Detergents: Types, Components and Uses : Types, Components and Uses [1 ed.] 9781617282416, 9781616689865

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Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Detergents: Types, Components and Uses : Types, Components and Uses, Nova Science Publishers, Incorporated, 2010.

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Detergents: Types, Components and Uses : Types, Components and Uses, Nova Science Publishers, Incorporated, 2010.

BIOCHEMISTRY RESEARCH TRENDS

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DETERGENTS: TYPES, COMPONENTS AND USES

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DETERGENTS: TYPES, COMPONENTS AND USES

EMILIE T. HAGEN EDITOR

Nova Science Publishers, Inc. New York

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Copyright © 2010 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.

LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Detergents : types, components, and uses / [edited by] Emilie T. Hagen. p. cm. Includes index. ISBN 978-1-61728-241-6 1.Detergents. I. Hagen, Emilie T. TP992.5.D38 2009 668'.14--dc22

Published by Nova Science Publishers, Inc.  New York

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2010014088

CONTENTS Preface Chapter 1

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Chapter 2

vii Biosurfactants and Their Uses in the Petroleum Industry and Hydrocarbon Pollution Remediation N. L. Olivera and M. L. Nievas Detergents in Molecular Biology: DNA Extraction and Purification Christian Alberto García-Sepúlveda, Sandra Elizabeth Guerra-Palomares and Diana Lorena Alvarado-Hernández

Chapter 3

Carbohydrases in Detergents Piamsook Pongsawasdi and Shuichiro Murakami

Chapter 4

The Influence of Different Detergents on the Preservation of Discrete Membrane Microdomains: A Comparative Ultrastructural Study on Whole-Mounted Colorectal Cancer Cells Kristina A. Jahn, Yingying Su, Tessa Smissaert van de Haere, Iris Benjamins and Filip Braet

Chapter 5

-Sulfo Fatty Methyl Ester Sulfonates ( -MES): A New Anionic Surfactant León Cohen, Fernando Soto, Francisco Trujillo, David W. Roberts and Claudio Pratesi

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49

71

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vi Chapter 6

Contents Acacia Caven (Mol.) Molina Pollen Proteases: Application to the Peptide Synthesis and to Laundry Detergents Cristina Barcia, Evelina Quiroga, Carlos Ardanaz, Gustavo Quiroga and Sonia Barberis

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Index

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PREFACE This new book reviews research in the field of detergent types, their components and uses. Discussed herein are biosurfactants and their uses in the petroleum industry and in hydrocarbon pollution remediation; detergent-based DNA extraction techniques in molecular biology; enzyme usage as an active ingredient in detergents and the influence of different detergents on the preservation of the cytoskeleton and detergent-resistant membranes on wholemounted cells. In this compilation, also explored, is the study on the synthesis, separation, analysis and performance of sulfo fatty methyl ester sulfonates as new anionic surfactants. Chapter 1 - Biosurfactants are amphipathic molecules which have the capability to reduce the medium surface tension. At low concentrations, surfactants in aqueous solution exist as monomers. As the surfactant concentration increases, the medium surface tension decreases. Above a threshold concentration known as the critical micelle concentration (CMC), biosurfactant molecules form micelles with a hydrophobic core and a hydrophilic surface. CMC values are characteristic of each biosurfactant, depending on the chemical structure and composition of the biosurfactant as well as the solution conditions (e.g. temperature, ionic strength, additives). There is an important diversity in chemical structures and functions among natural surfactants. A distinction between low and high-molecular-weight biosurfactants is accepted, the former are generally glycolipids or lipopeptides with surface activity, while the latter are exocellular polymeric substances with important emulsifying properties. This chapter focuses on microbial biosurfactants that on the context of degradation of lipophilic compounds, such as hydrocarbons, may enhance their bioavailability to microorganisms, either by increasing the apparent hydrocarbon solubility in the aqueous phase or by increasing the contact surface area due to emulsification. In

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Emilie T. Hagen

this regard, different biosurfactants have been applied to enhance the biodegradation of oily residues and to fight against oil pollution. In addition, biosurfactants possess the advantage of being biodegradable molecules which encourages its use in the development of bioremediation strategies. In the petroleum industry, biosurfactants also find application in MEOR (microbial enhanced oil recovery) improving hydrocarbon mobilization and consequently the recovery of crude oil from reservoirs. Chapter 2 - Over the last 20 years, epidemiologist have shifted their attention from the analysis of phenotypic features to evaluating the way genetic traits determine disease causation and severity. This trend has clearly benefitted from the coming of age of versatile molecular based techniques such as the Polymerase Chain Reaction (PCR) and the now widespread availability of high-throughput molecular screening assays. In this context, deoxyribonucleic acid (DNA) has rapidly become the biospecimen of choice as it is readily obtained, transported, processed and stored. Both molecular epidemiology and genomic characterization studies require the typing of large numbers of specimens in order to achieve statistical significance and enormous advances, such as microarray technology, have resulted as a consequence of this. Nevertheless a usually overlooked yet crucial aspect of nucleic acid based typing is the DNA extraction process. DNA extraction is the process by which nucleic acids are retrieved from their biological compartments and deprived of proteins, salts and other substances that might hinder their analysis. In their natural state nucleic acids are compartmentalized inside the cell nucleus or mitochondrion and protected by proteins and a lipid envelope. In order to extract these nucleic acids cells must be burst open and the contaminating proteins and lipids eliminated. Detergents of essentially every type have been used to disaggregate the lipid components of cell membranes and are common components of essentially all DNA extraction methods. In this chapter a brief historical review of the different DNA extraction techniques that have been developed is made followed by a detailed presentation of the most relevant applications of detergent-based DNA extraction techniques. Finally, we describe our experience at implementing and optimizing a simple and low-cost laundrydetergent-based DNA extraction method for the extraction of high-quality human genomic DNA from peripheral blood samples and the many uses that such material has been put to use for. Chapter 3 - The chapter starts with an introduction on detergents and the detergent industry. The history and development of enzyme usage as an active ingredient in detergents for laundry, dishes and other household products are listed. The importance and the main characteristics of enzymes that are required so as to be suitable for use in detergents are described. The properties and

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Preface

ix

functions of hydrolases (proteases, carbohydrases and lipases) that are beneficial for the detergent industry are overviewed. Then the carbohydrases that have specific functions in detergents are focused upon, especially α-amylase, cellulase and mannanase. For each enzyme type, the catalytic reaction, sources and production of the enzyme, enzyme specificity, biochemical characteristics, function and development of the enzyme for desired properties through protein engineering, are discussed. The work of our group on a new highly alkaline, thermostable α-amylase that is resistant to certain surfactant and bleaching reagents commomly used as additives to detergents, and so has a good potential in the detergent industry, is presented. The commercial carbohydrases that have been used in detergent industry are reviewed. Finally, this chapter ends with reviewing some of the future prospects on the use of enzymes in detergents. Chapter 4 - The cellular plasma membrane is partially resistant to solubilisation with mild and non-denaturing detergents allowing detergentresistant membranes (DRMs) to be isolated and biochemically analysed. The composition of DRMs however varies depending on the detergent applied. We previously developed a novel method to visualise DRMs on whole-mount cancer cells in which the soluble fraction of the plasma membrane is removed and the cytoskeleton is fixed and prepared for scanning electron microscopy. This method is less destructive than many others commonly used to study DRMs and it enables the direct visualisation of intact, cytoskeleton-associated DRMs. In this chapter we compare five commonly used detergents (TX-100, Brij96, CHAPS, Lubrol WX and OG) with respect to the achieved membrane solubilisation at different concentrations. Our results indicate that TX-100, Brij96 and CHAPS isolate similar DRMs whereas OG fully solubilised the plasma membrane. Interestingly, Lubrol WX poorly extracted cells even at high concentrations. This is the first ultrastructural study that systematically describes and visualises the effects of different detergents on the preservation of the cytoskeleton and DRMs on wholemounted cells. This approach delivers new and valuable ultrastructural information at the nanometer scale that did not exist to date. Chapter 5 - -Sulfo Fatty Methyl Esters sulfonates ( - MES) are new anionic surfactants obtained via sulfoxidation of fatty acid methyl esters (FAME) with SO2, O2, and ultraviolet light of appropriate wavelength. The designation of Φ refers to the random positioning of SO3 in the alkyl chain. In this work we summarize the most relevant results of our research started fifteen years ago, on the synthesis, separation, analysis and performance of sulfo fatty methyl ester sulfonates known as -MES, and we update our last findings . We have to point out that we are for the time being, the only research group in the world to have published our research on -MES.

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Emilie T. Hagen

This work describes the optimum batch conditions for the sulfoxidation of FAME and a reaction mechanism is proposed. The paper depicts an improved workup for the separation of reaction products from non reacted methyl ester and the GC-MS analysis of - sulfo fatty methyl ester sulfonate is shown. Besides, an interpretation of conversion and selectivity of sulfoxydation reaction is given. Finally, performance of water solutions based on sulfoxylated methyl ester of palmitic acid ( -MES C16) have been studied and compared to two leading types of surfactants used today: linear alkylbezene sulfonate (LAS) secondary alkane sulfonate (SAS) and to -sulfo fatty methyl ester sulfonate ( -MES) with regard to solubility, performance and skin compatibilty. The experimental results obtained indicate that -MES can be regarded as a potential component of detergent formulations and most likely of body care products. Chapter 6 - It is known that the proteases have applications in several industrial processes such us leather processing, laundry detergents, producing of protein hydrolysates and food processing, as well as in the peptide synthesis in non conventional media. The application of proteases as catalyst of short oligopeptides in aqueous-organic media, have received a great deal attention as a viable alternative to chemical approach because of their remarkable characteristics. On the other hand, alkaline proteases have also been used to improve the cleaning efficiency of detergents. Detergent enzymes account for about 30% of the total worldwide enzyme production and represent one of the largest and most successful applications of modern industrial biotechnology. The aim of this work was to study the performance of proteolytic enzymes of Acacia caven (Mol.) Molina pollen for its potential application as an additive in various laundry detergents formulations and as catalyst of the peptide synthesis in aqueous-organic media. Pollen grains (35 mg/ml) were suspended in 0.1M TrisHCl buffer pH 7.4 and slowly shaken for 2 h at 25 C. Then, the slurry was centrifugated for 30 min at 8000 rpm and the supernatant (crude enzyme extract, CE) was tested in protein content (Bradford’s method) and proteolytic activity (using BAPNA and Z-Ala-pNO as substrates). A partial characterization of Acacia caven CE was carried out: enzyme extract displayed maximum proteolytic activity at pH 8 and 35-40º C; it showed remarkable thermal stability after 1.5 h at 25-40º C but it decreased as long as temperature increased to 60º C. On the other hand, the enzyme extract was incubated with different surfactants and commercial laundry detergents at 25-60° C during 30 min and 1h; and it showed high stability and compatibility with them. The peptide synthesis catalyzed by Acacia caven CE was carried out in a mixture of 0.1M Tris-HCl buffer pH 8.5 and ethyl acetate (50:50 ratio) at 37° C using 2-mercaptoethanol as activator and TEA as neutralizing agent of the amino component (Phe-OMe.HCl). Carboxylic

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components were selected in base of the highest preference of CE. The identification of synthesized peptide products was carried out by HPLC-MS. According to the obtained results, this work contributes with a new variety of phytoprotease useful as catalyst of the peptide synthesis and as additive of laundry detergents.

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In: Detergents: Types, Components and Uses ISBN: 978-1-61668-986-5 Editor: Emilie T. Hagen © 2010 Nova Science Publishers, Inc.

Chapter 1

BIOSURFACTANTS AND THEIR USES IN THE PETROLEUM INDUSTRY AND HYDROCARBON POLLUTION REMEDIATION N. L. Olivera* and M.L. Nievas

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Centro Nacional Patagónico (CONICET), Blvd. Brown 2915, U9120ACF, Puerto Madryn, Chubut, Argentina

ABSTRACT Biosurfactants are amphipathic molecules which have the capability to reduce the medium surface tension. At low concentrations, surfactants in aqueous solution exist as monomers. As the surfactant concentration increases, the medium surface tension decreases. Above a threshold concentration known as the critical micelle concentration (CMC), biosurfactant molecules form micelles with a hydrophobic core and a hydrophilic surface. CMC values are characteristic of each biosurfactant, depending on the chemical structure and composition of the biosurfactant as well as the solution conditions (e.g. temperature, ionic strength, additives). There is an important diversity in chemical structures and functions among natural surfactants. A distinction between low and highmolecular-weight biosurfactants is accepted, the former are generally glycolipids or lipopeptides with surface activity, while the latter are *

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N. L. Olivera and M. L. Nievas exocellular polymeric substances with important emulsifying properties. This chapter focuses on microbial biosurfactants that on the context of degradation of lipophilic compounds, such as hydrocarbons, may enhance their bioavailability to microorganisms, either by increasing the apparent hydrocarbon solubility in the aqueous phase or by increasing the contact surface area due to emulsification. In this regard, different biosurfactants have been applied to enhance the biodegradation of oily residues and to fight against oil pollution. In addition, biosurfactants possess the advantage of being biodegradable molecules which encourages its use in the development of bioremediation strategies. In the petroleum industry, biosurfactants also find application in MEOR (microbial enhanced oil recovery) improving hydrocarbon mobilization and consequently the recovery of crude oil from reservoirs.

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1. INTRODUCTION Surfactants are amphipathic compounds that in solution, particularly in aqueous solution, tends to congregate at the boundaries of surfaces, such as the water-air interface, the walls of a recipient, or the liquid-liquid interface if a second immiscible liquid phase is present (Cullum, 1994). The surfactants used in the world comprise of many different substances, traditionally manufactured by the chemical industry from petroleum or its derivatives. These compounds are generally called “synthetic” or “chemical” surfactants, and their application spectrum includes industries as diverse as petrochemical, chemical, cosmetic, food, pharmaceutical, and environmental protection, in addition to their extended uses as domestic and industrial cleaners. Surfactants from biological sources or biosurfactants are compounds with a wide diversity of chemical structures and natural roles. In particular, microbial surfactants may increase the bioavailability of hydrophobic substrates, regulate the attachment-detachment of microorganisms to surfaces, promote biofilm formation, and exert antibacterial or antifungal activities (Ron and Rosenberg, 2001; Van Hamme et al., 2006). In previous decades, scientific and industrial interest in biosurfactants increased due to their novel intrinsic properties (Van Bogaert et al., 2007; Kitamoto et al., 2009) and the possibility of replacing some of the synthetic surfactant market. With its biological origin, biosurfactants are considered biodegradable and non-toxic substances (Rosenberg, 1986; Lin, 1996), which is especially advantageous for environmental applications (Desai and Banat, 1997).

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In this chapter, we focus on microbial biosurfactants, describing their main characteristics, chemical composition, as well as their potential and current applications associated to hydrocarbon pollution remediation and to the petroleum industry.

2. BASIC CONCEPTS

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The main characteristic of biosurfactants is the surface activity; “surfactant” is the contraction of the term surface-active agent. Thus they modify interfaces in multiphase systems, lowering the surface tension of the solutions or the interfacial surface tension in biphasic liquid systems (Rosen, 2004; Cullum, 1994). Biosurfactants share several properties with chemical surfactants. In this section, some basic concepts applying to both of them will be introduced. These characteristics are key features in biosurfactant application because they determine the behaviour of biosurfactants, and in consequence their usefulness for different applications.

hydrophilic moiety

hydrophobic moiety

Figure 1. Molecular structure and schematic configuration of hydrophobic and hydrophilic moieties of the biosurfactant surfactin produced by Bacillus subtilis O9

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2.1. Biosurfactant Nature As surfactants are amphipathic compounds, their molecules present two distinct parts, one hydrophilic and the other hydrophobic (Rosen, 2004). The chemical structure of the biosurfactant surfactin is shown in Figure 1 to illustrate this characteristic. Both synthetic and biological surfactants have an important diversity in the nature of their hydrophilic and hydrophobic groups. In general, the hydrophilic moiety contains anionic, cationic, non-ionic or amphoteric chemical structures, while the hydrophobic moiety consists usually of fatty acids.

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2.2. Surface and Interfacial Tension The surface tension is the measurement of the interfacial free energy per unit area between two fluid phases. The interfacial free energy is the minimum amount of work required to create a given interface between two fluid phases (Rosen, 2004). Generally, the term surface tension (ST) is used to denote the measurement at the liquid-air interface, while the term interfacial tension (IFT) is applied to the surface tension between two liquid phases at the interfacial area. Consequently, the ST represents the minimum amount of work required to create a unit area of a given interface or to expand it by a unit area. It also measures the different nature of the fluid phases; the ST increases with the dissimilarity nature of the fluids (Rosen, 2004). For example, the pure water ST at 25°C is 72.7 mN m-1 (ST between water and air), while the IFT of nhexadecane-water is about 53.5 mN m-1 (Perry, 1999; Dongmin and Hornof, 1999). The lower the IFT between two liquids is, the easier an interfacial area could be formed (e.g. in an emulsion formation). On the other hand, the lower the liquid ST is, the higher the liquid wettability on a given surface will be.

2.3. Reduction of Surface and Interfacial Tension by Biosurfactants and Critical Micelle Concentration Like chemical surfactants, biosurfactants in aqueous solutions reduce the ST at the water-air interface. This effect is caused by surface interactions of surfactants that decrease the cohesion force among the molecules of the liquid,

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75

65

Surface tension (mN/m)

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diminishing the ST and causing, at the same time, an increase in the liquid capability to bond to surfaces (Rosen, 2004). Figure 2 shows the behaviour of the ST measured as a function of the surfactin concentration in aqueous solution. At low concentrations, biosurfactant molecules are as monomers (Edwards et al., 1991). As the biosurfactant concentration increases, the ST decreases (Figure. 2). When the ST is plotted against the logarithm of the biosurfactant concentration, two straight lines are obtained, with a little curvature in the area where the lines crossed. Fitting the data, the interception of these lines can be found. This point corresponds to the critical micelle concentration (CMC) or critical aggregate concentration (CAC), the threshold concentration above which the biosurfactant molecules form micelles or aggregates with a hydrophobic core and a hydrophilic surface. Only the biosurfactant monomeric form is efficient in lowering the ST and the minimal ST will be registered at the CMC or CAC concentration. Further increases in biosurfactant concentration will not significantly affect the ST because the biosurfactant will be in the aggregate form (Volkering et al., 1998; Edwards et al., 1991). The ST that corresponds to the CMC (STCMC) and the CMC are two characteristic parameters of biosurfactants, which greatly depend on the composition and the chemical structure (Marqués et al., 2009).

55

45

35

25 1

10

100

1000

10000

Surfactin concentration (mg/l)

Figure 2. CMC determination of surfactin produced by B. subtilis O9

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N. L. Olivera and M. L. Nievas

Other important characteristics of surfactants are the aggregation number and the self-assembly structure. The former represents the number of molecules that form an aggregate structure. The self-assembly structure is the way in which the surfactant molecules arrange and includes different forms such as spherical micelles and lamellar structures (Rosen, 2004; Kitamoto et al., 2009). In previous years, the fast growing knowledge about biosurfactants has led to the description of new aggregates such as sponge, vesicles, and cubic phases of complex liquid crystals (Kitamoto et al., 2009; Dahrazma et al., 2008; Marqués et al., 2009). The aggregation number and the selfassembly structure are typical properties of a given biosurfactant, since they depend on the biosurfactant molecular structure and concentration, as well as the environmental conditions in which it is immersed. The medium conditions (e.g. temperature, pH, ionic strength, presence of stabilizer cations) also affect the value of CMC or CAC. In addition, such conditions not only influence the concentration above which the biosurfactant aggregate in structures, but also the own micelle conformation. Dahrazma et al. (2008) observed changes in the size and structure of biosurfactant aggregates (rhamnolipid), which augmented their size almost three times (up to 200 nm) when the pH values shifted from acid to alkaline (pH=13.0). Moreover, transitions between lamellar to hexagonal phases have been described in response to an increase of the medium NaCl concentration (Helvaci et al., 2004), or to changes in the biosurfactant concentration (Marqués et al., 2009).

2.4. Solubilization Capability Surfactants can increase the apparent solubility of hydrophobic compounds by pseudosolubilizing small droplets of them in the hydrophobic core of their micelles or aggregates. As well, the chemical composition and conformation of a biosurfactant influence its affinity for a given substrate, determining the mass ratio between the solute and the biosurfactant in the aggregates. This mass ratio indicates the biosurfactant capability to transport or mobilize a hydrophobic compound. The molar solubilization ratio (MSR) is the relative amount of moles of a hydrophobic compound that can be solubilized within the aggregates of one mol of surfactant, when its concentration is above the CMC or CAC. Analogously, the weight solubilization ratio (WSR) expresses a similar relative value, but the weight of each compound is considered instead of the number of moles. These two ratios are often used to define the effectiveness of

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a particular surfactant in the solubilization of a given solute (Edwards et al., 1991). The WSR is described by the following equation:

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WSR

Sx,mic Sx,CMC Csurf CMC

where Sx,mic is the total apparent solubility of the solute x at a surfactant concentration Csurf above the CMC, and Sx,CMC is the total apparent solubility of the solute x at the CMC (all the concentrations expressed as w/v). This equation results in the MSR when the molar concentrations (instead of the weight concentrations) are used in the calculation. From dissolution experiments, the MSR or the WSR can be obtained for a particular biosurfactant-hydrophobic compound combination (Edwards et al., 1991; Barkay et al., 1999). The total amount of solubilized hydrophobic solute (in aqueous solution + in aggregate form) is determined as a function of the surfactant concentration. An example of this procedure is shown in Figure 3, where the capability to solubilize phenanthrene of Bacillus subtilis O9 surfactin was assessed. In this experiment, above the CMC, a linear relationship between the biosurfactant concentration and the apparent solubility of the solute was obtained. WRS was calculated from the slope of the linear section of the graph. On the other hand, below the CMC, surfactants have usually negligible effect in solubilizing hydrophobic compounds (Edwards et al., 1991).

2.5. Emulsion Stabilization An emulsion is a dispersion of fluid droplets in another immiscible liquid. When surfactants situate at the interface between the liquids avoid the coalescence of the droplets (Rosen, 2004). However, not all the surfactants promote stable emulsification as emulsions may coalesce rapidly (Pekdemir et al.; 2005, Fingas and Fieldhouse, 2003). Some biosurfactants, usually called bioemulsifiers, show the ability to stabilize emulsions for long periods. The specific properties of the hydrophilic and hydrophobic phases along with the biosurfactant structure greatly condition the emulsion stability (Fukuoka et al., 2007; Fingas and Fieldhouse, 2003). Two main kinds of emulsions are known, oil in water (O/W) and water in oil (W/O). In O/W emulsions, the organic phase is dispersed and the water phase is in a continuous form. By contrast in

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W/O emulsions, the water droplets are stabilized in an oil continuous phase (Zhang and Que, 2008; Fingas and Fieldhouse, 2003; Pekdemir et al., 2005). Some surfactants form O/W or W/O emulsions depending on the proportion of the components involved in the mixture. This information is often summarized in ternary phase diagrams, in which the type of emulsion formed (O/W or W/O) is a function of the relative proportions of the water/hydrophobic/biosurfactant components (Marqués et al., 2009; Ábalos et al., 2004; Kitamoto et al., 2009; Worakitkanchanakul et al., 2009). The hydrophilic-lipophilic balance or HLB number is a semi-empirical parameter originally proposed by Griffin (1954) for non-ionic surfactants, as a guide for the selection of synthetic emulsifiers (Rosen, 2004). This number represents a way of predicting the emulsion type (O/W or W/O) that a surfactant can form according to its molecular composition. HLB varies in an arbitrary range from 0 to 20 and describes the relative contribution of the hydrophilic moiety to the weight of the surfactant molecule (Volkering et al., 1998). For non-ionic surfactants the equation is:

HLB

Mw 20 M

200

400

Phenanthrene solubilized (mg/l)

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4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -200

0

600

800

1000

1200

Surfactin concentration (mg/l)

Figure 3. Apparent phenanthrene solubility in aqueous solution with surfactin produced by B. subtilis O9. From the slope of the plot at values higher than the CMC, a WSR of 0.0027 is obtained

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where Mw is the molecular weight of the biosurfactant hydrophilic moiety and M the biosurfactant molecular weight. Other equations have been proposed for different types of surfactants (Pasquali et al., 2008; Fukuoka et al., 2007). Surfactants with low HLB numbers (around 4-7) are lipophilic and form W/O emulsions, whereas surfactants with high HLB numbers (around 9-18) are more hydrophilic and the formation of O/W emulsions can be expected (Zhang and Que, 2008; Volkering et al., 1998). Although the HLB number does not strictly reflect a physical property of the surfactant, it is useful as an indication of the expected emulsion type. Nevertheless, this parameter does not offer an accurate idea about the effectiveness of a surfactant to form an emulsion (weight ratio), neither of its stability during the time (Rosen, 2004).

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3. CLASSIFICATION OF BIOSURFACTANTS Synthetic surfactants are traditionally classified by the nature of the hydrophilic part of the molecule as anionic, cationic, non-ionic or amphoteric (Rosen, 2004; Cullum, 1994). Such classification is not usually used to describe biosurfactants, for which other features such as the molecular weight, the chemical composition of the biomolecule, the extent of the surface activity, the capability to form stable emulsions and the source are considered. Regarding microbial surfactants, a distinction between low and highmolecular-weigh compounds is generally accepted (Rosenberg and Ron, 1999; Lang, 2002; Rahman and Gakpe, 2008). The former are molecules such as glycolipids and lipopeptides with a significant surface activity, while the latter are exocellular polymeric substances with important emulsifying properties (Rosenberg and Ron, 1999; Ron and Rosenberg, 2001).

3.1. Low-Molecular-Weigh Biosurfactants This group of biosurfactants includes compounds such as glycolipids, lipopeptides, fatty acids, and phospholipids (Hommel, 1990; Lang, 2002, Mulligan, 2005; Kitamoto et al., 2009). One of the most studied biosurfactants are the glycolipids whose hydrophilic component contains sugar molecules and the hydrophobic part, long chain aliphatic acids or hydroxyl-aliphatic acids (Rosenberg, 1986). In particular, many studies have focused on the rhamnolipids produced by Pseudomonas spp. (Figure. 4a). In general,

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N. L. Olivera and M. L. Nievas

rhamnolipid biosurfactants are formed by one or two rhamnose molecules and one or two ß-hydroxy (3-hydroxy) fatty acids (Soberón-Chávez et al., 2005). Frequently, a single strain can produce a mixture of several rhamnolipids, whose predominance differs according to the culture conditions and the physiological state of the cells (Table 1). Rhamnolipds are potent bio surfactants which cause a decrease in the medium ST to about 30 mN m-1 and CMC values between 10 and 230 mg l-1 approximately (Mulligan, 2009). In addition, rhamnolipids show a high capability to form stable oil-water emulsions that traditionally are measured by an index which considers the magnitude of the emulsion formed after mechanically mixing oil and aqueous phases (Cooper and Goldenberg, 1987). As an example, emulsification indices produced by rhamnolipids of Pseudomonas aeruginosa Bs20 ranged between 59 % and 66 % against kerosene, diesel, and motor oil (Abdel-Mawgoud et al., 2009), while P. aeruginosa DAUPE 614 showed a maximum emulsification index against toluene of 86.4 % (Monteiro et al., 2007). Another remarkable characteristic of rhamnolipids is its stability to a wide pH (Yin et al., 2009), salinity (Abdel-Mawgoud et al., 2009; Yin et al., 2009) and temperature range (Abdel-Mawgoud et al., 2009). Above the CMC, biosurfactant molecules aggregate in a variety of microstructures including micelles, vesicles, and lamellas. Champion et al. (1995) observed that the morphology of rhamnolipid aggregates depends on pH, having morphology changes from lamellas, to vesicles, to micelles as pH increases. These changes of morphology also affect the dispersion and solubilization of lipophilic compounds such as hydrocarbons (Zhang and Miller, 1992; Bai et al., 1998; Shin et al., 2008). Other kinds of glycolipid biosurfactants are the sophorose lipids (Figure. 4b) produced by yeasts of the genus Candida (Hommel et al., 1994a; 1994b), the mannosylerythritol lipids produced by Pseudozyma yeasts (Morita et al., 2007; Arutchelvi et al., 2008) and the trehalose lipids (Figure. 4c) such as those characterized in Rhodococcus spp. (Rapp et al., 1979; Philp et al., 2002; Tuleva et al., 2008). Table 1 shows some examples of these biosurfactants and their properties. Alcanivorax strains, which are highly specialized hydrocarbon degraders, also produce biosurfactants (Harayama et al., 2004; Olivera et al., 2009). Alcanivorax borkumensis SK2T produces a glucose-lipid surfactant (Abraham et al., 1998; Yakimov et al., 1998). Golyshin et al. (2003) observed that cells harvested in late exponential phase produce the glucose-lipid surfactant in two forms: a glycine-containing cell-bound precursor that increases cell hydrophobicity and a glycine-lacking form that is released from the cell to the medium.

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Table 1. Low-Molecular-Weight Biosurfactants Biosurfactant

Rhamnolipids

Strain

Pseudomona s aeruginosa UW-1

Pseudomona s strain RLAT10

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Pseudomona s strain RL47T2

Pseudomona s aeruginosa DAUPE 614 Pseudomona s aeruginosa S6 Pseudoxanth o-monas sp. PNK-04 Sophorose lipids

Candida bombicola ATCC 22214 Candida bombicola

Chemical characterization

L-rhamnosyl-ß-hydroxy decanoyl-ßhydroxydecanoate L-rhamnosyl-Lrhamnosyl-ßhydroxydecanoyl-ßhydroxydecanoate Rha-Rha-C10-C10; Rha-Rha-C10-C12:1; Rha-Rha-C10-C12; Rha-C10-C12:1; RhaC10-C10; RhaC10C12:1; Rha-C10-C12; Rha-C8:1; Rha-C12:2 Rha-Rha-C8-C10; RhaRha-C8-C12.1; RhaRha-C10-C10; RhaRha-C10-C12.1; RhaRha-C10-C12; RhaRha-C12-C10; RhaRha-C12:1-C12; RhaRha-C10-C14:1; RhaC8-C10; Rha-C10-C8; Rha-C10-C10; RhaC10-C12:1; Rha-C10C12; Rha-C12-C10 6 mono-rhamnolipid homologues and 6 dirhamnolipid homologues RhaRhaC10C12:1; RhaC12:1 C10; RhaC10C10; RhaC8C10 mono- and dirhamnose units linked to ßhydroxy alkonic acids containing C8 to C12 the major constituent (81% relative abundance) of the sophorolipid mixture contains an oleoyl chain (SL-C18:1) sophorolipid mixture with a predominant compound of 716 Da

Surface activity

Reference

ST (mN m-1) 27.7-30.4

CMC (mg l-1) 40.0

26.8

150.0

Haba et al., 2003

32.8

108.8

Haba et al., 2003

27.3

13.9

Monteiro et al., 2007

33.9

50.0

Yin et al., 2009

29.0

n.d.

Nayak et al., 2009

38.0

13.0

Solaiman et al., 2004

34.1

59.4

Daverey & Pakshiraja n, 2008

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Sim et al., 1997

12

N. L. Olivera and M. L. Nievas Table 1. (Continued)

Biosurfactant

Strain

Candida batistae CBS 8550

Mannosylerythritol lipids

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Trehalose lipids

Pseudozyma antarctica JCM 10317 Rhodococcus erythropolis DSM 432 15

Rhodococcus ruber

Other glycolipids

Flavolipids

Rhodococcus wratislaviens is BN38 Burkholderia cenocepacia BSP3 Microbacteri um sp. Flavobacteri um sp. MTN11

Chemical characterization

Surface activity

Reference

ST (mN m-1) 43.2

CMC (mg l-1) 138.0

n.d.

n.d.

Morita et al., 2007

trehalose dicorynomycolate; αtrehalose as the sole non-reducing sugar; lipid moiety consists predominantly of saturated long-chain αbranched -ß-hydroxy fatty acids (mycolic acids) ranging from C32H6403 to C38H7603, of which C34H6803, and C35H7003 predominate surfactant complex consisting on 3 glycolipids, all having trehalose as the hydrophilic moiety 2,3,4,2'-trehalose tetraester

n.d.

n.d.

Rapp et al., 1979

27.4

n.d.

Philp et al., 2002

24.4

5.0

Tuleva et al., 2008

glycolipid biosurfactant

25.0

316.0

4 cell-associated glycoglycerolipids and 1 diphosphatidylglycerol 4-[[5-(7-methyl-(E)-2octenoylhydroxyamino) pentyl]amino]-2-[2-[[5(7-methyl-(E)-2octenoylhydroxyamino) pentyl]amino]-2oxoethyl]-2-hydroxy- 4oxobutanoic acid

33.0

n.d.

Wattanaph on et al., 2008 Wicke et al., 2000

26.0

300.0

mainly acid-form sophorolipids GL-A 18-L-[(2´-O-ß-Dglucopyranosyl-ß-Dglucopyranosyl)-oxy]octade-cenoic acid, 6´, 6´´-di-O- acetylate mannosylerythritol lipids

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Konoshi et al., 2008

Bodour et al., 2004

Biosurfactants and Their Uses in the Petroleum Industry…

13

Table 1. (Continued) Biosurfactant

Strain

Lipopeptides

Bacillus subtilis YB7 Bacillus subtilis BBK-1 Bacillus licheniformis BAS50 Bacillus licheniformis F2.2 Pseudomona s putida PCL 1445 Pseudomona s putida 267 Nocardiopsis alba MSA10

Chemical characterization surfactin-analog cyclic lipopeptides bacillomycin L, plipastatin, and surfactin

Surface activity 30.0

Refere nce 40.9

n.d.

n.d.

lichenysin A

28.0

12.0

plipastatin and surfactin, as well as a new nonlipopeptide biosurfactant called BL 1193 putisolvin I and II

n.d.

n.d.

Thaniyavar n et al., 2003

n.d.

n.d.

Kuiper et al., 2004

putisolvin-like cyclic lipopeptides lipopeptide

31.5

25.0

n.d.

n.d.

Kruijt et al., 2009 Gandhimat hi et al., 2009

Arutchelvi et al., 2009 Roongsaw ang et al., 2002 Yakimov et al., 1995

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n.d.: no data

Flavolipids are another class of biosurfactants, with a polar moiety that is due to a citric acid plus two cadaverine molecules and a nonpolar moiety composed of two branched chain acyl groups, ranging from 6 to 10 carbons (Bodour et al., 2004). Flavolipids exhibit strong surface activity as well as solubilization and emulsification capabilities (Bodour et al., 2004). Lipopeptides such as iturins, surfactins and lichenysins produced by Bacillus spp. are an extensively characterized group of biosurfactants (Figure. 4d; Table 1). Surfactin, produced by Bacillus subtilis strains, is formed by a heptapeptide linked via a lactone bond to a ß-hydroxy fatty acid with 13-15 C atoms (Peypoux et al., 1999). Surfactin produces a remarkable decrease of water ST to 27 mN m-1 at a concentration of 20 μM (Arima et al., 1968). Another lipopeptide produced by Bacillus licheniformis JF-2, which consists of a heterogeneous C15 fatty acid tail (mixture of normal, anteiso, and isobranched forms) linked to a peptide moiety, has a CMC as low as 10 mg l-1 and reduces the IFT against decane to 6 x 10-3 mN m-1 (Lin et al., 1994). Later, this strain was reidentified using molecular methods and assigned to Bacillus mojavensis (Folmsbee et al., 2006). Surfactin and related lipopeptides have not only a powerful surfactant activity, but also show a wide range of anti microbial properties (Vollenbroich et al., 1997; Seydlová and Svobodová,

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N. L. Olivera and M. L. Nievas

2008). Due to their significant stability even under extreme conditions (Makkar and Cameotra, 1997), lipopeptidic biosurfactants have been used in activities such as mobilization of oil from petroleum reservoirs (see Section 4.2.1). Other examples of lipopeptide-producing strains are in Table 1. The plant growth-promoting strain Pseudomonas putida 267, isolated from the rhizosphere of black pepper, produces lipopeptide biosurfactants which are involved in swarming motility and biofilm formation, and also have zoosporicidal and antifungal activities (Kuiper et al., 2004). Nocardiopsis alba MSA10, a sponge-associated marine actinomycetes, produces a lipopeptide with biosurfactant and antimicrobial activity (Gandhimathi et al., 2009). Fatty acid and phospholipid surfactants are also produced by several bacteria and yeasts (Desai and Banat, 1997). Acinetobacter sp. HO1-N produces vesicles characterized as phospholipid-rich, lipopolysaccharide-rich particles that enhance n-hexadecane solubilization (Käppeli and Finnerty, 1979). Rhodococcus erythropolis DSM43215 produces surfactant compounds using n-alkanes as a carbon source; the predominant was α,α-trehalose-6,6'dicorynomycolates which reduced the IFT between n-hexadecane and water from 44 to 18 mN m-1 (Kretschmer et al., 1982). In addition, this strain produces phosphatidylethanolamines which reduced the IFT below 1 mN m-1 in a water-hexadecane system (Kretschmer et al., 1982). A strain isolated from seaside soil, R. erythropolis 3C-9, produces two kinds of glycolipids (a glucolipid and a trehalose lipid) as well as free fatty acids (at least 12 free fatty acids of chain lengths from C9 to C22) as biosurfactants when growing on nhexadecane (Peng et al., 2007).

3.2. High-Molecular-Weigh Biosurfactants A variety of microbial species of different genera produce polymeric extracellular biosurfactants composed by polysaccharides, proteins, lipopolysaccharides, lipoproteins or complex mixtures of these polymers (Ron and Rosenberg, 2001). As their molecular masses vary in a wide range of up to 104 kDa, they are considered high-molecular-weight biosurfactants (NavonVenezia et al., 1995; Bach et al., 2003; Martínez-Checa et al., 2007).

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Biosurfactants and Their Uses in the Petroleum Industry… a-

bCH2OAc O

HO CH3

OH

15

CH3 O

O O CH CH C O 2

CH

(CH2)6

(CH2)6

CH3

CH3

OH

O

CH2 COOH

OH

CH (CH2)15

HO CH2OAc

COOH O O

OH HO OH CH3

c-

d-

(CH2)n

L-Val L-Asp

D-Leu

D-Leu

L-Leu

CH2O CO CH CHOH (CH2)m CH3 OH O OH OH HO

O

OH

O OH H3C (H2C)m HOHC CH (CH2)n

CO

L-Leu L-Glu O CH - CH2 - C = O ( CH2 )9

OCH2

CH3 - CH - CH3

m + n = 27 to 31

CH3

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Figure 4. Biosurfactant molecular structures; a: monorhamnolipid, b: acid form of a sophorose lipid, c: trehalolipid, d: lipopeptide (surfactin)

Several Acinetobacter strains produce protein-polysaccharide bio emulsifiers, being Alasan and Emulsan as the best characterized (NavonVenezia et al., 1995; Rosenberg et al., 1979a, 1979b). Alasan is produced by Acinetobacter radioresistens KA53 when it grows on ethanol. It stabilizes O/W emulsions in a wide pH (3 to 9) and temperature (ambient to 100°C) range for long periods (up to 3 months). Alasan shows emulsifying specificity toward individual aliphatic and aromatic hydrocarbons as well as oil complex mixtures, and enhances the apparent solubility of polycyclic aromatic hydrocarbons (PAHs), (Navon-Venezia et al., 1995; Barkay et al., 1999). Structurally Alasan, with a molecular weight of about 103 kDa, is a complex of protein and anionic polysaccharide containing covalently bound alanine (Navon-Venezia et al., 1995). The emulsifying activity showed by Alasan is associated to the proteic component; the deproteinized polysaccharide named apo-Alasan acts as a protein stabilizer (Navon-Venezia et al., 1998; Toren et al., 2001). Alasan proteic component consists of three proteins named AlnA, AlnB and AlnC (Toren et al., 2001). AlnA, which has a structure analogous to the outer membrane protein OmpA of E. coli, is the main component responsible for the emulsifying activity (Toren et al., 2002a; Toren et al., 2002c). Interestingly, AlnA was successfully expressed in E. coli and

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N. L. Olivera and M. L. Nievas

produced a higher emulsifying activity than Alasan. Further studies suggested that AlnA mechanisms of hydrophobic compound pseudosolubilization are not mediated by traditional micelle formation described for low-molecular-weight biosurfactants (Toren et al., 2002b; Toren et al., 2003). Moreover, an apparent solubility enhancement of PAHs with both, monomeric and oligomeric forms of Alasan was observed (Toren et al., 2002b; Toren et al., 2003). A complex with an apparent molecular mass of ca. 220 kDa (6 molecules of Alasan) was observed in Alasan-PAH systems, suggesting that interactions of hydrophobic compounds with the polymer hydrophobic regions are responsible for solubilization. In addition, AlnA changes from monomer to hexamer form in the presence of a variety of PAHs (Toren et al., 2003). Emulsan is produced at commercial scale and used in many industrial applications (Rosenberg and Ron, 2002). Acinetobacter venetianus RAG-1 produces this polyanionic amphipathic protein-polysaccharide complex (Zuckerberg et al., 1979), which is an effective hydrocarbon emulsifier at concentrations as low as 0.001 to 0.01% (Ron and Rosenberg, 2001). It possesses high specificity to form O/W emulsions and shows important activity with mixtures of aromatic and aliphatic hydrocarbons (Rosenberg et al., 1979a). Like Alasan, the deproteinized form of Emulsan (apo-Emulsan) shows severe reduction of emulsifying activity in experiments with some highly hydrophobic compounds (Bach et al., 2003). For this well characterized emulsifier, a model for emulsion stabilization due to the interaction between polysaccharide and protein parts has been proposed (Bach and Gutnik, 2006). The yeast Candida lipolytica produces the emulsifier Liposan, a heteropolysaccharide containing glucose, galactose, galactosamine, galacturonic acid and proteins (Cirigliano and Carman, 1985). This polymer forms stable emulsions with different aromatic and aliphatic hydrocarbons and retains its activity in a pH range between 2 and 5 and up to 70°C (Cirigliano and Carman, 1984; 1985). Recently, the production of another bioemulsifier by C. lipolytica that also reduces the ST to 25 mN m-1 was reported (Sarubbo et al., 2007; Rufino et al., 2008). Such polymer is a protein-lipidpolysaccharide complex with remarkable stability to pH (2-12), temperature (up to 120°C) and salinity (2-10 % NaCl). Other advantages are its effective emulsifier capability and its production using an optimized medium containing low-cost soybean oil from refinery residues. Halomonas eurihalina strains, which are moderately halophilic bacteria, produce extracellular exopolysaccharide (EPS) bioemulsifiers (MartínezCheca et al., 2002; 2007; Bouchotroch et al., 2000; Calvo et al. 1995; 1998; 2002) in hydrocarbon supplemented media and also on soluble carbon sources

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as glucose. EPS contain carbohydrates, proteins, uronic acids, acetyl residues, sulfates, and some cations (e.g. sodium, calcium, and magnesium). Its chemical composition is strongly influenced by the carbon sources used for microbial growth (Martínez-Checa et al., 2002; 2007; Calvo et al., 1998; 2002). EPS are effective in emulsifying n-alkanes and individual aromatic hydrocarbons such as xylene and toluene, but the best performance was found in crude oil emulsification (Martínez-Checa et al., 2002; 2007; Calvo et al., 2002). Another characteristic of EPS solutions is that the viscosity raises as the pH decreases, forming a kind of gel (Calvo et al. 1995; 1998). Recently, an EPS with emulsifying activity produced by the moderately halophilic marine bacterium Planococcus maitriensis Anita I was described (Suresh Kumar et al., 2007). Its chemical composition was similar to that of Halomonas eurihalina. Salt tolerant bacteria and their polymeric emulsifiers possess interesting potentials for petroleum recovery and environmental applications in saline sites. New studies performed by Fusconi et al. (2010) resulted in the characterization of EPS produced by Gordonia polyisoprenivorans CCT 7137 from soluble carbon sources. In addition, the aliphatic-hydrocarbon degrading Gordonia sp. BS29 also produces a high-molecular-weight lipopolysaccharide with emulsifying activity (Franzetti et al., 2009; 2008). This EPS enhanced the oil and PAHs removal during the washing of contaminated soils.

4. TOXICITY AND BIODEGRADABILITY OF BIOSURFACTANTS Considering the biosurfactant natural origin, some of the advantages generally assumed with respect to their chemically synthesized counterparts are a higher biodegradability and a lower toxicity (Rosenberg, 1986; Lin, 1996; Nitschkea and Costa, 2007). However, these characteristics have been less studied than the surface active properties described in the previous sections (see Section 2). In contrast, antimicrobial activities of some biosurfactants such as surfactin are well known (Singh and Cameotra, 2004; Rodrigues et al., 2006; Seydlová and Svobodová, 2008). Poremba et al. (1991) analyzed the toxicity of eight synthetic and nine biogenic surfactants because of their potential to be applied as oil dispersants. They found that most biogenic surfactants were less toxic than synthetic surfactants. Moreover, they did not detect toxicity in the glucose-lipid

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N. L. Olivera and M. L. Nievas

biosurfactant produced by Alcaligenes sp. MM1, a strain that thereafter would be identified as Alcanivorax borkumensis (Yakimov et al., 1998). In another experiment, toxicity to menidia and mysids of three commercial preparations containing biosurfactants (BioEM-rhamnolipids, Emulsan, and PES61) and three synthetic surfactants (PES51, Corexit 9500, and Triton X-100) were compared (Lepo et al., 1997). A high correlation between the bioassays of the two species was observed; the compounds were ranked by their increasing toxicity as PES61