ISOLATION OF SURFACTANTS SYNTHESIZED BY THE PSEUDOMONAS BACTERIA AND STUDY OF THEIR PROPERTIES

Background. The important problem of biosurfactants production is biosynthesis optimization. But lack of effective isolation methods elaboration with simultaneous new costeffective products is the greatest weaknesses of existing technologies. Objective. The aim of the study is rational technology elaboration for isolation of biosurfactants obtained from strains Pseudomonas sp. PS-17 and P. fluorescens 8573. Investigation of the influence of different acids and temperatures on the efficiency of isolation of the surfactant products, study the properties of the obtained products. Determination of the possible directions of the use of supernatant obtained after precipitation of the biocomplexes (SPL) as a new inexpensive product. Methods. Rhamnolipid surfactant concentrate was precipitated from culture liquid supernatant (CLS) by acidification to pH 3-4 with acid solutions (HCl, H2SO4, H3PO4, HNO3, CH3COOH), kept at 100 C for 25 min, cooled to room temperature, centrifuged before the phase separation, the supernatant was decanted. The surface tension of the SPL was determined by du Noüy method (with a platinum ring). The emulsification index of biosurfactants was determined regarding mineral oil and sunflower oil. The RLs were isolated from the SPL by extraction with a mixture of ethyl acetate and isopropanol, their composition was determined by thin layer chromatography. Influence on plants was assessed by their morphometric parameters after presowing seed treatment. Results. The rational technology for surfactants isolation from strains Pseudomonas sp. PS-17 and P. fluorescens 8573 was developed It was shown that the suitable method of the isolation of the biosurfactants of Pseudomonas sp. PS-17 and P. fluorescens 8573 is acidic precipitation from CLS with heating. As a result, the product yield was increased by 20%, and the duration of the process was reduced. The physico-chemical properties of the SPLs after the isolation of biosurfactants from the CLS were studied. SPLs have been shown to be effective oil emulsifiers, foaming and wetting agents for various surfaces. It was shown that SPLs (at dilutions 1:10) do not exhibit phytotoxic effects and stimulate the growth of watercress. Conclusions. The new wasteless technology for Pseudomonas strains biosurfactants isolation has been proposed, which provides for the elimination of the extraction stage with solvents, as a result, the yield of the target products has been increased. Thus, the technology has economic and environmental advantages. It was shown that SPLs, being inexpensive and effective products, can be used in environmentally friendly technologies: in agriculture (for stimulation of plant growth), for remеdiation of contaminated soils, production of detergent compositions.


Introduction
Nowadays environmental technologies have become a priority, so biogenic surfactants are considered as an alternative to synthetic [1]. Among the promising producers, the bacteria of the genus Pseudomonas deserve the attention, since they synthesize the rhamnolipids (RLs) with high surface activity. According to their chemical structure, RLs are the ethers of rhamnose and fatty acids. Due to their diphylic structure, the biosurfactants are characterized by the same properties as synthetic ones: surface and interfacial tension, critical mi-celle concentration, hydrophilic-lipophilic balance, stabilization of hydrocarbon emulsions, foaming ability, etc. Rhamnolipids are able to reduce the surface tension of solutions up to 29 mN/m, emulsify a number of hydrocarbons, vegetable oils, mineral oils, petroleum, etc. In aqueous solutions, biosurfactants are adsorbed on solid surfaces, changing their adhesion and wetting. RLs specifically influence the microorganisms and viruses: they provide antimicrobial effects in higher concentrations while increasing the permeability of cell membranes in lower concentrations. These properties of biosurfactants determine their ability to enhance the ac-

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tion of other substances while using them. They can be used in agriculture (complex plant protection and nutrition), in the food, pharmaceutical and cosmetic industries. Thus, due to physicochemical properties combined with the biocompatibility the rhamnolipid surfactants can replace synthetic products in environmentally priority technologies.
Nowadays the economic accessibility of biosurfactants is limited by the cost of their synthesis, and even more by the costs of isolation and purification of the products. In addition, the knowledge of physico-chemical and biological properties of biosurfactants, which are necessary for assessing their practical potential, is not always complete [2]. The most problematic stage in the production of RLs is their isolation from the culture liquid. Even with optimized biosynthesis, the production efficiency depends heavily on rational methods of isolation of the target products. In this connection, there is a growing need for the optimization of the processes of isolation of biosurfactants. For instance, the cost of raw materials is up to 20-40% of the total cost of production [3]. To replace traditional substrates (sugar, alcohol) it is proposed to use economically viable carbon sources (industrial wastes, renewable substrates, etc.) [4,5]. Previously, soybean, sunflower oils, technical glycerola by-product of biodiesel production were used for the rhamnolipid biosynthesis [6][7][8], the mathematical methods were applied for the synthesis optimization [9][10][11]. However, it is known, that the processes of biosurfactants allocation are up to 50-80% of the total cost of their production [2]. Currently, solvents are often used for extraction of pure biosurfactants, but they do not fully extract products, and also are toxic [12]. The economic availability of rhamnolipids technology can be increased via obtaining their complexes: culture liquid supernatant (CLS), rhamnolipid biocomplex, etc. The maximum use of components of the post-fermentative culture liquid is also a rational way to the production efficiency.
The aim of the present study is the rational technology elaboration for isolation of biosurfactants obtained from strains Pseudomonas sp. PS-17 and P. fluorescens 8573. The tasks of the study: to investigate the influence of different acids and temperature conditions on the efficiency of isolation of the surfactant products of Pseudomonas strains and to study the properties of the obtained products. To determine the possible directions of the use of supernatant obtained after precipitation of the biosurfactants as a new inexpensive product.

Materials and methods
The following strains were used for the study: Pseudomonas sp. PS-17 and P. fluorescens 8573producents of extracellular biosurfactants from the collection Department of Physico-Chemistry of Fossil Fuels of L.N. Litvinenko InPOCC, NAS of Ukraine. Cultivation was carried out on a rotary shaker, 220 rpm, (WL-2000, JV Electronic, Poland), 30 C, 5 days [6]. Rhamnolipid biocomplex was precipitated from CLS [13] by acidification to pH 3-4 with acid solutions (HCl, H2SO4, H3PO4, HNO3, CH3COOH), kept at 100 C 25 min, cooled to room temperature, centrifuged (8000 rpm, 20 min) before the phase separation, the supernatant obtained after precipitation of the biosurfactants (SPL 1) was decanted. For comparison, biosurfactant was isolated from the acidified CLS upon cooling (4 C) -SPL 2. The surface tension of the SPL was determined by du Noüy ring method with a platinum ring on the Tensiometer KRÜSS K6 ("KRÜSS" Gmbh, Germany) [14], the relative concentration of biosurfactants was determined by the critical micellar dilution (CMD) [15]. The emulsification index of SPL (E24) was determined with respect to vaseline oil and sunflower oil. The RLs were isolated from the SPL by extraction with a mixture of ethyl acetateisopropanol 2:1, their composition was determined by thin layer chromatography (Merck Silica gel 60, Germany), chloroform-methanol-water 65:2:4, visualizationby 5% phosphomolybdic acid [16]. Presowing treatment of watercress seeds was carried out by soaking in SPL, sprouting, germination was determined according to State Standard of Ukraine DSTU 4138-2002 [17]. The contact angles of wetting of the SPL were determined on the cathetometer KM-8, foaming formationaccording to State Standard of Ukraine DSTU 3789-98 [18]. The experiments were repeated three times. The results were statistically processed using Microsoft Excel software by the method of average error.

Results
The new technology for surfactants isolation of the strains Pseudomonas sp. PS-17 and P. fluorescens 8573 was developed.
The CLS was acidified (to a pH of 2.5-4.0), then heated (Method 1) or cooled (Method 2), centrifuged to obtain SPL 1 and SPL 2 and a concentrate of rhamnolipid surfactant (Table 1).  It was shown that when the acidified CLS of both strains was heated, the yield of the rhamnolipid surfactant concentrate increased by 7-28%, depending on the acid nature. The maximum yield was 6.6 g/L for Pseudomonas sp. PS-17 (with HCl), for P. fluorescens 8573 -6.2 g/L (with H2SO4). The properties of the obtained SPL were studied to assess their practical potential. It was fined that 3-5 g/L of the lipids have remained in the SPL (depending on the method used) (Tables 1, 2). SPLs, obtained by acidification and followed CLS cooling (method 2), are capable of emulsifying hydrophobic substances (E24 -45-59% -for sunflower oil, 40-50% -for vaseline oil), so they can be used as emulsifying agents of various oils, fats, hydrocarbons, etc.
SPLs promotes the wetting of surfaces ( Table 2). The contact angle of wetting the geranium leaves by solutions of SPLs, obtained by the method 1 was 75, and by the method 2 -15-30 (for water -110). A similar effect was obtained for a hydrophobic surface of polytetrafluorethene (PTF). Thus, SPLs can be used in the composition of agricultural agents and other means for wetting enhancement.
For practical application of SPLs of Pseudomonas sp. PS-17 strain, their phytotoxicity was assessed with the test plant -Lepidium sativum ( Table 3).
SPLs (1:10) do not exhibit phytotoxic effect; they even stimulate the growth of Lepidium sativum: its morphometric indices were increased. The length of the shoots was on 5-18% higher than control (4 days), the length of the rootson 6-17%, similar effects were observed after 9 days and 16 days. Therefore, SPL is a low-toxic product.

Culture Liquid Supernatant
Thus, a new wasteless technology for Pseudomonas strains biosurfactants isolation has been elaborated (the Figure). The technology includes the solvent extraction stage.
The identified physico-chemical properties (emulsification, wetting, foaming) of the obtained products, as well as their low toxicity, are a real basis for their use in agricultural technologies (e.g. plant growth stimulation), the remediation of contaminated soils, in detergent compositions as it was shown in our previous works [19,20].

Discussion
Rhamnolipids are effective surface-active products synthesized by the bacteria of the genus Pseudomonas. It is known, that the processes of biosurfactants isolation make up to 50-80% of the total cost of their production [2]. In addition, physico-chemical and biological properties of biosurfactants, which are necessary for the assessment of practical potential in specific industries, are still not sufficiently characterized.
Currently, solvents are often used for extraction and purification of biosurfactants, but they do not extract products completely, and also are toxic. The economic feasibility of rhamnolipids can be increased via obtaining their complexes: culture liquid supernatant, rhamnolipid biocomplex, etc. The maximum use of components of the postfermentative culture liquid can also be a rational method to the increase in production efficiency.
In this work, the methods of rhamnolipid surfactant isolation from the CLS of Pseudomonas strains using different acids and different temperature regimes were improved. The rational parameters for the deposition of biosurfactants of Pseudo-monas strains were developed. The advisability of surfactants isolation from the CLS of Pseudomonas sp. PS-17 and P. fluorescens 8573 by acid precipitation with obtaining of biosurfactant precipitate and supernatant was established. The optimum mode of acid precipitation of surfactants was determinedheating of the CLS to 100 C for 25 min. As a result, the yield of the rhamnolipid surfactant concentrate was increased up to 20%. Thus, the advantages of the developed method are the time saving, energy costs reduction due to the absence of the cooling stage of the acidified CLS as well as an increase in the yield of the product.
The physico-chemical properties of SPL (after the precipitation of biosurfactants from CLS of the Pseudomonas strains) for the evaluation of their practical potential were investigated. SPLs, obtained by acidification and followed cooling, are capable of emulsifying hydrophobic substances (various oils, fats, hydrocarbons).
The differences in emulsifying and foaming properties of SPL, obtained by various methods, are explained by a lower concentration of surfactants in SPL, obtained with heating, due to their more complete precipitation. Obviously, SPL obtained by this method are promising for composite detergents (with other surfactants), in cosmeticsfor emulsifying oils and fats, etc. SPL obtained with cooling can also be used for the remediation of oilcontaminated soils (due to emulsifying properties). The wetting properties of SPL are important for the removal of oily stains, soot, and metallic dust from hydrophobic surfaces. Moisturizing properties of SPL also can be used in the pharmaceutical, cosmetic means. It is also shown that SPL (in dilutions of 1:10 and more) do not show a phytotoxic effect, but even stimulate the growth of watercress.
Thus, a new wasteless technology for Pseudomonas strains biosurfactants isolation has been proposed. This technology has economic and environmental advantages.
Thanks to the strategic planning technique used to identify strengths, weaknesses, opportunities, and threats related to the obtained biosurfactant products (SWOT-analysis) [23] it has been shown that their advantages over synthetic ones are high efficiency, heat resistance, non-toxicity, biocompatibility and biodegradability (Table 4). Renewable raw materials including production waste can be used for the biosynthesis. It is shown that their predicted cost is 70-180 USD/kg depending on the concentration, whereas for commercial biosurfactant (Urumqi Unite Bio-Technology, AGAE Technologies) -320-1600 USD/kg [24]. Thus, the unique physicochemical and biological properties of the obtained substances, their low toxicity, and the mild effect on the cells of microorganisms and plants are a powerful argument for their use as environmentally safe products in modern industrial and agricultural technologies. So, thanks to polyfunctional properties and environmental safety, the obtained surfactants of Pseudomonas strains will be competitive in the modern market as independent products and components of complex products for modern industrial and agricultural technologies.

Conclusions
The rational technology for Pseudomonas strains biosurfactants isolation has been proposed. It was shown that an effective way to isolate biosurfactants Pseudomonas sp. PS-17 and P. fluorescens 8573 is acidic precipitation from CLS with heating. As a result, the yield of the biosurfactant concentrate is increased by 20%, and the duration of the process is reduced.
The physic-chemical properties of SPL after isolating the rhamnolipid concentrate from the culture liquid supernatant were studied. SPLs have been shown to be effective oil emulsifiers, foaming and wetting agents for various surfaces.
It was found that SPL (at 1:10 dilutions) do not show phytotoxic effects and stimulate the growth of watercress.
SPL as a cheap effective product can be used in environmentally friendly technologies: for agriculture, remediation of contaminated soils, detergents, etc.