Two schemes for production of biosurfactant from Pseudomonas aeruginosa MR01: Applying residues from soybean oil industry and silica sol–gel immobilized cells

https://doi.org/10.1016/j.colsurfb.2017.01.024Get rights and content

Highlights

  • Pseudomonas aeruginosa MR01 was used to study on economic production of biosurfactant.

  • Different wastes of soybean oil refinery lead to similar rhamnolipid homologues.

  • Sol–gel technique was used to entrap the P. aeruginosa cells in mold silica gels.

  • Pseudomonas aeruginosa MR01 was entrapped by silica gel for biosurfactant production.

  • Silica gel entrapped cells showed the efficient reusability and activity preservation.

Abstract

Rhamnolipids are the most common biosurfactants and P. aeruginosa strains are the most frequently studied microorganisms for the production of rhamnolipids. Eco-friendly advantages and promising applications of rhamnolipids in various industries are the major reasons for pursuing the economic production of these biosurfactants. This study shows that cultivation of P. aeruginosa MR01 in medium contained inexpensive soybean oil refinery wastes which exhibited similar levels and homologues of rhamnolipids. Mass spectrometry indicated that the Rha-C10-C10 and Rha-Rha-C10-C10 constitute the main rhamnolipids in different cultures of MR01 including one of oil carbon source analogues. Moreover, rhamnolipid mixtures extracted from different cultures showed critical micelle concentrations (CMC) in the range of ≃24 to ≃36 mg/l with capability to reduce the surface tension of aqueous solution from 72 to ≃27–32 mN/m. However, the sol–gel technique using tetraethyl orthosilicate (TEOS) was used as a gentler method in order to entrap the P. aeruginosa MR01 cells in mold silica gels. Immobilized cells can be utilized several times in consecutive fermentation batches as well as in flow fermentation processes. In this way, reusability of the cells may lead to a more economical fermentation process. Approximately 90% of cell viability was retained during the silica sol-gel immobilization and ≃84% of viability of immobilized cells was preserved for 365 days of immobilization and storage of the cells in phosphate buffer at 4 °C and 25 °C. Moreover, mold gels showed good mechanical stability during the seven successive fermentation batches and the entrapped cells were able to efficiently preserve their biosurfactant-producing potential.

Introduction

Biosurfactants are the biocompatible surface active molecules that are produced by several living cells, particularly by microorganisms. However, the high cost of biosurfactant production is the greatest deterrent for their use in large industries [1]. Therefore, any successful alteration, however small, may improve the efficiency and economy of process. The end production cost of a product is one of the determining factors for industry owners when making important production decisions. Provided that the cost of producing the biosurfactants becomes competitive with chemical surfactants, industries which deal with a mass consumption of surfactant for different processes may be persuaded to replace the biosurfactants and benefit from their eco-friendly and biocompatibility features. Saving on raw material costs is a traditional way to achieve reduced end costs.

Culture medium composition and living cells are the two mainstays for production of biological surfactants and therefore economic considerations in each case can improve production economics. In this regard, our previous studies dealt with the production of rhamnolipid type surfactants from waste carbon sources, i.e. soybean oil refinery wastes, by P. aeruginosa MR01 [2]. Similarly, the effect of fatty acid composition of wastes on rhamnolipid structure and surfactant activity of biosurfactants is also examined in the present work.

Moreover, the immobilization of P. aeruginosa MR01 is studied in order to improve reusability resulting in cost savings in biosurfactant production. Cell immobilization is considered the second technique for economical production due to the fact that there is no need to restock microorganism cells. Immobilization of microorganisms for industrial applications first described by Chibata et al. [3] continues to be employed for different bioprocesses. High cell density, high stability, absence of cell washout and extended reaction times are some of the advantages of cell immobilization [4]. Immobilized bacteria can remain viable for a long period of time, appear resistant to harsh culture conditions and can be reused a number of times which makes cell immobilization a promising alternative for free cells in flow bioreactors [5].

The sol–gel technique has emerged as an interesting alternative among the numerous immobilization methods because it allows materials to be obtained with more desirable chemical and mechanical features [6], [7]. In recent decades, sol–gel encapsulation techniques have been applied for the immobilization of bacteria [8], mammalian cells [9], biomolecules and enzymes [10] and other biological agents for different applications. Among different encapsulation materials, silica matrices have shown many advantageous traits such as high porosity, desired mechanical strength, high chemical and thermal stability [11], [12] as well as non-toxicity and eco-friendliness [10], [13]. Moreover, the immobilization process in silica matrices involves neither high temperatures nor harsh chemical reactions. From the time Carturan et al. [14] first introduced the encapsulation of living microorganisms in sol–gel silica matrices, other researchers reported the extension of this process to other cell types [15], [16] such as bacteria, yeast, algae and mammalian cells. These were all successfully immobilized in silica matrices [9], [17], [18], [19], [20], [21].

However, the sol–gel technique often requires the use of tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS) as precursors for synthesis of silica matrices causing accumulation of relatively high concentrations of alcohol that may be harmful for the biological activity of cells (growth, metabolic activity and enzymatic activity) [22], [23]. In some cases, biocompatible additives such as glycerol, polyethylene glycol or glycine betaine were used to improve the cytocompatibility of the silica matrices for cell immobilizations [22], [24], [25], [26]. However, Meunier et al. [15] summarized the considerable progress made in cell entrapment using silica matrixes and presented silica as a promising host for cell immobilization.

In the present study, we examined the immobilization of Pseudomonas aeruginosa MR01 in sol–gel silica matrices and its application for biosurfactant production. This strain previously demonstrated good potential to produce biosurfactant under controlled conditions [1], [27], [28] and, therefore, may be considered a suitable candidate for this purpose. Herein, silica gel matrixes were synthesized with TEOS as the precursor and used for entrapping the P. aeruginosa MR01 in the presence of high molar ratio of water to alkoxy silicate. Viability and surfactant-producing ability of bacterial cells are compared before and after immobilization.

Section snippets

Chemicals and feedstocks

All chemicals were purchased from the companies Merck and Sigma and used without further purification. Reagent grade tetraethyl orthosilicate (TEOS: Si(OC2H5)4) was purchased from Sigma-Aldrich (Sigma-Aldrich Corp. St. Louis, MO, USA). KH2PO4, MgSO4, NaNO3, yeast extract, ethanol and glutaraldehyde were purchased from Merck Chemical Co. (Germany). The samples of refined soybean oil and refinement wastes including acid oil, deodorizer distillate and soapstock were provided by Behshahr Industrial

Fatty acid patterns for oily carbon sources

The gas chromatography profile of fatty acids (Table 1) reveals that two saturated fatty acids, palmitic (C16:0) and stearic acids (C18:0) and three unsaturated fatty acids, oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) acids are the highest constituents of each oil source (∼95.7 ± 0.9). This result had previously been observed by Demirbaş [36] that stearic, palmitic, oleic, linoleic and linolenic acids are the most common fatty acids found in vegetable oils. Table 1 shows approximately

Conclusion

Rhamnolipids, natural and eco-friendly surfactants which have shown many applications in a wide range of industries, are faced with economic challenges in order to be more widely commercialized. Therefore, the economic aspects of the rhamnolipid production process have been the main focus of this work. The present study attempts to demonstrate that P. aeruginosa MR01 can grow effectively in medium of soybean oil refinery wastes and produce rhamnolipids with similar surfactant features, such as

Acknowledgements

The authors gratefully acknowledge the financial support granted by the National Institute of Genetic Engineering and Biotechnology [NIGEB, Grant No. 430] as well as the Iran National Science Foundation [INSF, Grant No. 91003071] which made this research possible. They would also like to thank the Behshahr Industrial Company for providing the soybean oil wastes and other materials used in this study.

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