. [24]. More recently, extreme environments were described as sources of biosurfactants, microbial producers were isolated from desert and arid soils or from the cryosphere (polar soils and lakes) [24, 25]. The first report concerning the atmospheric environment was made by Ahern [26]. This team showed that 70 fluorescent *Pseudomonas* strains isolated from cloud and rain waters in Scotland were producing biosurfactants; among them, 43 isolates were high producers. More recently, our group screened 480 microbial strains isolated from cloud water collected at the Puy de Dôme station [27]. This microbial collection was composed mainly of Gammaproteobacteria (23.3%), with a majority of *Pseudomonas*; Alphaproteobacteria (19.8%), with a majority of *Sphingomonas*; Actinobacteria (24.2%); and Basidiomycota (19.6%). Using the pending drop method, we measured the decrease of the surface tension of water droplets (12 μL in volume) induced by the addition of crude culture medium of the different strains. Up to 41% of the tested strains were producing biosurfactants (σ ≤ 55

), 7% of them (*Pseudomonas* and *Xanthomonas* strains) were very active

for concentrations 5 or 6 orders of magnitude lower

amu) such as

**74**

mN.m<sup>−</sup><sup>1</sup>

producers (σ ≤ 30 mN.m<sup>−</sup><sup>1</sup>

).

These results show that the biodiversity present in clouds and rain can be a very interesting, still unexplored, source of biosurfactants. As atmospheric environments are cold habitats, these results confirm that cold-adapted organisms are good candidates to produce biosurfactants [25].

Biosurfactants may constitute very valuable compounds of industrial interest as they are promising substitutes for synthetic surfactants with higher biodegradability and lower toxicity. They reach such low surface tensions, even for trace concentrations. Typical desirable properties include solubility enhancement, surface tension reduction, and low critical micelle concentrations, higher foaming, higher selectivity, and specific gravity at extreme temperature, pH, and salinity. In terms of economics, biosurfactants can be synthetized from a renewable stock; however, large-scale production remains challenging [28, 29]. The enormous diversity of biosurfactants also makes them an interesting group of materials for application in many areas such as agriculture, public health, food, health care, medicine, cleaning, textiles, nanotechnologies, waste utilization, and environmental pollution control such as in degradation of hydrocarbons present in soil or extraction of heavy metals [15, 16, 18, 19, 28, 30–35].

Considering the potential industrial interest of biosurfactants, we decided to go further in investigating our unique collection of microbial strains isolated from clouds as a source of biosurfactants. The objective of this work was thus to isolate biosurfactants produced by some of the best producers as determined from our previous screening in order to study their structure and their critical micelle concentration (CMC) properties. In addition, we were interested in modeling their conformation at the water-air interface to understand better their behavior in making cloud droplets. To reach these goals, we selected three strains isolated from clouds (*Pseudomonas* sp. PDD-14b-2, *Xanthomonas campestris* PDD-32b-52, and *Pseudomonas syringae* PDD-32b-74) that were high biosurfactant producers [27].
