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This project is supported by the European Union under the Food Agriculture and Fisheries and Biotechnology theme of the 7th Framework Programme for Research and Technological Development under GA no 312139

KillbullSpill

Integrated Biotechnological Solutions for Combating Marine Oil Spills

Deliverable D39

Evaluation of novel dispersants

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Work package WP3 Impact assessment of developed technologies

Deliverable no D39

Deliverable title Evaluation of novel dispersants surface characterization in presence of oil and dispersant

Due date Month 36

Actual submission date Month 48

Start date of project 2013-01-01

Deliverable Lead Beneficiary (Organisation name)

University at Buffalo

Participant(s) (Partner short names) UB

Author(s) in alphabetic order Alexandridis Paschalis Placek Tess Tsianou Marina

Contact for queries Paschalis Alexandridis Dept of Chemical and Biological Engineering University at Buffalo Buffalo NY 14260-4200 USA T +1 716 6451183 E palexandbuffaloedu

Dissemination Level (PUblic Restricted to other Programmes Participants REstricted to a group specified by the consortium COnfidential only for members of the consortium)

PU

Deliverable Status final

Grant Agreement no 312939 Deliverable D39 Evaluation of novel dispersants

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Table of Content

1 About this deliverable 1

2 Introduction 1

21 Surfactants 1

22 Synthetic Surfactants versus Natural Biosurfactants 2

221 Synthetic Surfactants 2

222 Biosurfactants 3

23 Surfactant Properties 6

231 Self-Assembly in Solution 6

232 Micellization and Solution Conductivity 7

233 Surfactant Adsorption on Surfaces 8

24 Oil Spills and Dispersants 10

25 Rationale for this work 12

3 Materials and Methods 13

31 Materials 13

32 Methods 13

321 Sample Preparation 13

322 Fluorescence Spectroscopy 13

323 Conductivity 15

324 Quartz Crystal Microbalance with Dissipation 16

4 Results 17

41 Solution Assembly of Synthetic Surfactants 17

42 Solution Assembly of Biosurfactants 22

43 Adsorption of Synthetic Surfactants on Surfaces 32

44 Adsorption of Biosurfactants on Surfaces 38

45 Conclusions 51

5 References 52


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List of Figures

Figure 21 Chemical structures of A sodium dodecyl sulfate and B dioctyl sulfosuccinate sodium salt 3

Figure 22 Chemical structures of A mono-rhamnolipid B di-rhamnolipid C sophorolipid D Surfactin 5

Figure 23 Fluorescence emission spectra of pyrene in A surfactant solutions below the CMC and B surfactant aqueous solutions above the CMC 6

Figure 24 Ionic surfactant conductivity versus surfactant concentration and determination of CMC 7

Figure 25 Amount of oil recovered per remediation method listed on the right in regards to a remote oil spill of 10000 tons 11

Figure 31 Figure taken from Aguiar et al(Aguiar et al 2003) depicts the locations of CMC1 and CMC2 values obtained from the I1I3 pyrene ratio 15

Figure 32 Schematic of Quartz Crystal Microbalance instrument 16

Figure 41 Pyrene I1I3 fluorescence intensity ratios of SDS in 0 2 and 35 wt NaCl aqueous solution 17

Figure 42 Pyrene I1I3 fluorescence intensity ratios of SDS in 0 2 and 35 wt NaCl aqueous solution and derivatives identifying CMC1 18

Figure 43 Conductivity data of SDS in deionized water 18

Figure 44 Conductivity data and pyrene I1I3 fluorescence intensity ratios for SDS in deionized water 19

Figure 45 Pyrene I1I3 fluorescence intensity ratios for AOT in 0 2 and 35 wt NaCl aqueous solutions 19

Figure 46 Pyrene I1I3 fluorescence intensity ratios for AOT in 0 2 and 35 wt NaCl aqueous solutions and derivatives identifying CMC1 20

Figure 47 Conductivity data of AOT in deionized water 20

Figure 48 Conductivity data and pyrene I1I3 fluorescence intensity ratios of aqueous AOT in deionized water 21

Figure 49 Pyrene I1I3 fluorescence intensity ratios of SDS and AOT in 0 and 35 wt NaCl aqueous solutions 21

Figure 410 Pyrene I1I3 fluorescence intensity ratios for R95M90 and R95D90 in deionized water 22

Figure 411 Pyrene I1I3 fluorescence intensity ratios for R95M90 and R95D90 in NaCl aqueous solution 23

Figure 412 Plot of I1I3 versus surfactant concentration demonstrating how CMC1 and CMC2 were determined using the R95D90 data as an example 23

Figure 413 Conductivity data at 20 degC of mono-rhamnolipid dominant and di-rhamnolipid dominant samples in deionized water corresponding to curves 1 and 2 respectively 24


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Figure 414 Pyrene I1I3 fluorescence intensity ratios for R95M90 and R95D90 in aqueous solutions with and without NaCl 25

Figure 415 Pyrene I1I3 fluorescence intensity ratios for crude rhamnolipids obtained from Burkholderia thailandensis and crude rhamnolipids obtained from Pseudomonas aeruginosa in deionized water 26

Figure 416 Conductivity of crude rhamnolipids obtained from Pseudomonas aeruginosa in deionized water 27

Figure 417 Conductivity data and pyrene I1I3 fluorescence intensity ratios of aqueous rhamnolipids obtained from Pseudomonas aeruginosa in deionized water 27

Figure 418 Pyrene I1I3 fluorescence intensity ratios for the crude rhamnolipids obtained from Pseudomonas aeruginosa in aqueous solution with and without NaCl 28

Figure 419 Pyrene I1I3 fluorescence intensity ratios for the crude sophorolipids in aqueous solution with and without NaCl 28

Figure 420 Pyrene I1I3 fluorescence intensity ratios for the crude Surfactin in aqueous solutions with and without NaCl 29

Figure 421 Conductivity of Surfactin in deionized water 30

Figure 422 Conductivity data and pyrene I1I3 fluorescence intensity ratios of Surfactin in deionized water 30

Figure 423 Pyrene I1I3 fluorescence intensity ratios for four crude biosurfactants R95M90 R95D90 sodium dodecyl sulfate (SDS) and dioctyl sodium sulfosuccinate (AOT) in deionized water 31

Figure 424 Pyrene I1I3 fluorescence intensity ratios for four crude biosurfactants R95M90 R95D90 sodium dodecyl sulfate (SDS) and dioctyl sodium sulfosuccinate (AOT) in NaCl aqueous solution 31

Figure 425 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 05 wt SDS aqueous solution 33

Figure 426 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 05 wt SDS in 2 wt NaCl aqueous solution 34


Figure 428 A Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 05 wt SDS aqueous solution B Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 05 wt SDS in 35 wt NaCl aqueous solution 35

Figure 429 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 05 wt AOT aqueous solution 36

Figure 430 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 05 wt AOT in 2 wt NaCl aqueous solution 36

Figure 431 A Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 05 wt AOT aqueous solution B Frequency and dissipation


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changes over time of a gold sensor surface responding to exposure to a 05 wt AOT in 2 wt NaCl aqueous solution 37

Figure 432 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt R95M90 aqueous solution 38

Figure 433 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt R95M90 in 35 wt NaCl aqueous solution 39

Figure 434 A Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt R95M90 aqueous solution B Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt R95M90 in 35 wt NaCl aqueous solution 40

Figure 435 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt R95D90 aqueous solution 41

Figure 436 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt R95D90 in 35 wt NaCl aqueous solution 42

Figure 437 A Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt R95D90 aqueous solution B Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt R95D90 in 35 wt NaCl aqueous solution 43

Figure 438 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt crude rhamnolipid obtained from Pseudomonas aeruginosa aqueous solution 44

Figure 439 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt crude rhamnolipid obtained from Pseudomonas aeruginosa in 35 wt NaCl aqueous solution 44

Figure 440 A ∆ Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt crude rhamnolipid obtained from Pseudomonas aeruginosa aqueous solution B Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt crude rhamnolipid obtained from Pseudomonas aeruginosa aqueous solution 45

Figure 441 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt sophorolipid aqueous solution 46

Figure 442 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt sophorolipid in 35 wt NaCl aqueous solution 47

Figure 443 A Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt sophorolipid aqueous solution B Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt sophorolipid in 35 wt NaCl aqueous solution 48

Figure 444 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt Surfactin aqueous solution 49

Figure 445 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt Surfactin in 35 wt NaCl aqueous solution 49


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Figure 446 A Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt Surfactin aqueous solution B Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt Surfactin in 35 wt NaCl aqueous solution 50

List of Tables

Table 1 Summary of surfactants used and CMCs identified using different analysis methods The abbreviations for surfactants in Table 1 are sodium dodecyl sulfate (SDS) dioctyl sulfosuccinate sodium salt (AOT) Pseudomonas aeruginosa rhamnolipid crude extract (PARh) Burkholderia thailandensis rhamnolipid crude extract (BTRh) 95 pure rhamnolipids containing 90 mono-rhamnolipid (R95M90) 95 pure rhamnolipids containing 90 di-rhamnolipid (R95D90) sophorolipid (SPL) and crude Surfactin (LotActySURCE001) (Surfactin) 32

Table 2 Summary of mass of surfactant adsorbed to gold surfaces during QCM-D experiment The abbreviations for surfactants in Table 1 are sodium dodecyl sulfate (SDS) dioctyl sulfosuccinate sodium salt (AOT) Pseudomonas aeruginosa rhamnolipid crude extract (PARh) Burkholderia thailandensis rhamnolipid crude extract (BTRh) 95 pure rhamnolipids containing 90 mono-rhamnolipid (R95M90) 95 pure rhamnolipids containing 90 di-rhamnolipid (R95D90) sophorolipid (SPL) and crude Surfactin (LotActySURCE001) (Surfactin) 50


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1 About this deliverable

Surfactants are amphiphilic molecules that are widely applied to disperse oil into small droplets by lowering the interfacial tension between oil and water thus facilitating the degradation of oil This study aims to investigate biosurfactant micellization properties in deionized water and seawater-like conditions for potential use in environmental applications as opposed to the traditionally used petroleum-based synthetic surfactants sodium dodecyl sulfate (SDS) and dioctyl sodium sulfosuccinate (AOT) Glycolipids such as sophorolipids and rhamnolipids as well as a lipopeptide Surfactin are the biosurfactants examined in this study Multiple rhamnolipid samples were studied including samples that contain 90 mono-rhamnolipids and 90 di-rhamnolipids separately and crude extracts Fluorescence spectroscopy and conductivity were used to determine surfactant aqueous solution self-assembly properties Quartz Crystal Microbalance with Dissipation (QCM-D) was used to investigate surface adsorption of surfactants on gold surfaces The biosurfactants studied here exhibited critical micelle concentrations (CMC) significantly lower than those of typical synthetic surfactants These results indicate that smaller amounts of biosurfactants may be effective in dispersing oil QCM-D measurements show that in deionized water surfactants adsorb in thin relatively rigid films and in salt solutions the adsorbed surfactant layer exhibits more viscoelastic behavior A better understanding of biosurfactant self-assembly and adsorption properties is essential to improving the methods currently employed to reduce the adverse effects and overall impact on the environment of oil spills

2 Introduction

21 Surfactants

Surfactants short for surface active agents are amphiphilic molecules meaning that they contain both hydrophilic and hydrophobic parts Surfactants absorb spontaneously at an oil-water interface causing a decrease in interfacial surface tension and consequently an increase the solubilization of two immiscible liquids (The Essential Chemical Industry - online - Surfactants 2013) Surfactants are either synthetic derived from petrochemical compounds or bio-based produced naturally by microorganisms

Surfactant properties such as critical micelle concentration (CMC) self-assembly structure and adsorption to surfaces can be affected by many factors The CMC is the concentration at which surfactant monomers start to assemble in solution to form a micelle This property is what allows surfactants to emulsify oils (Pornsunthorntawee et al 2009) Surfactants properties can be affected by factors such as salt temperature and pH The addition of salt to aqueous surfactant solutions can sometimes cause an increase in the micelle aggregation number the number of surfactant monomers in a micelle and significantly reduce the CMC(Miura and Kodama 1972) The aggregation number of surfactants can increase so much that in order to accommodate a larger number of monomers the shape and structure of assembled surfactants in solution can change (Dey et al 2010) Salt effects are less prominent for nonionic surfactants however an increase in the alkyl chain length can result in similar effects (Evans and Wennerstroumlm 1999) At solid surfaces the addition of salt to a surfactant solution can increase the amount of surfactant adsorbed (Long et al 2013 Somasundaran and Krishnakumar 1997)

Surface adsorption can be affected by the nature of the surface itself in regard to its charge or degree of hydrophobicity These effects can change how surfactants adsorb on surfaces in different ways (Tiberg et al 1999) Biosurfactants have been shown to have higher micellization stability than synthetic


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surfactants across a range of temperatures and salinities (Pornsunthorntawee et al 2009) Further the addition of solvents and cosurfactants affects the properties of micellization such as critical micelle concentration (CMC) surface tension hydrophilic-lipophilic balance (HLB) and the overall emulsion structure and interactions(Nursakinah et al 2014)

The properties of surfactants allow them to self-assemble in solution under different conditions making them useful in a wide range of applications The focus of this work is to assess the self-assembly properties of biosurfactants in comparison to traditional surfactants

22 Synthetic Surfactants versus Natural Biosurfactants

Surfactants can be either produced naturally in the environment by fungi plants bacteria and yeast or from chemical reactions of petroleum based substances Synthetic surfactants are separated and synthesized from petroleum or other organic compounds by fractional distillation and hydrogenation as well as other chemical processes (Sjoumlblom 2006 Madsen et al 2015) While both bio-based and traditional surfactants are made of organic molecules synthetic surfactants tend to be more homogeneous and less bulky with smaller head groups than the biosurfactants (Cohen and Exerowa 2007) For traditionally produced synthetic surfactants the end product of a reaction is quite uniform in comparison to the many forms and homologues of molecules that a living organism will produce (Monteiro et al 2007) Factors affecting the type of biosurfactants produced can be the strain of organism culture media and time of growth Many studies have shown that under different conditions different mixtures of surfactants are formed (Monteiro et al 2007) Biosurfactants can additionally possess biocide or anticancer properties(Felse et al 2007 Desai and Banat 1997) The differences in physical properties purity and consistency of surfactants produced can affect the self-assembly of surfactants

221 Synthetic Surfactants

Synthetic surfactants are produced by a series of chemical reactions starting with organic materials from the petrochemical industry resulting in ionic non-ionic or polymeric surfactants (Evans and Wennerstroumlm 1999) Many surfactants are available commercially such as Triton-X-100 sodium dodecyl sulfate (SDS) and dioctyl sulfosuccinate sodium salt (AOT) as well as Polysorbate 80 (TWEENreg 80) SDS and AOT are two examples of synthetic surfactants used in this study

SDS sodium dodecyl sulfate is an ionic surfactant containing a hydrocarbon chain and an ionic head group The sulfate is a negatively charged head group balanced by a positively charged sodium counter ion in solution making SDS an anionic surfactant (Mitsionis and Vaimakis 2012) The molecular weight is low at only 28837 gmol on average with a CMC in water reported in literature around 8 mM at 25 degC (Shanks and Franses 1992 Quina et al 1995) At this concentration SDS self-assembles into micelles with the hydrophilic head groups partitioning to the polar solvent and the hydrophobic tails make up the non-polar interior or the micelle The addition of SDS to water reduces its surface tension to 32 mNm from 72 mNm in 292 wt NaCl at 25 degC (Umlong and Ismail 2007) The structure of SDS is shown below in Figure 21A SDS is additionally used in many household detergents cleaning and hygiene products on the market today (Delimaassociates 2016)


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Figure 21 Chemical structures of A sodium dodecyl sulfate and B dioctyl sulfosuccinate sodium salt

Similar assembly is seen for synthetic surfactant dioctyl sulfosuccinate sodium salt AOT as described for SDS at the CMC in water the head group of AOT partitions to the polar solvent and the hydrocarbon tail makes up the interior of the micelle AOT shown in Figure 21B is a synthetic surfactant consisting of an anionic sulfonate head group balanced with a sodium counter ion in solution Its hydrophobic region contains two saturated hydrocarbon chains with a molecular weight of 44456 gmol (Mitsionis and Vaimakis 2012) The CMC from our work of AOT is slightly higher than reported values of roughly 3 mM in water at room temperature (Grillo and Penfold 2011 Thavorn et al 2014) Water with a surface tension of 72 mNm at 25 degC can be reduced to 26 mNm when 275 mM AOT is dissolved in a 11 wt NaCl aqueous solution (Umlong and Ismail 2005) AOT is also used in household cleaners and hygiene products as well as pet shampoos (Delimaassociates 2016)

222 Biosurfactants

Biosurfactants can be produced by a number of organisms and come in four main classes lipopeptides phospholipids polymeric surfactants and glycolipids (Healy et al 1996) Rhamnolipids sophorolipids and Surfactin will be discussed here

Rhamnolipids are a type of glycolipid which consist of a carbohydrate sugar head group which is more hydrophilic and a hydrocarbon fatty acid chain as the hydrophobic segment The head group for rhamnolipids is the rhamnose sugar and can be either mono- or di- in the compound (Pornsunthorntawee et al 2009) This variability in biosurfactants structure is due to production by different stains and culture conditions Various strains of the common bacteria species pathogenic Pseudomonas aeruginosa and Burkholderia thailandensis are utilized for rhamnolipid production (Marchant and Banat 2012) Many rhamnolipid homologues are extracted from one culture and can affect the biosurfactant properties For example the surface tension of water can be reduced to between 283 and 40 mNm with the addition of 25 to 120 mgL of rhamnolipid biosurfactants depending on strain used and culture purity (Pornsunthorntawee et al 2008a Guerra-Santos et al 1984) Montiero et al (Monteiro et al 2007) reported there to be 28 different forms of rhamnolipids with the most common being Rha-C10-C10 and Rha-Rha-C10-C10 depicted in Figure 22A and B The mono- and di- rhamnolipid structures in Figure 21 are redrawn based on the paper by Marchant and Banat (Marchant and Banat 2012)

A

B


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Sophorolipids like rhamnolipids are a type of glycolipid The sophorolipids consist of the sophorose sugar bonded to a fatty acid chain 14 to 22 carbons long (Kang et al 2010) Among others the two most common forms of sophorolipids are acidic as shown in Figure 22C and lactonic where the acid on the end of the fatty acid bonds to the sophorose sugar and forms a cyclic ester(Hirata et al 2009) Acidic forms are more useful for dispersant purposes and reportedly reduce the surface tension of water to between 336 and 372 mNm at a CMC of 30 to 43 mgL (Daverey et al 2011 Hirata et al 2009) Sophorolipids are produced by the non-pathogenic yeast Candida bombicola which is more advantageous when working on applications on the industrial scale compared to the pathogenic bacteria that produce rhamnolipids(Kang et al 2010) The sophorolipid structure in Figure 22C was drawn based on the paper by De Oliveira et al (De Oliveira et al 2015)

Surfactin commonly produced by Bacillus subtilis is a biosurfactant of the lipopeptide category (Mulligan 2009 Nitschke and Pastore 2006) Surfactin consists of cyclically linked amino acids bonded to a hydrophobic fatty acid Other lipopeptides can be ionic or non-ionic comprising of a head group of a carbohydrate alcohol amino acid or phosphate (Mulligan 2009) Multiple forms of Surfactin exist in the extracted sample from bacteria and the types found are influenced by the amino acids available in the culture media (Marti et al 2014) At 25 degC the surface tension of water at 72 mNm can be reduced to 266 mNm and to 27 mNm in 33 mgL and in 20 mgL Surfactin aqueous solutions as reported by Nitschke et al(Nitschke and Pastore 2006) and Marti et al(Marti et al 2014) respectively The structure of a typical Surfactin molecule shown in Figure 22D was drawn from the structure described by Marti et al(Marti et al 2014)

2221 Production of Biosurfactants

Biosurfactants are produced by bacteria yeast and other organisms in the environment that can then be isolated and cultivated in a laboratory(Pornsunthorntawee et al 2008a Baelum et al 2012) Due to the presence of oils in the environment some organisms can metabolize them by producing surfactant molecules in order to be able to break them down Samples of oil contaminated sea water and soil are collected in the environment and brought back to the lab Serial dilutions are made and cultures are grown on plates with selective media to isolate the surfactant producing microorganisms (Pornsunthorntawee et al 2008a) Once identified palm soybean corn or another oil is used as the carbon source in the media for cell cultivation After the growth phase the produced surfactants are isolated by means of centrifugation chromatography columns or extraction and washing methods(Pornsunthorntawee et al 2008a Nitschke et al 2005 Felse et al 2007)

Large scale production is necessary in making biosurfactants a viable alternative to traditional surfactants The prevalent factor restricting the use of biosurfactants in a wider range of applications is the cost of production and purification Batch cultures of organisms result in very low amounts of biosurfactants extracted(Pornsunthorntawee et al 2008b Houmlrmann et al 2010) An increase in biosurfactant yield is necessary in making this process cost effective

Methods have been investigated to not only reduce waste but make biosurfactant production less expensive To accomplish this scientists have used industrial and food waste products as growth media for microorganism cultivation and surfactant production(Felse et al 2007 Nitschke et al 2005) Monteiro et al(Monteiro et al 2007) Nitschke et al(Nitschke et al 2005) and Felse et al(Felse et al 2007) have shown that different types of growth media can affect the extracted surfactant Typically many forms or homologues of a biosurfactant are produced from microorganisms The main


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components of biosurfactants produced can also be influenced by factors such as organism strain and culture time in addition to the media components Nearly 60 of the production costs of biosurfactants can come from purification methods such as solvent extraction centrifugation chromatography and others(Desai and Banat 1997 Muthusamy et al 2008) Due to the cost and time of purification crude biosurfactants that have undergone minimal purification and therefore contain more impurities are sometimes examined to determine self-assembly properties (Muthusamy et al 2008) While a pure product may be ideal often these impurities or different homologues found in the crude biosurfactant samples contribute to lowering the CMC (Monteiro et al 2007)

Figure 22 Chemical structures of A mono-rhamnolipid B di-rhamnolipid C sophorolipid D Surfactin

A

B

C

D


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23 Surfactant Properties

231 Self-Assembly in Solution

At the CMC surfactant systems experience a change in many physical properties that can be measured in order to identify the concentration where micellization starts The self-assembly of surfactants can be investigated using surface tension viscosity fluorescence spectroscopy and conductivity methods Surface tension detects a decrease in measured surface tension at the CMC when plotted against the surfactant concentration(Mańko et al 2014 Marti et al 2014 Pornsunthorntawee et al 2008a) Measuring the viscosity of surfactant solutions can also help identify the CMC Above the CMC viscosity will increase significantly as a result of micellization and an increased number of micelles in solutions changing their interactions and transport properties(Wang et al 2004) The inflection point of the plot of viscosity versus concentration can be taken as the CMC(Mańko et al 2014) This work will utilize fluorescence spectroscopy and conductivity to determine the CMC of surfactant solutions as described in sections 231 and 232

The concept behind using fluorescence spectroscopy to determine the CMC involves the pyrene peak intensity ratio and how it relates to the solvent polarity Pyrene a hydrophobic molecule has five characteristic peaks that can vary in intensity as pyrene interacts with different solvents to produce different intensity profiles when excited from the ground state(Madsen et al 2015 Mitsionis and Vaimakis 2012 Nivaggioli et al 1995) The Py scale of solvent polarity is the ratios of intensities from the pyrene emission spectrum A plot of the I1I3 ratios versus surfactant concentration results in the characteristic sigmoidal curve used to identify the CMC(Kalyanasundaram and Thomas 1977 Dong and Winnik 1984 Glushko et al 1981)

Figure 23 Fluorescence emission spectra of pyrene in A surfactant solutions below the CMC and B surfactant

aqueous solutions above the CMC Peaks labeled 1 and 3 indicate the two peaks from the pyrene emission spectrum that the I1I3 ratios are obtained from

In polar environments such as water-surfactant solutions below the CMC pyrene undergoes vibronic coupling with the solvent and the intensity of band 1 is significantly enhanced whereas band 3 is not as affected by solvent polarity (Kalyanasundaram and Thomas 1977) In non-polar solvents above the CMC pyrene-solvent dipole interactions are minimal as the hydrocarbon interior of the micelle is not charged now hydrogen bonding is the main interaction and the peak intensity ratios of I1I3 decrease (Kalyanasundaram and Thomas 1977 Glushko et al 1981 Infelta and Graumltzel 1979) Figure 23 shows


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the I1I3 ratio change as pyrene moves to the micelle interior at concentrations above the CMC These vibronic band perturbations can be used as a probe for CMC determination because pyrene is very solvent sensitive Complex interactions with dipoles solvent polarizabilty dielectric constants excited transition energies hydrogen bonding and quenching can play a part in pyrene excitation emission (Dong and Winnik 1984 Kalyanasundaram and Thomas 1977)

The intensity ratios of pyrene have accurately identified the CMCs of surfactants in several solvents (Mitsionis and Vaimakis 2012 Dong and Winnik 1984 Glushko et al 1981) The CMC of SDS is 0008 M in water using fluorescence spectroscopy this value for the CMC additionally agreed with what is observed using surface tension and conductivity at 25 degC (Kalyanasundaram and Thomas 1977 Pineiro et al 2015) SDS and AOT surfactant solutions have been investigated with fluorescence spectroscopy in methanol and other mediums (Mitsionis and Vaimakis 2012) Rhamnolipids in a tris buffer solution at 20 degC have been reported to have a CMC around 01 to 1 mM or approximately or 00043 to 00434 wt (Madsen et al 2015)

232 Micellization and Solution Conductivity

The conductivity of a surfactant solution is influenced by the surfactant structure aggregation number counter ion size hydration number in water as well as concentration in solution(Pinazo et al 1999 Loacutepez-Diacuteaz and Velaacutezquez 2007) Below the CMC conductivity of ionic surfactant solutions increases as the surfactant concentration increases due to dissolution of the surfactants and counter ions resulting in more molecules overall moving freely in solution Once the CMC is reached and the surfactants assemble into micelles the increase in conductivity will become lower because the slightly charged micelles partially bind counter ions in solution causing the overall number of charged species in solution to be less than below the CMC when counter ions and surfactant monomers are not assembled(Shanks and Franses 1992 Inoue et al 2007) This results in a change of slope in the conductivity data indicative of the CMC This method is very useful for surfactant systems that are strong electrolytes to clearly observe the CMC by the change in slope of the plot of concentration vs conductivity Figure 24 depicts the change in slope as the surfactant concentration increases and the solution changes from free monomers in solution to micelles

Figure 24 Ionic surfactant conductivity versus surfactant concentration and determination of CMC

Conductivity equations such as the mixed electrolyte model in equation (1) developed by Shanks and Franses (Shanks and Franses 1992) describe the molar conductivities of surfactant solutions Λ This equation is expressed as the contributions from free surfactants and micelles as well as takes into


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account the molar ionic conductivities of the ions in solution and the total concentration of the solution (Cahan 1989 Shanks and Franses 1992) Conductivity can be calculated from the conductivities contributed by the micelle and free surfactants individually (Inoue et al 2007 Shanks and Franses 1992) At concentrations below micellization there are free surfactant monomers similar to free ions in solution At concentrations where micellization occurs the charges in the system are redistributed around the larger micelle aggregates where counter-ions frequently bind to the head groups of the micelle (Mitsionis and Vaimakis 2012 Shanks and Franses 1992 Inoue et al 2007 Aleiner and Usyarov 2010)

In Equation (1) which describes conductivity of surfactants in solution Λ1eq and Λmeq are equivalent molar conductance values of the monomers and the micelles respectively The equivalent molar conductivities result from the multiplication of the valence electron charge and the quantity of each species in solution β is the degree of counter ion binding Variables ct and c0 are the total surfactant concentration and monomer surfactant concentration respectively By using the CMC value to approximate c0 (c0ct) and ((ctminusc0)ct) represent the fraction of monomeric and aggregated surfactant in the solution respectively (Inoue et al 2007)

Conductivity has been used to determine the CMC of many ionic surfactant systems At room temperature SDS has a reported CMC of 02308 wt corresponding to about 8 mM in water by conductivity (Mohsenipour and Pal 2013) SDS and AOT have been investigated in methanol systems and determined a CMC of 67 mM and 87 mM respectively at 25degC(Mitsionis and Vaimakis 2012) Further some biosurfactants have been tested using conductivity in an effort to determine their CMC Mańko et al(Mańko et al 2014) has reported two samples one of di-rhamnolipid dominant and the other mono-rhamnolipid dominant to have CMCs between 00024-00026 wt at 20 degC These CMC findings were supported by other methods such as surface tension and viscosity measurements (Mańko et al 2014)

233 Surfactant Adsorption on Surfaces

Surfactants in solution adsorb onto surfaces and can form different structures based on the surfactant structure concentration environmental conditions and surface properties Many surfaces such as hydrophilic aluminum gold and silica surfaces have been investigated with synthetic surfactants to examine adsorption behavior (Thavorn et al 2014 Soares et al 2007 Dudaacutešovaacute et al 2008) Hydrophobic surfaces including gold with a carbonaceous layer and silica with reacted silanol groups to be hydrophobic have shown strong interactions with surfactant carbon chains(Thavorn et al 2014) Surfactant desorption from surfaces is also useful in determining how strongly a surfactant will adsorb on a surface resulting in either a rigid or soft layer(Dudaacutešovaacute et al 2008) This is particularly important for surfactants in environmental applications where there are many types of sediment plants and other materials to adsorb on

Surfactants adsorbed on surfaces can be studied by techniques that allow for visualization of the structures such atomic force microscopy (AFM) and scanning electron microscopy (SEM) in addition to methods such as Quartz Crystal Microbalance with Dissipation (QCM-D) which is a very sensitive tool useful in determining the amount of a substance adsorbing and desorbing from surfaces (Picas et al 2012) AFM can provide a topographic image of the surface and depth which can be very helpful in

(1)


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determining the structure of the adsorbed surfactant layer (Dedinaite et al 2004) SEM provides detailed images of the surface structures of adsorbed surfactants (Said and Alwi 2014 Akhavan et al 2013) QCM-D will be used in this work

QCM-D is a highly sensitive instrument which can measure changes in adsorbed mass as well as dissipation of solutions over a quartz crystal coated with various materials that take real time measurements of adsorption properties (Dudaacutešovaacute et al 2008 Plunkett et al 2003) In QCM-D a voltage is applied across the crystal sensor with electrodes and oscillation of the crystal is measured As solution flows across the sensor and mass is adsorbed the frequency measured decreases (Dudaacutešovaacute et al 2008 Knock and Sanii 2010) The decrease in frequency of the oscillating crystal is proportional to the adsorbed mass if the film is thin and rigid In this case the Sauerbrey relation shown in equation (2) can be used to determine the amount of adsorbed mass (Knock and Sanii 2010)

Here Δm is the adsorbed mass C is a constant characteristic of the gold coated crystal equal to 177 ngcm2middotHz n is the overtone number and Δf is the difference between the resonance frequency of the crystal sensor exposed to a solution and its value without the adsorbing molecules from solution (Dudaacutešovaacute et al 2008) Further the area per surfactant molecule A can be calculated from the Sauerbrey relation shown in Equation (3) with the Δm from equation (2) and the molecular weight of the surfactant (Knock and Sanii 2010)

Surfactant dissipation D calculated by equations (4) and (5) is an important measure of the surfactant solutions softness (Dudaacutešovaacute et al 2008) For surfactants that do not adsorb rigidly the Sauerbrey equation does not accurately reflect the actual adsorbed amount (Thavorn et al 2014 Knock and Sanii 2010)

In equation (4) Edissipated is the dissipated energy over one oscillation and Estored is the stored energy over one oscillation cycle Equation (5) is the change in dissipation of D in the pure solvent minus D throughout the experiment with the surfactant solution (Thavorn et al 2014 Merta et al 2004) ρq and tq are the thickness and the density of the crystal quartz sensor respectively and ρ l and nl are the viscosity and density of the fluid respectively (Dudaacutešovaacute et al 2008 Thavorn et al 2014) If dissipation is large then the viscoelastic model should be used to calculate the adsorbed mass Adsorption of rigid layers with no change in dissipation will follow equation (5) and for soft layers an increase in dissipation will be observed (Dudaacutešovaacute et al 2008 Ekholm et al 2002)

fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10

The Voigt viscoelastic model is given in equation (6) (Slavin et al 2012) The modeling center of the Q-Sense program software uses the Voigt model to determine properties of the layer adsorbed to the sensor such as the thickness(Liu and Kim 2009) In equation (6) G Gʹ and Gʺ are the complex shear modulus the storage modulus and the loss modulus respectively(Liu and Kim 2009) micro f and η are the elasticity frequency and shear viscosity coefficient respectively (Slavin et al 2012 Liu and Kim 2009) Given the effective density ρeff equation (7) can be used by the software to relate the determined thickness heff to the mass of the layer adsorbed (Slavin et al 2012 Liu and Kim 2009)

Previous work has studied many synthetic surfactant systems that adsorb at surfaces using QCM-D AOT adsorption on gold surfaces was researched by Thavorn et al(Thavorn et al 2014) and found that below the CMC the mass adsorbed continues to increase with increasing concentration of AOT Above the CMC adsorption remains fairly constant at about 140 ngcm2 with a very low dissipation less than 210-6 therefore the Sauerbrey relation was used to calculate the adsorbed mass(Thavorn et al 2014) For SDS at concentrations above the CMC adsorption on aluminum showed amounts of 131 to 142 ngcm2 and was readily washed off during rinsing with water (Karlsson et al 2008) At concentrations below the CMC the hydrocarbon tails of SDS adsorb to the gold surface and anionic head groups interact with the water due to polarity preferences (Soares et al 2007)

24 Oil Spills and Dispersants

As the transport of oil around the world has increased the risk and occurrence of oil spilling into the environment has also increased Traditional mechanical methods of oil spill clean-up used typically only recovers about 20 of the oil spilt (Lessard and Demarco 2000) These methods include mechanical removal oil burning and natural bioremediation(Chandrasekar et al 2006) Disadvantages to these methods are that there is a limited window of opportunity for these actions to be truly effective and often times are limited by severe weather or accessibility in implementing these methods (Li et al 2009b) Figure 25 depicts a few methods for oil recovery and the ratio of oil that could be recovered by each method in relation to the whole oil spill represented by the entire shaded area (Lessard and Demarco 2000)

From Figure 25 it is clear that dispersants which are formulated with surfactants are the most effective means to manage an oil spill by effectively treating a larger area over the same amount of time as the other remediating methods The shaded areas represent the area of oil spill treated with each method Owing to the amphiphilic nature of surfactants they aggregate at the interface of oil and water to reduce the surface tension between the two liquids (Liu et al 2015 Gong et al 2014) At sufficient surfactant concentrations self-assembly in aqueous media results in the formation of micelles In the hydrophobic interior of the surfactant micelle oil can be solubilized (Gong et al 2014 Pacwa-Plociniczak et al 2011) While at the interface between oil and water the surfactant molecules can encapsulate some of the oil into the micelle and disperse it within the aqueous phase (Liu et al 2015 De Oliveira et al 2015 Pacwa-Plociniczak et al 2011)

(6)

(7)


11

Figure 25 Amount of oil recovered per remediation method listed on the right in regards to a remote oil spill

of 10000 tons Oil spill is represented by the total shaded area Solid black area represents untreated oil From Reference 74 (Lessard and Demarco 2000)

Surfactants have many advantages to consider in determining its usefulness over more traditional clean-up procedures The ability of surfactants to assemble solubilize and disperse oil makes them particularly important for reducing the impacts of spilled oil on shoreline habitats by significantly dissipating it into the water column (Chandrasekar et al 2006 Pacwa-Plociniczak et al 2011) Dilution of oil into the water column has the added effect that it reduces the chance of droplet collision recoalescence and resurfacing into the oil slick (Li et al 2009a Gong et al 2014) Additionally surfactants can be applied over large areas in harsh weather conditions where their effectiveness is often increased The less impact the oil and surfactants have on the environment the faster it will be able to recover (Lessard and Demarco 2000 Lewis and Pryor 2013)

Following the Deepwater Horizon oil spill in the Gulf of Mexico the impacts of oil and dispersants on the environment have been studied (Beyer et al 2016) COREXITreg dispersant was predominantly used to minimize environmental impact of the oil spill As a result of dispersant used large underwater plumes of emulsified oil formed Oil also sunk to the sea bed and rose to formed surface slicks (Thibodeaux et al 2011 Beyer et al 2016) Due to increased amount of oil in the sea the dominant microorganisms present also changed The microbes capable of degrading hydrocarbons played a significant role in degrading the underwater plume and saw population spikes as a result of available oil to metabolize (Baelum et al 2012) Initially bacteria such as Oceaniserpentilla and Pseudomonas species capable of degrading heavy petroleum hydrocarbons were present and after some time aromatic degrading microbes became dominant such as Colwellia and Cycloclasticus species (Baelum et al 2012 Beyer et al 2016 Hamdan and Fulmer 2011)

The Deepwater Horizon oil spill had many lasting effect on the environment both due to the oil itself as well as dispersant use (Beyer et al 2016) AOT a component of COREXIT does not undergo rapid degradation in the environment when incorporated in the subsea level plume (White et al 2014


12

Kujawinski et al 2011) Additionally AOT has been found at higher concentrations at the seafloor after having settled there in a mixture of oil sediment and dispersant These coagulates of sand oil and dispersant have been known to wash up on shore could contribute to further toxicity to the environment if not removed (White et al 2014 Gray et al 2014)

Microorganism and animal species have also been seriously impacted by the Deepwater Horizon spill Starting at the bottom of the food web plankton has been shown to be affected by the hydrocarbons which can further propagate through the food web at high levels oil and dispersants can be toxic to plankton (Beyer et al 2016 Abbriano et al 2011) Coral species at the seabed experienced toxic effects as a result of Deepwater Horizon Additionally after the spill shrimp exposed to the chemicals experienced limited growth rates (Hamdan and Fulmer 2011) Some coastal fish were reported to have skin growths and lesions as well as reproductive issues even though coastal fish received minimal oil exposure and the tested levels of polycyclic aromatic hydrocarbons (PAH) in fish remained below public health levels of concern (Beyer et al 2016) Finally mortality rates of seabirds and turtles increased as a result of Deepwater Horizon oil spill (Beyer et al 2016)

The issue with current surfactant use in dispersants is the level of impact that pouring these chemical agents onto spill will have on the environment While today chemical dispersants are significantly lower in toxicity than most common household cleaning products the concentrations required to disperse oil can still be toxic to an ecosystem (Song et al 2013) Much effort is going into a ldquogreenrdquo alternative to traditional surfactants thus ideally surfactants will have little to no effects on the environment or its inhabitants and yet still be effective at dispersing oil as small droplets (Li et al 2009a) Bio-based and biodegradable surfactants that will be broken down with the oil by oceanic microorganisms is the best aftermath for dispersants and these are found in the form of naturally produced biosurfactants from microorganisms (Baelum et al 2012 Song et al 2013)

25 Rationale for this work

Oil pollution causes big problems for marine costal and terrestrial ecosystems(Day et al 1989) New developments have resulted in superior surface active properties of biosurfactants produced from naturally isolated and cultured organisms from the environment over synthetic surfactants (Kang et al 2010 Pornsunthorntawee et al 2008a Pornsunthorntawee et al 2008b) Biosurfactants have high environmental compatibility lesser toxicity and increase oil availability for biodegradation (Hirata et al 2009 Kang et al 2010 Day et al 1989) Additionally there is increasing demand for ldquogreenrdquo surfactants and remediation methods that are less harmful to the environment (Marti et al 2014) Synthetic surfactants pose many threats not only to plants and animals but also to humans Due to the many advantages of biological surfactants they are favored as the future for many cosmetic food oil and other industrial applications (Nitschke and Costa 2007 Rodrigues and Teixeira 2010) In order to take advantage of the unique properties of biosurfactants it is important to first understand how they assemble in solutions and adsorb on surfaces in order to create technologies that benefit us

The objective of this work is to determine and understand the physical properties of crude biosurfactants in comparison with purified biosurfactants and synthetic surfactants using fluorescence conductivity and QCM-D Biosurfactants have the ability to reduce the interfacial tension between oil and water for dispersant development and oil spill treatment As biosurfactants are still a developing field with very little literature available on their adsorption to surfaces using QCM-D we aim to study this behavior on solid surfaces as well (Muherei et al 2009)


13

3 Materials and Methods

31 Materials

Various surfactants were investigated in this study dioctyl sulfosuccinate sodium salt (Sigma BioXtra ge99) sodium dodecyl sulfate (Sigma BioXtra ge985) crude Surfactin lotActySURCE001 (Actygea) Pseudomonas aeruginosa rhamnolipid crude extract (University of Ulster) Burkholderia thailandensis rhamnolipid crude extract (University of Ulster) 95 pure rhamnolipids containing 90 mono-rhamnolipid (R95M90) (Sigma AGEA Technologies) 95 pure rhamnolipids containing 90 di-rhamnolipid (R95D90) (Sigma AGEA Technologies) sophorolipid (Actygea 66 HPLC calculated strength) and used as received

The solvent used in this study was Milli-Q grade (18 MΏ cm) water The sodium chloride (NaCl) (EMD Millipore Sigma gt99) aqueous solution also prepared with Milli-Q water Pyrene (Sigma gt99) was used for fluorescence spectroscopy QCM-D sensor cleaning solutions of ammonium hydroxide (Sigma Aldrich 25) and hydrogen peroxide (Fisher Scientific 30) were used as received

32 Methods

This section describes the use of fluorescence spectroscopy conductivity and quartz-crystal microbalance with dissipation methods to determine surfactant aggregation properties All experiments were conducted at room temperature and at various surfactant concentrations above and below the CMC

321 Sample Preparation

Surfactant stock solutions are made at various wt concentrations above and below the CMC that vary depending on the surfactant of interest and the value of its CMC Stock solutions are made by dissolving the surfactant crude or pure in Milli-Q water and allowing them to mix on a rotator for 24 hours If necessary other samples can be prepared from the stock solution by dilutions with Milli-Q water until the desired wt concentration is attained The new samples were allowed to mix again for 24 hours before conducting the experiments giving time for the solutions to equilibrate

To analyze the surfactant behavior in an environment similar to that of sea water containing 35 ppt (parts per thousand) salt small masses of concentrated NaCl solution were added to each sample in order to achieve a salt content of 35 wt without significantly altering their original surfactant concentration (Skop et al 1993) These samples were again vortexed briefly allowed to mix on a rotator and analyzed in the fluorescence spectrophotometer

322 Fluorescence Spectroscopy

A Hitachi Fluorescence Spectrophotometer F-2500 was used to conduct the fluorescence experiments at room temperature An excitation beam of 335 nm was used and data was recorded between 340 and 460 nm The emission excitation of pyrene consists of five peaks between 340 and 460 nm (Dong and Winnik 1984) In fluorescence an excitation wavelength interacts with the pyrene molecule in the samples to cause an electron to jump to a higher vibronic level As that excess energy is released and the electron relaxes to a lower vibronic state it emits that excess energy in the form of a photon (Madsen et


14

al 2015 Mitsionis and Vaimakis 2012) Pyrene is used because it is sensitive to solvent polarity and useful in measuring the CMC of surfactant systems as discussed in section 231

3221 Pyrene Solution Preparation and Experimental Method

A 1 millimolar solution of pyrene is made by dissolving 001 grams of pyrene in 50 milliliters of ethanol This solution is mixed for at least 24 hours before addition to surfactant samples prepared as described in section 321 (Nivaggioli et al 1995 Sarkar et al 2013) 2 microL of pyrene is added to each surfactant solution vortexed and allowed to mix on a rotator before running in the spectrophotometer The final concentration ranges for all biosurfactants and AOT start at 000025 wt through 10 wt solutions SDS sample concentrations ranged from 00025 wt up to 10 wt The R95M90 sample range went from 000025wt to 01wt in an effort to consume less of the sample (Mańko et al 2014) Sophorolipid crude surfactant concentrations for fluorescence ranged from 0000025wt to 01wt Surfactant concentrations were analyzed on a log scale and spaced equally with three concentrations per order of magnitude for all surfactants Concentration ranges were selected based on previous literature data and centering the sample concentrations around the CMC (Mitsionis and Vaimakis 2012)

Samples were analyzed starting with the low surfactant concentrations to prevent possible higher concentrations from contaminating the readings of lower surfactant concentration samples Small volumes of the sample were first added to the cuvette and used to rinse the inside of any previous sample These portions were then collected as waste and another small portion of the sample was pipetted into the cuvette the 14 mL cuvette used has a thin channel in the middle The cuvette was then wiped down to remove any residue or fingerprints on the outside and inserted into the spectrophotometer to be analyzed

3222 Pyrene Fluorescence Data Analysis

The results were analyzed and the intensity maximum of peaks one and three were identified from the data These values were then recorded and the ratios of intensity peaks 13 were plotted against the surfactant concentration to determine the critical micelle concentration (CMC) when the pyrene is in a hydrophobic environment The same analysis was used for the data of surfactants in 35 wt NaCl aqueous solution

CMC determination was done using two methods similar to the work done by Aguiar et al(Aguiar et al 2003) for fluorescence measurements through analysis of the I1I3 ratios (Aguiar et al 2003 Regev and Zana 1999) The first method takes the derivate of the I1I3 versus concentration data and uses the point at which the derivative is at a minimum as the CMC value this is typical of nonionic surfactants The minimum of the derivative corresponds to the change in concavity of the sigmoid curve of I1I3 versus surfactant concentration Another method involves fitting lines to the vertical portion of the data and to the lower limit of the data and finding where these two line intersect this method is typically more accurate for ionic surfactant CMCs (Aguiar et al 2003) Igor Pro software was used to differentiate the data and find the minimum value of the curve as the CMC For the later method the data was manually fitted to linear equations and the intersection point was identified as the CMC


15

Figure 31 Figure taken from Aguiar et al(Aguiar et al 2003) depicts the locations of CMC1 and CMC2 values

obtained from the I1I3 pyrene ratio

A visual of the I1I3 data versus surfactant concentration is shown in Figure 31 as well as the CMC values that can be obtained depending on the method used to evaluate the data A2 refers to the lower limit of the data A1 the upper limit and f(x) is the linear fit of the data over the decreasing portion of the sigmoid

323 Conductivity

Conductivity is used to determine the CMC of ionic surfactant solutions where there will be a change in conductivity designated by a change in slope of the plotted data Below the CMC ionic surfactants and their counter ions dissolve in the water or solvent increasing the number of free conducing species in solution (Chakraborty et al 2007) Above the CMC surfactants assemble into micelles and partially bind the ions in solution to decrease the overall number of conducting species causing a more gradual increase in the slope of the conductivity vs concentration curve above the CMC (Aleiner and Usyarov 2010)

An Accumet Model 20 pHConductivity Meter with a 01cm cell constant probe was used to perform the conductivity experiments The instrument was calibrated with a 100 microScm standard at 25 degC (RICCA 472 ppm TDS as NaCl) before beginning the experiment The probe was submerged in the sample with continuous stirring a plastic syringe was used for dilutions after each stable meter reading Conductivity meter readings were plotted against the sample concentrations in order to identify the CMC

To preform conductivity experiments each surfactant was analyzed individually and was conducted using the same procedure described as follows Small volumes of concentrated surfactant solutions were diluted with water to analyze a large range of concentrations (due to limited amount of sample available) Surfactin (Lot ActySURCE001) concentration range was 01 wt to 0 01344 wt Pseudomonas aeruginosa rhamnolipid concentration ranges from 002 to 000267 wt These ranges were selected do to prior fluorescence testing for CMC determination Additionally SDS and AOT were measured for comparison


16

To determine the CMC a plot of concentration versus conductivity was constructed The CMC of the surfactant is designated by a change in slope of the plotted data Determination of the CMC from conductivity data was done by fitting the majority of the data into two linear equations one for data at a steep slope and one for the data that fit a less steep slope and finding the intersection of these two lines of best fit The intersection of these lines is taken as the CMC value The molar conductivity from equation (1) was not used to analyze the conductivity data obtained here

324 Quartz Crystal Microbalance with Dissipation

A Quartz-Crystal Microbalance from Q-Sense Biolin Scientific was the instrument used to measure the adsorption of surfactants on gold aluminum and silica surfaces Once the base line was established using Milli-Q water then surfactants solution was then passed over the sensor until the frequency measurement becomes stable within 02-05 Hz Then washing with Milli-Q water begins until the frequency readings again become stable All experiments were run at 25degC with a flow rate of 01 mLmin for the baseline and surfactant solutions QCM-D tracks the mass changes of molecules on a surface by measuring the frequency of the oscillating crystal as solution is passed over it QCM-D also determines the dissipation factor which identifies whether or not the adsorbed film is rigid or flexible in relation to the voltage across the crystal surface (Tavakkoli et al 2013) A schematic of the QMC-D instrument is shown in Figure 32

Figure 32 Schematic of Quartz Crystal Microbalance instrument

The resulting frequency and dissipation data can be analyzed using one of two methods based on the change in dissipation (ΔD) as described in section 233 If ΔD is very small or less than 210-6 the Sauerbrey relation can be used to analyze the data (Thavorn et al 2014) If the dissipation is small then the adsorbed surfactant layer follows three assumption made by the Sauerbrey relation in equation (2) the mass adsorbed is small relative to the crystal it is evenly distributed and it forms a rigid layer (Plunkett et al 2003) For a large ΔD these assumptions are not satisfied and the viscoelastic model can be utilized for data analysis The area per molecule of adsorbed surfactant can additionally be found from equation (3) using the mass adsorbed surfactant molecular weight and Avogadrorsquos number

The gold coated quartz crystal sensor was cleaned before and after each use The sensor was first inserted into a chamber below a UV lamp for 10 minutes Next a 5 to 1 to 1 mixture of water 25 ammonia and 30 hydrogen peroxide was heated to 75degC (Mivehi et al 2011) The sensor was submerged in this solution for five minutes at 75degC and then rinsed thoroughly with Milli-Q water Nitrogen gas was then used to completely dry the sensor and remove any rinse water (Thavorn et al


17

2014) Finally the sensor was again put under the UV lamp for 10 minutes before placing in the module (Mivehi et al 2011)

The QCM-D module was cleaned first by a 2 wt SDS solution by flowing the solution through the module at 03 mLmin flow rate for 30 minutes The solution was then changed to Milli-Q water for an additional 30 minutes of cleaning The module was then disassembled dried with nitrogen gas and a clean sensor was inserted into the module for measurements

4 Results

The results of the surfactant micellization and adsorption experiments are reported and discussed in this chapter Six biosurfactants 95 pure rhamnolipids containing 90 mono-rhamnolipid (R95M90) 95 pure rhamnolipids containing 90 di-rhamnolipid (R95D90) Burkholderia thailandensis rhamnolipid crude extract Pseudomonas aeruginosa rhamnolipid crude extract sophorolipids and Surfactin (Lot ActySURCE001) were analyzed in comparison to the synthetic surfactants sodium dodecyl sulfate (SDS) and dioctyl sulfosuccinate sodium salt (AOT) (chemical structures for the various surfactants depicted in figures 21) Experiments were carried out at 25degC in deionized water and in NaCl aqueous solutions

41 Solution Assembly of Synthetic Surfactants

Pyrene I1I3 fluorescence intensity ratio results for SDS aqueous solutions are shown in Figure 41 The lower I1I3 ratios of the solid circle data points in Figure 41 for SDS show a CMC of about 021-025 wt which is equivalent to a CMC of about 8-9 mM which corresponds to the literature CMC for SDS (Shanks and Franses 1992 Knock and Sanii 2010) Our CMC is between 023-025 wt which is consistent with the literature value of 025 wt CMC for SDS (Knock and Sanii 2010) These values were obtained from conductivity and fluorescence CMC2 fitting methods as discussed in section 322 Figure 42 shows the value obtained from CMC1 fitting methods as indicated by the derivative line According to the derivative method CMC1 of SDS deionized water is 021 wt

Figure 41 Pyrene I1I3 fluorescence intensity ratios of SDS in 0 2 and 35 wt NaCl aqueous solution


18

Figure 42 Pyrene I1I3 fluorescence intensity ratios of SDS in 0 2 and 35 wt NaCl aqueous solution and

derivatives identifying CMC1

Figure 43 shows the conductivity for SDS in deionized water A CMC value of 023 wt was identified from fitting the data There is a sharp definition at the break point in the conductivity data where the CMC is located as expected of geometrically linear and ionic surfactant molecules (Mohsenipour and Pal 2013 Mitsionis and Vaimakis 2012) SDS has a reported CMC of 02308 wt corresponding to about 8 mM in water by conductivity at 25 degC in agreement with our results (Mohsenipour and Pal 2013)

Figure 44 shows the break point of the conductivity data matches up well with the CMC obtained by the fluorescence data confirming CMC The correspondence between the higher conductivity breakpoint and the I1I3 fluorescence data for CMC2 shows a better agreement for determining the CMC This agreement is additionally confirmed in Figure 48

Figure 43 Conductivity data of SDS in deionized water

With the addition of sodium chloride salt solution to SDS samples a significant reduction in the CMC is observed Figure 41 shows the effects at 2 and 35 wt NaCl on the CMC of synthetic surfactant SDS The CMC of SDS drops from 025 wt to about 0026 wt in 2 wt salt and 019 wt in 35 wt salt


19

based on the analysis for CMC2 The CMC of SDS is reported to drop to concentrations as low as 0011 wt presence of 29 wt salt at 25 degC (Umlong and Ismail 2005 Umlong and Ismail 2007)

Figure 44 Conductivity data and pyrene I1I3 fluorescence intensity ratios for SDS in deionized water

Figure 45 Pyrene I1I3 fluorescence intensity ratios for AOT in 0 2 and 35 wt NaCl aqueous solutions

For the second synthetic surfactant considered here AOT the CMC in deionized water is at about 017-018 wt this value is a little higher than the value reported by Grille et al(Grillo and Penfold 2011) of 313 mM or 014 wt The CMC of AOT at 017 wt was found by using the CMC2 determination method and is in agreement with the CMC found from conductivity at 018 wt as shown in Figures 45 and 47 respectively The CMC obtained by CMC1 fitting methods from section 322 for AOT in deionized would result in a CMC at 012 wt shown in Figure 46 Agreement between the higher conductivity breakpoint and the I1I3 fluorescence data for CMC2 again occurs for AOT which is shown in Figure 48


20

Figure 46 Pyrene I1I3 fluorescence intensity ratios for AOT in 0 2 and 35 wt NaCl aqueous solutions and


Similar to the results observed for SDS AOT in NaCl aqueous solutions has a decrease in the CMC The CMC reduces from 017 wt in deionized water to 0015 wt and 0018 wt for both 2 and 35 wt salt respectively and the results are shown in Figure 45 The CMC of AOT is reported to drop to as low as 0009 wt for AOT in the presence of 11 wt salt at 25 degC (Umlong and Ismail 2005 Umlong and Ismail 2007) Figure 48 shows the comparison between fluorescence and conductivity data CMCs determined from each method are fairly consistent

Figure 47 Conductivity data of AOT in deionized water


21

Figure 48 Conductivity data and pyrene I1I3 fluorescence intensity ratios of aqueous AOT in deionized water

In Figure 49 we plot the pyrene I1I3 fluorescence intensity ratios of both SDS and AOT in deionized water and in 35 wt NaCl aqueous solution While both surfactants exhibit a reduction in CMC as a result of added electrolyte the AOT micellar solution changes to a different structure In concentrations of salt solutions from 003 to 009 wt the micelles of AOT become elongated into cylindrical rod-like structures (Dey et al 2010) This change in shape of the aggregates causes a thickening of the surfactant solution and a cloudy like appearance The decrease in CMCs of both SDS and AOT as charged synthetic surfactants in the presence of salt is consistent with literature (Umlong and Ismail 2007 Umlong and Ismail 2005)

Figure 49 Pyrene I1I3 fluorescence intensity ratios of SDS and AOT in 0 and 35 wt NaCl aqueous solutions


22

42 Solution Assembly of Biosurfactants

Pyrene I1I3 fluorescence intensity ratios versus R95M90 and R95D90 surfactant concentrations are shown in Figure 410 The R95M90 surfactant in deionized water has CMCs at 0018 wt and 0059 wt using the CMC1 and CMC2 methods respectively Similar to our results for R95M90 R95D90 had a CMC1 of 0033 wt and CMC2 of 0062 wt in deionized water

Figure 410 Pyrene I1I3 fluorescence intensity ratios for R95M90 and R95D90 in deionized water

Figure 411 shows the results of both R95M90 and R95D90 in 47 wt and 35 wt NaCl aqueous solutions respectively To some of the R95M90 samples the added NaCl was too high and caused them to become concentrated above 35 wt NaCl these samples were measured in an effort to preserve the limited sample available and the average of the NaCl concentrations in the samples were reported in Figures 411 and 414 Upon adding NaCl the R95M90 semi-purified biosurfactant showed a significant decrease in the CMC which was observed from 0018 wt to 0003 wt at CMC1 This reduction in CMC is larger than what will be seen from the crude biosurfactants in Figures 415 419 and 420 and is more similar to the trends of synthetic surfactants from deionized to salt water environments The reduction in CMC of the semi-purified R95M90 and R95D90 samples in salt water could be due to shielding of the partially charged head groups by the salt ions (Dey et al 2010) Similarly R95D90 sample showed a very comparable trend to the R95M90 sample with a CMC at 0033 wt in deionized water and 0005 wt in 35 wt NaCl aqueous solution Figure 412 uses R95D90 data to identify how the CMC values were obtained by finding the derivative of the sigmoid I1I3 data for CMC1 and the lower limit of the sigmoid curve for CMC2


23

Figure 411 Pyrene I1I3 fluorescence intensity ratios for R95M90 and R95D90 in NaCl aqueous solution

Figure 412 Plot of I1I3 versus surfactant concentration demonstrating how CMC1 and CMC2 were determined

using the R95D90 data as an example

The CMCs obtained in our study are higher than those reported in the literature Mańko et al(Mańko et al 2014) uses similar samples of 95 pure rhamnolipids and obtained a CMC of 00024 to 00026 wt from conductivity surface tension and viscosity measurements The R95M90 has 90 mono-rhamnolipid homologues in the sample compared to the R95D90 which is 90 di-rhamnolipid Having two rhamnose molecules attached to the surfactant makes for a much larger head group and can sometimes cause a change in aggregation depending on how the surfactant molecules pack together resulting in a lower CMC Our results do not reflect this expectation the R95M90 and R95D90 rhamnolipid samples have similar CMCs in regards to each other The literature as does not report a difference in CMCs between mono- and di-rhamnolipid samples (Mańko et al 2014)

Even in a high salt environment the R95M90 and R95D90 samples are not reaching the CMC values for mono- and di-rhamnolipids previously reported in deionized water (Weckman et al 2014 Mańko et al 2014) Figure 413 is taken from Mańko et al(Mańko et al 2014) showing the conductivity data and the


24

CMC obtained of about 00025 wt for mono-rhamnolipids and di-rhamnolipids Conductivity experiments were conducted at 20 degC and the rhamnolipid samples were dissolved in deionized water (Mańko et al 2014) Figure 413 includes very few data points compared to the conductivity experiments conducted in this study Additionally the rhamnolipid samples in deionized water from Mańko et al(Mańko et al 2014) had much higher conductivities just below 20 mScm while our crude rhamnolipid samples in deionized water conducted less than 04 mScm The discrepancy between the CMCs identified and those reported in literature could be attributed to the variation in abundance of congeners of rhamnolipid molecules in reference to their hydrocarbon chain lengths and experimental error A comparison of both the mono-rhamnolipid and di-rhamnolipid samples in deionized and 35 wt NaCl aqueous solution is shown in Figure 414

Figure 413 Conductivity data at 20 degC of mono-rhamnolipid dominant and di-rhamnolipid dominant samples in

deionized water corresponding to curves 1 and 2 respectively Graph copied from reference 47(Mańko et al 2014) Copyright (2014) Elsevier


25

Figure 414 Pyrene I1I3 fluorescence intensity ratios for R95M90 and R95D90 in aqueous solutions with and

without NaCl

The results from the pyrene I1I3 fluorescence intensity ratio experiments of the crude rhamnolipid obtained from Burkholderia thailandensis are depicted in Figure 415 Burkholderia thailandensis rhamnolipid crude extract has a 0004 wt CMC1 and 0011 wt CMC2 Although the data does not seem to level consistently at the higher rhamnolipid concentrations from 0025 to 05 wt making it more difficult to determine the CMC analysis was subsequently carried out as previously described for the Burkholderia thailandensis extract As the pyrene is solubilized in the biosurfactant micelles the intensity ratios of pyrene peaks should become close to 10 however this does not happen for Burkholderia thailandensis rhamnolipid extract perhaps indicating a higher level of impurities A CMC of 00026 wt was reported by Costa et al (Costa et al 2011) determined from surface tension measurements of a Burkholderia glumae strain producing rhamnolipids The rhamnolipid sample obtained from Burkholderia thailandensis considered in our study does not appear to have a well-defined CMC and was therefore only studied in deionized water and not in NaCl aqueous solution


26

Figure 415 Pyrene I1I3 fluorescence intensity ratios for crude rhamnolipids obtained from Burkholderia

thailandensis and crude rhamnolipids obtained from Pseudomonas aeruginosa in deionized water

The Pseudomonas aeruginosa rhamnolipid crude extract has CMC1 and CMC2 at 0004 wt and 0006 wt respectively also seen in Figure 415 The conductivity of crude rhamnolipids obtained from Pseudomonas aeruginosa in Figure 416 shows a CMC at 0006 wt which matches fairly well with CMC2

for Pseudomonas A however the range of concentrations does no go low enough to be able to detect a change at CMC1 Mańko et al(Mańko et al 2014) reported that mono-rhamnolipid and di-rhamnolipid dominant samples both have CMCs between 00024-00026 wt using conductivity again just outside of the range examined in this study Also note that the break point of the conductivity profile for the crude rhamnolipid is not as clearly defined as for the synthetic surfactants

The data for SDS and AOT conductivity measurements shown in figures 43 and 47 have much more defined slope changes at the CMCs than biosurfactants One possible explanation for the less defined conductivity profile has to do with the size and charge on the surfactants that affects attractive and repulsive forces during assembly For the synthetic surfactants the chemical structures are fairly linear with a small negatively charged head group comparably The biosurfactants are both bulky and large without a single point charged head group (Loacutepez-Diacuteaz and Velaacutezquez 2007) The poor conductivity of biosurfactants is due to their non-electrolytic character and do not dissociate in water (Senese and Brady 2009) These molecules have partial charges but do not conduct electricity to the same magnitude strong electrolytic solutions do which almost completely dissociate in water (Senese and Brady 2009) A study using another biosurfactant flavolipid showed a comparable conductivity profile to those obtained in this work for biosurfactants (Kim and Vipulanandan 2006) Conductivity equations take into account the molar ionic conductivities of the ions in solution and the total concentration of the surfactant solutions (Cahan 1989)


27

Figure 416 Conductivity of crude rhamnolipids obtained from Pseudomonas aeruginosa in deionized water

Figure 417 shows the comparison between fluorescence and conductivity data and the CMCs that can be determined using each method for the crude rhamnolipids obtained from Pseudomonas aeruginosa Figure 418 shows the reduction in CMC of the crude Pseudomonas A when in 35 wt NaCl aqueous solution this drop in CMC is not very significant The CMCs of several rhamnolipid samples with various homologue concentrations obtained from various Pseudomonas and Burkholderia bacteria strains have been reported across a range of concentrations between 5 and 200 mgL or 00005 wt to 002 wt using methods including surface tension viscosity density conductivity and fluorescence (Kosaric and Sukan 1993 Mańko et al 2014)

Figure 417 Conductivity data and pyrene I1I3 fluorescence intensity ratios of aqueous rhamnolipids obtained

from Pseudomonas aeruginosa in deionized water


28

Figure 418 Pyrene I1I3 fluorescence intensity ratios for the crude rhamnolipids obtained from Pseudomonas

aeruginosa in aqueous solution with and without NaCl

Figure 419 shows the pyrene fluorescence intensity I1I3 ratio data for sophorolipids From the analysis of the I1I3 ratios CMC1 is found at 000016 wt and CMC2 at 00002 wt in deionized water The pyrene intensity ratios of the sophorolipid samples in deionized water and in 35 wt salt solution are shown for comparison in Figure 419 The CMC1 and CMC2 values are quite similar both in deionized water and in the salt solution The sophorolipid surfactant shows a CMC considerably lower than any other surfactant considered here at 00002wt and this value stays fairly consistent in the salt aqueous solution Daverey et al(Daverey et al 2011) reported a CMC for sophorolipids of 00045 wt in deionized water at 25 degC by surface tension measurements and is in agreement with other work (Daverey et al 2011 Hirata et al 2009)

Figure 419 Pyrene I1I3 fluorescence intensity ratios for the crude sophorolipids in aqueous solution with and

without NaCl

Surfactin pyrene I1I3 fluorescence intensity ratio data are shown in Figure 420 Crude Surfactin (LotActySURCE001) sample has CMC1 at 0004 CMC2 at about 003 wt as the fluorescence I1I3 ratios


29

decrease at the higher Surfactin concentrations Surfactin produced by Bacillus subtilis and purified to greater than 95 purity was found to have a CMC of 00015 wt at 25 degC an order of magnitude lower than the crude Surfactin (Lot ActySURCE001) sample studied in the present work (Marti et al 2014) In comparison to literature the CMC values of the biosurfactants in this study suggest that CMC1 (see section 322) may be a more accurate measure of the critical micelle concentration of biosurfactants

From the conductivity measurements of Surfactin shown in Figure 421 the CMC looks to be at 004 wt however again the range of surfactant concentrations does not drop low enough to encompass the concentration range from well below CMC1 Although the CMC values for fluorescence and conductivity match up fairly well it is difficult to discern the true CMC until a full range of conductivity measurements can be made Still the conductivity profile of Surfactin as a biosurfactant is much less defined at the break point Another reason for the less defined conductivity data is that the crude extracts may contain other charged molecules that interfere more when preforming conductivity experiments than with fluorescence Because SDS and AOT are commercially manufactured and relatively pure they will not show these contaminants that are in crude biosurfactants extracted from cell cultures Furthermore crude biosurfactants may contain different concentrations of homologues and other chemical components from the growth culture that can reduce the CMC and affect assembly at the CMC (Monteiro et al 2007)

Figure 420 Pyrene I1I3 fluorescence intensity ratios for the crude Surfactin in aqueous solutions with and

without NaCl


30

Figure 421 Conductivity of Surfactin in deionized water

Figure 422 Conductivity data and pyrene I1I3 fluorescence intensity ratios of Surfactin in deionized water

Figure 423 shows the pryene I1I3 fluorescence intensity ratio data in deionized water for all the synthetic and biosurfactants used in this study According to these data that surfactants typically have much lower CMCs even than some nonionic surfactants (Mańko et al 2014) Among the several biosurfactants considered here sophorolipids unmistakably has the lowest CMC nearly three orders of magnitude lower than the CMCs of SDS and AOT


31

Figure 423 Pyrene I1I3 fluorescence intensity ratios for four crude biosurfactants R95M90 R95D90 sodium

dodecyl sulfate (SDS) and dioctyl sodium sulfosuccinate (AOT) in deionized water

All critical micelle concentration data obtained in this study are summarized in Table 1 From the data in Figure 424 the three crude biosurfactants examined in both deionized water and aqueous NaCl solutions do not appear to have a significant change in CMC at 35 wt NaCl These results are in line with findings from Pornsunthorntawee et al(Pornsunthorntawee et al 2009) who measured rhamnolipid surfactant solution surface tensions above and below the CMC at 25 to 27 degC and observed no change after the addition of NaCl indicating that the rhamnolipids tested have a high tolerance for NaCl concentrations of 04 M or 23 wt equivalent (Pornsunthorntawee et al 2009)


dodecyl sulfate (SDS) and dioctyl sodium sulfosuccinate (AOT) in NaCl aqueous solution

Depending on which method is used to determine the CMC different values may be obtained Some work suggests that CMC1 is more indicative of the CMC of non-ionic surfactants and CMC2 is more accurate for ionic surfactants (Aguiar et al 2003) Based on the CMCs found in this work CMC2 is


32

appropriate for the synthetic surfactants and CMC1 could be better fit for the biosurfactants because the conductivity results of the crude rhamnolipid obtained from Pseudomonas aeruginosa and Surfactin samples were inconclusive

Table 1 Summary of surfactants used and CMCs identified using different analysis methods The abbreviations for surfactants in Table 1 are sodium dodecyl sulfate (SDS) dioctyl sulfosuccinate sodium salt (AOT) Pseudomonas aeruginosa rhamnolipid crude extract (PARh) Burkholderia thailandensis rhamnolipid crude extract (BTRh) 95 pure rhamnolipids containing 90 mono-rhamnolipid (R95M90) 95 pure rhamnolipids containing 90 di-rhamnolipid (R95D90) sophorolipid (SPL) and crude Surfactin (LotActySURCE001) (Surfactin)

Surfactant Type NaCl (wt)

Technique Conductivity Fluorescence Spectroscopy Break Point CMC (wt)

Derivative CMC (wt)

Linear Intercept CMC (wt)

SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011

BT Rh Natural 0 0004 0011 PA Rh Natural 0 001 0004 0006

35 0002 0005 SPL Natural 0 00002 00002

35 00001 00002 Surfactin Natural 0 004 0004 0031

35 0005 0025

43 Adsorption of Synthetic Surfactants on Surfaces

The QCM-D frequency and dissipation data discussed in sections 43 and 44 show surfactant adsorption on gold surfaces After a baseline is established on the frequency measurements of Figures 425 through 446 the surfactant solution is flown across the sensor surface indicated by a decrease in Δf Once the frequency shift is stabilized indicated by constant frequency measurements rinsing will begin with Milli-Q deionized water The dissipation shift is shown is measured along the right y-axis


33

Figure 425 shows the QCM-D frequency and dissipation shifts of the gold sensor over time responding to exposure to SDS in deionized water The reduction in frequency shift is minimal to about 10 Hz and returns to zero after rinsing Additionally the dissipation change was very small less than 210-6 when the surfactant solution was flowing over the crystal suggesting that a thin rigid layer forms These assumptions are in agreement with the Sauerbrey model and therefore this method was used to calculate the adsorbed mass Roughly 1116 ngcm2 was adsorbed to the crystal surface using equation (2) in section 233 Using the Sauerbrey relation in addition to the stable frequency shift measurement the gold coated quartz crystal constant C of 177 ngcm2middotHz and the overtone numbers 5 through 11 can be used to calculate the mass adsorbed

Similarly for SDS in salt solutions small changes in the frequency and dissipation shifts were observed and the Sauerbrey relation was used to find the adsorbed mass as described in section 233 Figures 426 and 427 show the frequency and dissipation changes of the gold sensor over time responding to exposure to 05 wt SDS in 2 wt and 35 wt NaCl aqueous solutions respectively Slightly more SDS surfactant is adsorbed when in the 2wt solution than in the 35 wt NaCl solution In the 2 wt NaCl aqueous solution SDS adsorbed at a steady rate and was removed completely by rinsing with deionized water In 35 wt salt SDS adsorbed to a mass of 1147 ngcm2 and stabilized very soon after the surfactant solution was introduced After rinsing as indicated by the second black arrow in Figure 427 all the mass adsorbed was removed from the gold surface

Figure 425 Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a

05 wt SDS aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

For the baseline of the SDS solutions in 2 wt and 35 wt NaCl aqueous solution pure salt solutions at each concentration were used The baseline frequency shift was slightly positive during this time and can account for the slightly higher frequency shift above zero after the rinsing has been completed Adsorbed mass is summarized in Table 2 Figure 428 shows a comparison of the frequency and dissipation of the gold sensor over time responding to exposure to SDS in deionized water and SDS in 35 wt NaCl aqueous solution The time scales are different based on the amount of time it took to consistently and accurately obtain a stable frequency reading on the sensor

Δ

Δ


34


05 wt SDS in 2 wt NaCl aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated



SDS in deionized water has reported to have adsorbed molecular areas between 420 and 590 Aring2molecule (Knock and Sanii 2010) In the present work the calculated molecular area from equation (3) in section 233 using the molecular weight of SDS at 28837 gmol resulted in 4291 Aring2molecule which is in agreement with the published data

Δ

Δ

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Δ


35

Figure 428 A Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a

05 wt SDS aqueous solution B Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 05 wt SDS in 35 wt NaCl aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

Frequency and dissipation changes of the gold sensor over time responding to exposure to AOT in deionized water are shown in Figure 429 Similar to the results for SDS AOT adsorbs minimally in deionized water to the gold surface and is completely removed during rinsing The dissipation is less than 210-6 and therefore the Sauerbrey relation from equation (2) was used to calculate an adsorbed mass of 905 ngcm2 (Thavorn et al 2014) This calculation came from the use of the stable frequency shift measured the gold coated quartz crystal constant C of 177 ngcm2middotHz and the overtone numbers 5 through 11 At concentrations above the CMC the mass of adsorbed AOT in water was found to be as high as 146 ngcm2 on gold (Thavorn et al 2014) The molecular area of 8157 calculated in this study is slightly higher than published data of 60 to 71 Aring2molecule indicating looser packing of surfactant molecules and that the monolayer adsorbed on the surface was not complete (Knock and Sanii 2010)


36


05 wt AOT aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated


05 wt AOT in 2 wt NaCl aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

AOT in 2wt salt water adsorbs much more significantly to gold surfaces as can be seen from the frequency and dissipation changes over time of the gold sensor in Figure 430 The adsorbed mass is calculated using the Voigt viscoelastic model from equations (6) and (7) because the change in dissipation is larger than 210-6 The resulting mass calculated is 22187 ngcm2 After rinsing with deionized water the adsorbed mass is able to be removed immediately and the frequency changes stabilize quickly Figure 431 shows the comparison of the frequency and dissipation shifts of the gold sensor over time responding to exposure to AOT in deionized water and in 2 wt NaCl aqueous solution The time it took to reach stable frequency measurements on the sensor were longer for AOT in the salt solution in addition to across different overtone numbers

Δ

Δ

Δ

Δ


37


05 wt AOT aqueous solution B Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 05 wt AOT in 2 wt NaCl aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

The difference in adsorption between SDS and AOT can be accounted for by the change in structure of AOT in NaCl aqueous solutions SDS and AOT surfactant molecules in salt solutions form micelles at lower concentrations than in deionized water Salt can also change the shape and size of the micellar structure In NaCl aqueous solutions as low as 5 to 15 mM AOT micelles are reported to become elongated and cylindrical which explains why the amount adsorbed to the gold surface increases (Dey et al 2010) Larger structures of AOT are adsorbing to the gold surface in NaCl aqueous solutions through the same physical interactions as AOT did in deionized water however now larger structures are adsorbing on the surface beyond the simple surface coverage which makes the dissipation important to measure (Somasundaran and Krishnakumar 1997) The molecules adsorbed per centimeter squared and area per molecule were calculated for both SDS and AOT using equation (3) of section 233 these values are found in Table 2

Δ

Δ

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Δ


38

44 Adsorption of Biosurfactants on Surfaces

The adsorption frequency and dissipation shifts of the gold sensor over time responding to exposure to R95M90 in deionized water are shown in Figures 432 In Figure 432 R95M90 is dissolved in deionized water and shows similar frequency and dissipation changes to the synthetic surfactant adsorption in deionized water at concentrations above the CMC The dissipation is less than 210-6 and the Sauerbrey relation is applicable to calculate the adsorbed mass as well as the mass remaining after rinsing Before rinsing there is 1781 ngcm2 on the gold surface and 786 gcm2 remains after washing with deionized water The mass calculated came from the use of the stable frequency shift measured before and after rinsing the gold coated quartz crystal constant C of 177 ngcm2middotHz and the overtone number from 5 through 11

The frequency and dissipation changes of the gold sensor over time responding to exposure to R95M90 in 35 wt NaCl aqueous solution is depicted in Figure 433 There is a significant increase in the dissipation above 210-6 indicating that the adsorbed layer is viscoelastic (Thavorn et al 2014) The mass adsorbed using the Voigt viscoelastic model from equations (6) and (7) in section 233 is 6977 ngcm2 adsorbed before rinsing with deionized water 903 ngcm2 remains on the sensor surface after rinsing


01 wt R95M90 aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

Δ

Δ


39


01 wt R95M90 in 35 wt NaCl aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

Somasundaran et al(Somasundaran and Krishnakumar 1997) discuss bonding to surfaces based on the strength and type of interactions electrostatic bonding being weak and easily removed whereas hydrophobic bonding of surfactant hydrocarbon tails more strongly adheres to surfaces and are not easily removed The semi-purified biosurfactants may be experiencing a mixture of these interactions to have mass remaining adsorbed to the sensor after rinsing (Somasundaran and Krishnakumar 1997) Figure 434 plots the QCM-D frequency and dissipation shifts of the gold sensor over time responding to exposure to R95M90 in deionized water and in 35 wt NaCl aqueous solution The time it took to reach stable readings were different for the R95M90 in deionized water and the NaCl aqueous solution The R95M90 salt solution was given extra time to ensure stable measurements

Δ

Δ


40


01 wt R95M90 aqueous solution B Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt R95M90 in 35 wt NaCl aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

Figures 435 and 436 show the frequency and dissipation shifts of the gold sensor over time responding to exposure to 01 wt R95D90 in deionized water and in 35 wt NaCl aqueous solution respectively R95D90 has an adsorbed mass of 126 ngcm2 before rinsing and 361 ngcm2 remains after rinsing using the Sauerbrey relation from equation (2) in section 233 The dissipation is less than 210-6 therefore the Sauerbrey relation is used to calculate the adsorbed mass on the sensor This calculation used the stable frequency shift measured before and after rinsing the gold coated quartz crystal constant C of 177 ngcm2middotHz and the overtone number from 5 through 11


41


01 wt R95D90 aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

In a 35 wt NaCl aqueous solution the adsorbed R95D90 surfactant molecules behaves as a viscoelastic layer based on the increased dissipation above 210-6 (Thavorn et al 2014) Using the Q-Sense modeling center a mass of 7372 ngcm2 adsorbed to the surface After rinsing with deionized water 670 ngcm2 remained adsorbed to the sensor surface

Figure 437 shows the sensor frequency and dissipation changes of the gold sensor over time responding to exposure to R95D90 in deionized water and in 35 wt NaCl aqueous solution After having conducted the experiments for R95M90 in both salt and deionized water we were able to see that the frequency changes remained stable over a long period of time Therefore for the R95D90 sample after the frequency stabilized rising was initiated and the time scales matched up for better comparison The molecules per centimeter squared were additionally calculated for the sigma R95M90 and R95D90 surfactants based on equation (3) The molecular weights used for R95M90 and R95D90 necessary for this calculation are 5047 gmol and 6508 gmol respectively The results are summarized in Table 2 The results of mass adsorbed and molecules per area are again similar for both R95M90 and R95D90 While the molecular areas on the surface found for R95M90 and R95D90 in NaCl aqueous solutions are quite similar in deionized water the R95D90 surfactant molecules take up about double the amount of space at 8523 Aring2molecule as the R95M90 surfactant molecules at 4705 Aring2molecule on the gold surface

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42


01 wt R95D90 in 35 wt NaCl aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

Both the R95M90 and R95D90 experienced an increased absorbance in salt water As a result increased electrostatic interaction in the salt solutions may contribute to decreasing the head group repulsions and increased surfactant adsorption on the surface After rinsing surfactants remaining adsorbed on the gold surface may be due to Van der Waals and lateral hydrogen bonding as electrostatic interactions play less of a role when rinsing with deionized water Stronger hydrophobic bonding must occur to account for the small amount of mass that remains on the gold surface after rinsing (Somasundaran and Krishnakumar 1997)

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43


01 wt R95D90 aqueous solution B Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt R95D90 in 35 wt NaCl aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

Figure 438 shows the frequency and dissipation shifts of the gold sensor over time responding to exposure to crude rhamnolipids obtained from Pseudomonas aeruginosa in deionized water There is a steady reduction in the frequency of the crystal sensor as the crude rhamnolipid solution flows across and rhamnolipids adsorb Although the surfactant adsorbs much more readily the dissipation still remains to be very small below 210-6 indicating that a thin rigid film is forming Using the Sauerbrey relation from section 233 to calculate the mass adsorbed 10186 ngcm2 is obtained After rinsing 3039 ngcm2 is calculated as the surfactant mass irreversibly adsorbed to the sensor surface From the mass adsorbed before rinsing using the molecular weight of the sample which was taken to be an average of mono-rhamnolipids and di-rhamnolipids at 5777 gmol the molecules per area adsorbed was calculated and is shown in Table 2


44


01 wt crude rhamnolipid obtained from Pseudomonas aeruginosa aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

Figure 439 shows the frequency and dissipation changes of the gold sensor over time responding to exposure to crude rhamnolipids in 35 wt NaCl aqueous solution In 35wt NaCl aqueous solution the adsorption of rhamnolipids obtained from Pseudomonas aeruginosa behaved according to the viscoelastic model due to the increase in dissipation above 210-6 The Q-Sense modeling center was used along with equation (6) and (7) in section 233 to determine viscoelastic parameters of the adsorbed layer and determine the mass adsorbed The adsorbed mass before rinsing was 21398 ngcm2 and 3381 ngcm2 after rinsing


01 wt crude rhamnolipid obtained from Pseudomonas aeruginosa in 35 wt NaCl aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

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45

The Pseudomonas aeruginosa rhamnolipid both in deionized water and in 35 wt NaCl aqueous solution saw increased adsorption and longer time was required for the frequency of the sensor to reach a steady state Figure 440 shows the comparison of the frequency and dissipation shifts of the gold sensor over time responding to exposure to crude rhamnolipids in deionized water and in 35 wt NaCl aqueous solution Stable frequency measurements were obtained in a similar time frame for the surfactant in deionized water and in the salt solution From the molecules per area adsorbed shown in Table 2 we can see that more of the crude rhamnolipid surfactant adsorbed to the surface compared to the partially purified R95M90 and R95D90 samples from Sigma Using SEM Meylheuc et al found that a biosurfactants produced by Pseudomonas fluorescens strain 495 with a CMC of 1 when pretreated to hydrophobic and hydrophilic steel adsorbed to the surfaces and reduced the microbial adherence to the surfaces (Meylheuc et al 2001)

Figure 440 A ∆ Frequency and dissipation changes over time of a gold sensor surface responding to exposure to

a 01 wt crude rhamnolipid obtained from Pseudomonas aeruginosa aqueous solution B Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt crude rhamnolipid obtained from Pseudomonas aeruginosa aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated


46

From the calculated molecular areas it is clear to see that the molecules of the crude rhamnolipids take up less space per molecule than the R95M90 and R95D90 samples in deionized water and in 35wt NaCl aqueous solution upon adsorption In deionized water the molecular area found for the crude rhamnolipids is 942 Aring2molecule For crude rhamnolipids in the 35 wt NaCl aqueous solution 448 Aring2molecule is calculated In the salt solution the amount adsorbed to the surface only increases slightly Indicating that head group repulsions may be reduced and the increased polarity of the solvent may drive the hydrophobic tails to the gold surface (Somasundaran and Krishnakumar 1997)

The frequency and dissipation shifts of the gold sensor over time responding to exposure to sophorolipids in deionized water can be seen in Figures 441 Due to low dissipation below 210-6 the Sauerbrey relation from equation (2) was used to calculate the mass adsorbed to the sensor surface 2622 ngcm2 of the sophorolipid surfactant adsorbed to the gold coated sensor After rinsing with deionized water very little of the adsorbed mass was removed There may be strong hydrogen bonding occurring at the gold surface as well as between the hydrocarbon chains of the surfactants to result in such small amount of surfactant removed (Somasundaran and Krishnakumar 1997) The molecules per area and molecular area were also calculated using the adsorbed mass as described in equation (3) The molecular weight used for the sophorolipids is 6227 gmol based on the structure in Figure 22C as described by De Oliveira et al (De Oliveira et al 2015) The molecular area found for sophorolipids in deionized water adsorbed to the gold sensor surface is 3944 Aring2molecule


01 wt sophorolipid aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

The frequency and dissipation shifts of the gold sensor over time responding to exposure to sophorolipids in 35 wt NaCl aqueous solution can be seen in Figures 442 The viscoelastic model in the Q-Sense modeling center was used to calculate the mass adsorbed in the salt solution because the dissipation increases above 210-6 (Thavorn et al 2014) In 35 wt NaCl aqueous solution sophorolipids have a significant increase in adsorption to 82295 ng cm2 After rinsing however 2478 ng cm2 remains irreversibly adsorbed to sensor surface From equation (3) 126 Aring2molecule was calculated for the adsorbed sophorolipids from the 35 wt NaCl aqueous solution As is observed for the pervious

Δ

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47

biosurfactants from deionized water to the NaCl aqueous solutions the molecular area of the adsorbed crude sophorolipid molecules also decreases


01 wt sophorolipid in 35 wt NaCl aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

Figure 443 shows the comparison of the frequency and dissipation shifts of the gold sensor over time responding to exposure to 01 wt sophorolipids in deionized water and in 35 wt NaCl aqueous solution The time required for steady frequency measurements in deionized water was much less than that of the frequency measurements in salt water The frequency measurements did not even become stable over 85 hours and rinsing with deionized water was initiated at about 500 minutes

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01 wt sophorolipid aqueous solution B Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt sophorolipid in 35 wt NaCl aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

Figure 444 shows the frequency and dissipation shifts of the gold sensor over time responding to exposure to 01 wt Surfactin in deionized water Surfactin in deionized water adsorbs quickly on gold with a dissipation remaining below 210-6 indication a thin rigid and evenly distributed surfactant layer adsorbed (Thavorn et al 2014) Using the Sauerbrey relation in equation (2) in addition to the stable frequency shift measurement the gold coated quartz crystal constant C of 177 and the overtone number the mass adsorbed can be calculated The resulting mass adsorbed is 1169 ngcm2 and 742 ngcm2 remains irreversibly adsorbed after rinsing with deionized water Surfactin produced by Bacillus subtilis was used to modify the hydrophilic nature of the cell surface and impact its ability to be retained on hydrophobic gel media(Ahimou et al 2000) It was found that the amphiphilic Surfactin molecules can adsorb to the hydrophilic cell surface by the polar head groups and the surfactant tails allow for greater retention on hydrophobic surfaces (Ahimou et al 2000)


49


01 wt Surfactin aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

Figure 445 shows the frequency and dissipation shifts of the gold sensor over time responding to exposure to 01 wt Surfactin in 35 wt NaCl aqueous solution The viscoelastic model from equations (6) and (7) and the Q-Sense modeling center resulted in 74162 ngcm2 adsorbed to the surface before rinsing because the dissipation was above 210-6 (Thavorn et al 2014) 7740 ngcm2 is irreversibly adsorbed to the sensor surface after rinsing with deionized water Additionally Table 2 shows the molecules per area adsorbed and molecular area calculated in both deionized and salt water for the Surfactin molecules using the molecular weight of 10363 gmol (Marti et al 2014) This calculation comes from equation (3) in section 233 using the adsorbed mass molecular weight and Avogadrorsquos number


01 wt Surfactin in 35 wt NaCl aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

Δ

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Figure 446 shows the comparison of the frequency and dissipation shifts versus time of the gold sensor responding to exposure to 01 wt Surfactin in deionized water and in 35 wt NaCl aqueous solution The frequency measurements in deionized water stabilized quickly and those in salt solution did not stabilize over 55 hours when rinsing with deionized water was initiated The calculated molecular area for adsorbed Surfactin in deionized is 14721 Aring2molecule this is larger than and other surfactant used in this study Surfactin is the largest molecule with the largest head group examined in this work explaining the lager molecular area The molecular area of the adsorbed Surfactin in the aqueous salt solution is 232 Aring2molecule


01 wt Surfactin aqueous solution B Frequency and dissipation changes over time of a gold sensor surface responding to exposure to a 01 wt Surfactin in 35 wt NaCl aqueous solution The first arrow indicates the time at which the solution was injected and the second arrow indicates the time at which rinsing was initiated

Of all the surfactants SDS and AOT clearly adsorb the least to the gold surface and can be rinsed off completely Table 2 shows the number of molecules adsorbed per centimeter squared in addition to the molecular areas in Aring2molecule While SDS and AOT are removed completely after rinsing the number of molecules adsorbed before rinsing are comparable to the molecules of biosurfactants remaining after


51

rinsing In salt water all surfactants except SDS form a structure different from a monolayer indicated by the increased amount of surfactant adsorbed on the surface and use of the viscoelastic model to determine the mass adsorbed (Thavorn et al 2014) The biosurfactants in deionized water due to minimal electrostatic repulsions of the head groups because the biosurfactant head groups not ionize in solution to the same degree that ionization occurs for synthetic surfactants (Somasundaran and Krishnakumar 1997) The biosurfactants head groups instead are larger spatially When salt is added to the solutions the adsorption increases due to the increased hydrophilicity of the solution which drives the hydrocarbon surfactant tails to the surface but the biosurfactants can subsequently be rinsed for significant removal from the surface (Somasundaran and Krishnakumar 1997)


50

Table 2 Summary of mass of surfactant adsorbed to gold surfaces during QCM-D experiment The abbreviations for surfactants in Table 1 are sodium dodecyl sulfate (SDS) dioctyl sulfosuccinate sodium salt (AOT) Pseudomonas aeruginosa rhamnolipid crude extract (PARh) Burkholderia thailandensis rhamnolipid crude extract (BTRh) 95 pure rhamnolipids containing 90 mono-rhamnolipid (R95M90) 95 pure rhamnolipids containing 90 di-rhamnolipid (R95D90) sophorolipid (SPL) and crude Surfactin (LotActySURCE001) (Surfactin)

Surfactant

NaCl (wt)

Model Areal Mass Adsorption on Gold (ngcm2)

Molecules Adsorbed Before Rinsing cm2

Area per molecule (Aring2molecule)

Irriversibly Adsorbed Mass on Gold (ngcm2)

Molecules Adsorbed After Rinsing cm2


SDS 0 Sauerbrey 1116 plusmn 316 233E+14 4291

2 Sauerbrey 1722 plusmn 181 326E+14 3072

35 Viscoelastic 1147 plusmn 82 235E+14 4260

AOT 0 Sauerbrey 905 plusmn 190 123E+14 8157


R95M90 0 Sauerbrey 1781 plusmn 193 213E+14 4705 786 plusmn 68 938E+13 10662

47 Viscoelastic 6977 plusmn 758 833E+14 1201 903 plusmn 39 108E+14 9280

R95D90 0 Sauerbrey 1268 149 117E+14 8523 361 plusmn 130 334E+13 29936

35 Viscoelastic 7372 718 682E+14 1466 670 plusmn 289 620E+13 16130

PA Rh 0 Sauerbrey 10185 plusmn 56 106E+15 942 3039 plusmn 374 317E+14 3157


SPL 0 Sauerbrey 2622 462 254E+14 3944 2622 plusmn 462 254E+14 3944


Surfactin 0 Sauerbrey 1169 plusmn 40 679E+13 14721 742 plusmn 191 431E+13 23193

35 Viscoelastic 74162 3828 431E+15 232 7740plusmn 24 450E+14 2223


51

45 Conclusions

The use of surfactants is very important for applications in oil spill cleanup in oceanic environments Synthetic surfactants can be toxic to ecosystems and are driving the demand for ldquogreenerrdquo and environmentally friendly alternatives (Marti et al 2014) In the present work the aqueous solution self-assembly and surface adsorption of biosurfactants were investigated against the traditionally used synthetic surfactants SDS and AOT Pyrene fluorescence spectroscopy and conductivity were employed to determine the critical micelle concentration of several surfactants The results showed that biosurfactants in this work have CMCs one or more orders of magnitude lower than those of the synthetic surfactants investigated This is significant in that smaller concentrations of biosurfactants are required in order to form micelles and solubilize hydrophobic substances in water

The critical micelle concentration of surfactants was also examined in aqueous solutions at 35 wt NaCl which is representative of the oceanic salt content The added salt reduces the critical micelle concentration values and promotes micellization at lower surfactant concentrations The CMC reduction for crude biosurfactant samples in NaCl aqueous solutions was not as pronounced as what was observed for SDS AOT R95M90 and R95D90 this is in agreement with literature and indicates higher stability in high salt environments

The behavior of R95M90 and R95D90 is very similar in both deionized and salt water Mono-rhamnolipids have one rhamnose surgar molecule attached as the head group and di-rhamnolipids have two sugar molecules at the head group We can conclude that one or two rhamnose molecules attached to the surfactant head group have little effect on the self-assembly properties of rhamnolipids (Mańko et al 2014)

Surfactant adsorption was investigated using QCM-D to determine how surfactants adsorb on surfaces in deionized water and salt water environments We found that the synthetic surfactants and biosurfactants in deionized water adsorb to gold surfaces in a thin relatively rigid film In both salt and deionized water the adsorption of SDS and AOT on gold is reversible while the biosurfactants all experience irreversible adsorption on gold to some degree Ideally adsorption to surfaces should be low reversible and easily rinsed away In salt water solutions the mass of adsorbed biosurfactants does increase as it does for AOT but the vast majority of it can once again be rinsed away This significant ability to remove adsorbed biosurfactants from surfaces makes biosurfactants again comparable to the behavior of synthetic surfactants in salt solutions

In conclusion the superior properties from self-assembly to biodegradability of biosurfactants make them viable alternatives to traditional surfactants Biosurfactants exhibited lower CMC values higher stability and comparable adsorption profiles With innovative cost effective and large scale production and purification methods biosurfactants can come to be an effective and ldquogreenrdquo alternative to petroleum based surfactants This work with biosurfactants adsorption on surfaces is an emerging field of interest that will help determine the practicality of using biosurfactants in environmental applications


52

5 References

Abbriano R M Carranza M M Hogle S L Levin R A Netburn A N Seto K L Snyder S M amp Franks P J S 2011 Deepwater Horizon oil spill a review of the planktonic response Oceanography 24 294-301

Aguiar J Carpena P Molina-Bolıvar J A amp Carnero Ruiz C 2003 On the determination of the critical micelle concentration by the pyrene 13 ratio method Journal of Colloid and Interface Science 258 116-122

Ahimou F Jacques P amp Deleu M 2000 Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity Enzyme and Microbial Technology 27 749-754

Akhavan B Jarvis K amp Majewski P 2013 Hydrophobic plasma polymer coated silica particles for petroleum hydrocarbon removal ACS Applied Materials amp Interfaces 5 8563-8571

Aleiner G S amp Usyarov O G 2010 Conductivity of micellar solutions of ionic surfactants and surface conductivity of micelles Colloid Journal 72 588-594

Baelum J Borglin S Chakraborty R Fortney J L Lamendella R Mason O U Auer M Zemla M Bill M Conrad M E Malfatti S A Tringe S G Holman H-Y Hazen T C amp Jansson J K 2012 Deep-sea bacteria enriched by oil and dispersant from the Deepwater Horizon spill Environmental Microbiology 14 2405-2416

Beyer J Trannum H C Bakke T Hodson P V amp Collier T K 2016 Environmental effects of the Deepwater Horizon oil spill A review Marine pollution bulletin 110 28-51

Cahan D 1989 Kohlrausch and electrolytic conductivity instruments institutes and scientific innovation Osiris 5 166-185

Chakraborty A Chakraborty S amp Saha S K 2007 Temperature dependant micellization of AOT in aqueous medium Effect of the nature of counterions Journal of Dispersion Science amp Technology 28 984-989

Chandrasekar S Sorial G A amp Weaver J W 2006 Dispersant effectiveness on oil spills - impact of salinity ICES Journal of Marine Science 63 1418-1430

Cohen R amp Exerowa D 2007 Surface forces and properties of foam films from rhamnolipid biosurfactants Advances in Colloid and Interface Science 134ndash135 24-34

Costa S G V a O Deacuteziel E amp Leacutepine F 2011 Characterization of rhamnolipid production by Burkholderia glumae Letters in Applied Microbiology 53 620-627

Daverey A Pakshirajan K amp Sumalatha S 2011 Sophorolipids production by Candida bombicola using dairy industry wastewater Clean Technologies amp Environmental Policy 13 481-488

Day J W Hall C a S Kemp W M amp Yaacutentildeez-Arancibia A 1989 Estuarine Ecology Toronto John Wiley amp Sons

De Oliveira M R Magri A Baldo C Camilios-Neto D Minucelli T amp Celligoi M a P C 2015 Review Sophorolipids a promising biosurfactant and itrsquos applications International Journal of Advanced Biotechnology and Research 6 161-174

Dedinaite A Meacuteszaros R amp Claesson P M 2004 Effect of sodium dodecyl sulfate on adsorbed layers of branched polyethylene imine Journal of Physical Chemistry B 108 11645-11653

Delimaassociates 2016 Household Products Database [Online] US Department of Health and Human Service National Institutes of Health Health amp Human Services US National Library of Medicine Available httpshouseholdproductsnlmnihgovcgi-binhouseholdbrandstbl=chemampid=78 [Accessed December 5 2016]

Desai J D amp Banat I M 1997 Microbial production of surfactants and their commercial potential Microbiology and Molecular Biology Reviews 61 47-64

Dey J Bhattacharjee J Hassan P A Aswal V K Das S amp Ismail K 2010 Micellar shape driven counterion binding Small-angle neutron scattering study of AOT micelle Langmuir 26 15802-15806


53

Dong D C amp Winnik M A 1984 The py scale of solvent polarities Canadian Journal of Chemistry 62 2560-2565

Dudaacutešovaacute D Silset A amp Sjoumlblom J 2008 Quartz crystal microbalance monitoring of asphaltene adsorptiondeposition Journal of Dispersion Science and Technology 29 139-146

Ekholm P Blomberg E Claesson P Auflem I H Sjoumlblom J amp Kornfeldt A 2002 A quartz crystal microbalance study of the adsorption of asphaltenes and resins onto a hydrophilic surface Journal of Colloid and Interface Science 247 342-350

The Essential Chemical Industry - online - Surfactants [Online] 2013 CIEC Promoting Science at the University of York York UK Joomla Available httpwwwessentialchemicalindustryorgmaterials-and-applicationssurfactantshtml [Accessed November 22 2016]

Evans D F amp Wennerstroumlm H 1999 The colloidal domain where physics chemistry biology and technology meet Wiley-VCH

Felse P A Shah V Chan J Rao K J amp Gross R A 2007 Sophorolipid biosynthesis by Candida bombicola from industrial fatty acid residues Enzyme and Microbial Technology 40 316-323

Glushko V Thaler M S R amp Karp C D 1981 Pyrene fluorescence fine structure as a polarity probe of hydrophobic regions Behavior in model solvents Archives of Biochemistry and Biophysics 210 33-42

Gong Y Zhao X Cai Z Orsquoreilly S E Hao X amp Zhao D 2014 A review of oil dispersed oil and sediment interactions in the aquatic environment Influence on the fate transport and remediation of oil spills Marine pollution bulletin 79 16-33

Gray J L Kanagy L K Furlong E T Kanagy C J Mccoy J W Mason A amp Lauenstein G 2014 Presence of the Corexit component dioctyl sodium sulfosuccinate in Gulf of Mexico waters after the 2010 Deepwater Horizon oil spill Chemosphere 95 124-130

Grillo I amp Penfold J 2011 Self-assembly of mixed anionic and nonionic surfactants in aqueous solution Langmuir 27 7453-7463

Guerra-Santos L Kaumlppeli O amp Fiechter A 1984 Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source Applied and Environmental Microbiology 48 301-305

Hamdan L J amp Fulmer P A 2011 Effects of Corexit (R) EC9500A on bacteria from a beach oiled by the Deepwater Horizon spill Aquatic Microbial Ecology 63 101-109

Healy M G Devine C M amp Murphy R 1996 Microbial production of biosurfactants Resources Conservation and Recycling 18 41-57

Hirata Y Ryu M Oda Y Igarashi K Nagatsuka A Furuta T amp Sugiura M 2009 Novel characteristics of sophorolipids yeast glycolipid biosurfactants as biodegradable low-foaming surfactants Journal of Bioscience and Bioengineering 108 142-146

Houmlrmann B Muumlller M M Syldatk C amp Hausmann R 2010 Rhamnolipid production by Burkholderia plantarii DSM 9509T European Journal of Lipid Science and Technology 112 674-680

Infelta P P amp Graumltzel M 1979 Statistics of solubilizate distribution and its application to pyrene fluorescence in micellar systems A concise kinetic model Journal of Chemical Physics 70 179-186

Inoue T Ebina H Dong B amp Zheng L 2007 Electrical conductivity study on micelle formation of long-chain imidazolium ionic liquids in aqueous solution Journal of Colloid and Interface Science 314 236-241

Kalyanasundaram K amp Thomas J K 1977 Environmental effects on vibronic band intensities in pyrene monomer fluorescence and their application in studies of micellar systems Journal of the American Chemical Society 99 2039-2044


54

Kang S-W Kim Y-B Shin J-D amp Kim E-K 2010 Enhanced biodegradation of hydrocarbons in soil by microbial biosurfactant sophorolipid Appl Biochemistry and Biotechnology 160 780-790

Karlsson P M Palmqvist A E C amp Holmberg K 2008 Adsorption of sodium dodecyl sulfate and sodium dodecyl phosphate on aluminum studied by QCM-D XPS and AAS Langmuir 24 13414-13419

Kim J amp Vipulanandan C 2006 Removal of lead from contaminated water and clay soil using a biosurfactant Journal of Environmental Engineering 132 777-786

Knock M M amp Sanii L S 2010 Effect of hydrophobization of gold QCM-D crystals on surfactant adsorption at the solid-liquid interface In Nagarajan R (ed) Amphiphiles Molecular Assembly and Applications New York American Chemical Society

Kosaric N amp Sukan F V 1993 Biosurfactants Production Properties Applications New York Marcel Dekker Inc

Kronberg B Holmberg K amp Lindman B 2014 Surface Chemistry of Surfactants and Polymers 1 ed Somerset Wiley

Kujawinski E B Kido Soule M C Valentine D L Boysen A K Longnecker K amp Redmond M C 2011 Fate of dispersants associated with the deepwater horizon oil spill Environmental Science amp Technology 45 1298-1306

Lessard R R amp Demarco G 2000 The significance of oil spill dispersants Spill Science amp Technology Bulletin 6 59-68

Lewis M amp Pryor R 2013 Toxicities of oils dispersants and dispersed oils to algae and aquatic plants Review and database value to resource sustainability Environmental pollution 180 345-367

Li Z Lee K King T Boufadel M C amp Venosa A D 2009a Evaluating crude oil chemical dispersion efficacy in a flow-through wave tank under regular non-breaking wave and breaking wave conditions Marine pollution bulletin 58 735-744

Li Z K Lee K King T Boufadel M C amp Venosa A D 2009b Evaluating chemical dispersant efficacy in an experimental wave tank 2-significant factors determining in situ oil droplet size distribution Environmental Engineering Science 26 1407-1418

Liu J-F Mbadinga S M Yang S-Z Gu J-D amp Mu B-Z 2015 Chemical structure property and potential applications of biosurfactants produced by Bacillus subtilis in petroleum recovery and spill mitigation Int J Mol Sci 16 4814-4837

Liu S X amp Kim J-T 2009 Application of KevinmdashVoigt model in quantifying whey protein adsorption on polyethersulfone using QCM-D Journal of the Association for Laboratory Automation 14 213-220

Long Y Wang T Liu L Liu G amp Zhang G 2013 Ion specificity at a low salt concentration in waterndashmethanol mixtures exemplified by a growth of polyelectrolyte multilayer Langmuir 29 3645-3653

Loacutepez-Diacuteaz D amp Velaacutezquez M M 2007 Variation of the critical micelle concentration with surfactant structure A simple method to analyze the role of attractive-repulsive forces on micellar association The Chemical Educator 12 327-330

Madsen J K Pihl R Moslashller A H Madsen A T Otzen D E amp Andersen K K 2015 The anionic biosurfactant rhamnolipid does not denature industrial enzymes Frontiers in Microbiology 6 292

Mańko D Zdziennicka A amp Jańczuk B 2014 Thermodynamic properties of rhamnolipid micellization and adsorption Colloids and Surfaces B Biointerfaces 119 22-29

Marchant R amp Banat I M 2012 Biosurfactants a sustainable replacement for chemical surfactants Biotechnology Letters 34 1597-1605


55

Marti M E Colonna W J Patra P Zhang H Green C Reznik G Pynn M Jarrell K Nyman J A Somasundaran P Glatz C E amp Lamsal B P 2014 Production and characterization of microbial biosurfactants for potential use in oil-spill remediation Enzyme and Microbial Technology 55 31-39

Merta J Tammelin T amp Stenius P 2004 Adsorption of complexes formed by cationic starch and anionic surfactants on quartz studied by QCM-D Colloids and Surfaces A Physicochemical and Engineering Aspects 250 103-114

Meylheuc T Oss C J V amp Bellon‐Fontaine M N 2001 Adsorption of biosurfactant on solid surfaces and consequences regarding the bioadhesion of Listeria monocytogenes LO28 Journal of Applied Microbiology 91 822-832

Mitsionis A I amp Vaimakis T C 2012 Estimation of AOT and SDS CMC in a methanol using conductometry viscometry and pyrene fluorescence spectroscopy methods Chem Phys Lett 547 110-113

Miura M amp Kodama M 1972 The second CMC of the aqueous solution of sodium dodecyl sulfate I conductivity Bulletin of the Chemical Society of Japan 45 428-431

Mivehi L Bordes R amp Holmberg K 2011 Adsorption of cationic Gemini surfactants at solid surfaces studied by QCM-D and SPR Effect of the rigidity of the spacer Langmuir 27 7549-7557

Mohsenipour A A amp Pal R 2013 Synergistic effects of anionic surfactant and nonionic polymer additives on drag reduction Chemical Engineering Communications 200 935-958

Monteiro S A Sassaki G L De Souza L M Meira J A De Arauacutejo J M Mitchell D A Ramos L P amp Krieger N 2007 Molecular and structural characterization of the biosurfactant produced by Pseudomonas aeruginosa DAUPE 614 Chemistry and Physics of Lipids 147 1-13

Muherei M A Junin R amp Bin Merdhah A B 2009 Adsorption of sodium dodecyl sulfate Triton X100 and their mixtures to shale and sandstone A comparative study Journal of Petroleum Science and Engineering 67 149-154

Mulligan C N 2009 Recent advances in the environmental applications of biosurfactants Current Opinion in Colloid amp Interface Science 14 372-378

Muthusamy K Gopalakrishnan S Ravi T K amp Sivachidambaram P 2008 Biosurfactants Properties commercial production and application Current Science 94 736-747

Nitschke M amp Costa S G V a O 2007 Biosurfactants in food industry Trends in Food Science amp Technology 18 252-259

Nitschke M Costa S G V a O Haddad R Gonccedilalves L a G Eberlin M N amp Contiero J 2005 Oil wastes as unconventional substrates for rhamnolipid biosurfactant production by Pseudomonas aeruginosa LBI Biotechnology Progress 21 1562-1566

Nitschke M amp Pastore G M 2006 Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater Bioresource Technology 97 336-341

Nivaggioli T Alexandridis P Hatton T A Yekta A amp Winnik M A 1995 Fluorescence probe studies of Pluronic copolymer solutions as a function of temperature Langmuir 11 730-737

Nursakinah I Ismail A R Rahimi M Y amp Idris A B 2014 Evaluation of HLB values of mixed non-ionic surfactants on the stability of oil-in-water emulsion system AIP Conf Proc 850-856

Pacwa-Plociniczak M Plaza G A Piotrowska-Seget Z amp Cameotra S S 2011 Environmental applications of biosurfactants Recent advances International Journal of Molecular Sciences 12 633-654

Picas L Milhiet P-E amp Hernandez-Borrell J 2012 Atomic force microscopy A versatile tool to probe the physical and chemical properties of supported membranes at the nanoscale Chem Phys Lipids 165 845-860


56

Pinazo A Wen X Peacuterez L Infante M-R amp Franses E I 1999 Aggregation behavior in water of monomeric and gemini cationic surfactants derived from arginine Langmuir 15 3134-3142

Pineiro L Novo M amp Al-Soufi W 2015 Fluorescence emission of pyrene in surfactant solutions Adv Colloid Interface Sci 215 1-12

Plunkett M A Claesson P M Ernstsson M amp Rutland M W 2003 Comparison of the adsorption of different charge density polyelectrolytesthinsp A quartz crystal microbalance and x-ray photoelectron spectroscopy study Langmuir 19 4673-4681

Pornsunthorntawee O Arttaweeporn N Paisanjit S Somboonthanate P Abe M Rujiravanit R amp Chavadej S 2008a Isolation and comparison of biosurfactants produced by Bacillus subtilis PT2 and Pseudomonas aeruginosa SP4 for microbial surfactant-enhanced oil recovery Biochemical Engineering Journal 42 172-179

Pornsunthorntawee O Chavadej S amp Rujiravanit R 2009 Solution properties and vesicle formation of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa SP4 Colloids and Surfaces B Biointerfaces 72 6-15

Pornsunthorntawee O Wongpanit P Chavadej S Abe M amp Rujiravanit R 2008b Structural and physicochemical characterization of crude biosurfactant produced by Pseudomonas aeruginosa SP4 isolated from petroleum-contaminated soil Bioresource Technology 99 1589-1595

Quina F H Nassar P M Bonilha J B S amp Bales B L 1995 Growth of sodium dodecyl sulfate micelles with detergent concentration Journal of Physical Chemistry 99 17028-17031

Regev O amp Zana R 1999 Aggregation behavior of Tyloxapol a nonionic surfactant oligomer in aqueous solution Journal of Colloid and Interface Science 210 8-17

Rodrigues L R amp Teixeira J A 2010 Biomedical and therapeutic applications of biosurfactants Advances in Experimental Medicine and Biology 672 75-87

Said Z amp Alwi H 2014 SEM study of oil adsorption on the surface of Khaya Senegalensis dried leaves APCBEE Procedia 9 108-112

Sarkar B Ravi V amp Alexandridis P 2013 Micellization of amphiphilic block copolymers in binary and ternary solvent mixtures Journal of Colloid and Interface Science 390 137-146

Senese F amp Brady J E 2009 Chemistry Matter and its Changes Hoboken NJ John Wiley amp Sons Shanks P C amp Franses E I 1992 Estimation of micellization parameters of aqueous sodium dodecyl

sulfate from conductivity data Journal of Physical Chemistry 96 1794-1805 Sjoumlblom J 2006 Emulsions and Emulsion Stability Boca Raton Taylor amp Francis Skop R A Tseng R-S amp Brown J W 1993 Effects of salinity and surface tension on microbubble-

mediated sea-to-air transfer of surfactants Journal of Geophysical Research Oceans 98 8489-8494

Slavin S Soeriyadi A H Voorhaar L Whittaker M R Becer C R Boyer C Davis T P amp Haddleton D M 2012 Adsorption behaviour of sulfur containing polymers to gold surfaces using QCM-D Soft Matter 8 118-128

Soares D M Gomes W E amp Tenan M A 2007 Sodium dodecyl sulfate adsorbed monolayers on gold electrodes Langmuir 23 4383-8

Somasundaran P amp Krishnakumar S 1997 Adsorption of surfactants and polymers at the solid-liquid interface Colloids and Surfaces A Physicochemical and Engineering Aspects 123ndash124 491-513

Song D Liang S Zhang Q Wang J amp Yan L 2013 Development of high efficient and low toxic oil spill dispersants based on sorbitol derivants nonionic surfactants and glycolipid biosurfactants J Environ Prot 4 16-22

Tavakkoli M Panuganti S R Vargas F M Taghikhani V Pishvaie M R amp Chapman W G 2013 Asphaltene deposition in different depositing environments Part 1 Model oil Energy amp Fuels 28 1617-1628


57

Thavorn J Hamon J J Kitiyanan B Striolo A amp Grady B P 2014 Competitive surfactant adsorption of AOT and Tween 20 on gold measured using a quartz crystal microbalance with dissipation Langmuir 30 11031-9

Thibodeaux L J Valsaraj K T John V T Papadopoulos K D Pratt L R amp Pesika N S 2011 Marine oil fate Knowledge gaps basic research and development needs a perspective based on the Deepwater Horizon spill Environmental Engineering Science 28 87-93

Tiberg F Brinck J amp Grant L 1999 Adsorption and surface-induced self-assembly of surfactants at the solidndashaqueous interface Current Opinion in Colloid amp Interface Science 4 411-419

Umlong I M amp Ismail K 2005 Micellization of AOT in aqueous sodium chloride sodium acetate sodium propionate and sodium butyrate media A case of two different concentration regions of counterion binding Journal of Colloid and Interface Science 291 529-536

Umlong I M amp Ismail K 2007 Micellization behaviour of sodium dodecyl sulfate in different electrolyte media Colloids and Surfaces A Physicochemical and Engineering Aspects 299 8-14

Wang S-C Wei T-C Chen W-B amp Tsao H-K 2004 Effects of surfactant micelles on viscosity and conductivity of poly(ethylene glycol) solutions Journal of Chemical Physics 120 4980-4988

Weckman N E Olsson A L J amp Tufenkji N 2014 Evaluating the binding of selected biomolecules to cranberry derived proanthocyanidins using the quartz crystal microbalance Biomacromolecules 15 1375-1381

White H K Lyons S L Harrison S J Findley D M Liu Y amp Kujawinski E B 2014 Long-term persistence of dispersants following the deepwater horizon oil spill Environmental Science amp Technology Letters 1 295-299


2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References

ii

Work package WP3 Impact assessment of developed technologies

Deliverable no D39

Deliverable title Evaluation of novel dispersants surface characterization in presence of oil and dispersant

Due date Month 36

Actual submission date Month 48

Start date of project 2013-01-01

Deliverable Lead Beneficiary (Organisation name)

University at Buffalo

Participant(s) (Partner short names) UB

Author(s) in alphabetic order Alexandridis Paschalis Placek Tess Tsianou Marina

Contact for queries Paschalis Alexandridis Dept of Chemical and Biological Engineering University at Buffalo Buffalo NY 14260-4200 USA T +1 716 6451183 E palexandbuffaloedu

Dissemination Level (PUblic Restricted to other Programmes Participants REstricted to a group specified by the consortium COnfidential only for members of the consortium)

PU

Deliverable Status final


iii

Table of Content


2 Introduction 1

21 Surfactants 1











31 Materials 13

32 Methods 13



323 Conductivity 15


4 Results 17





45 Conclusions 51

5 References 52


iv

List of Figures






















v




















vi

















vii


List of Tables




1



2 Introduction

21 Surfactants





2









3



222 Biosurfactants



A

B


4








5



A

B

C

D


6









7








8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


iii

Table of Content


2 Introduction 1

21 Surfactants 1











31 Materials 13

32 Methods 13



323 Conductivity 15


4 Results 17





45 Conclusions 51

5 References 52


iv

List of Figures






















v




















vi

















vii


List of Tables




1



2 Introduction

21 Surfactants





2









3



222 Biosurfactants



A

B


4








5



A

B

C

D


6









7








8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


iv

List of Figures






















v




















vi

















vii


List of Tables




1



2 Introduction

21 Surfactants





2









3



222 Biosurfactants



A

B


4








5



A

B

C

D


6









7








8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


v




















vi

















vii


List of Tables




1



2 Introduction

21 Surfactants





2









3



222 Biosurfactants



A

B


4








5



A

B

C

D


6









7








8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


vi

















vii


List of Tables




1



2 Introduction

21 Surfactants





2









3



222 Biosurfactants



A

B


4








5



A

B

C

D


6









7








8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


vii


List of Tables




1



2 Introduction

21 Surfactants





2









3



222 Biosurfactants



A

B


4








5



A

B

C

D


6









7








8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


1



2 Introduction

21 Surfactants





2









3



222 Biosurfactants



A

B


4








5



A

B

C

D


6









7








8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


2









3



222 Biosurfactants



A

B


4








5



A

B

C

D


6









7








8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


3



222 Biosurfactants



A

B


4








5



A

B

C

D


6









7








8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


4








5



A

B

C

D


6









7








8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


5



A

B

C

D


6









7








8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


6









7








8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


7








8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


8







(1)


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


9






fn

Cm ∆minus=∆1

sdot

∆

=minus

2

16

92

10

110

1cm

molmoleculeN

gmol

WMngg

cmngm

A

Avogadro

stored

dissipated

EE

Dπ2

=

ftD ll

qq πηρ

ρ 21

=∆

(2)

(3)

(4)

(5)


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


10






(6)

(7)


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


11







12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


12








13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


13


31 Materials



32 Methods








14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


14









15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


15




323 Conductivity





16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


16








17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


17



4 Results






18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


18








19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


19






20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


20






21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


21





22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


22






23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


23







24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


24





25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


25


without NaCl



26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


26







27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


27






28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


28





without NaCl



29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


29




without NaCl


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


30





31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


31








32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


32





Derivative CMC (wt)


SDS Synthetic 0 023 0218 0250 2 0021 0026 35 0017 0019

AOT Synthetic 0 019 0124 0176 2 0007 0016 35 0010 0019

R95M90 Natural 0 0018 0059 47 0003 0011

R95D90 Natural 0 0034 0063 35 0006 0011


35 0002 0005 SPL Natural 0 00002 00002


35 0005 0025




33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


33






Δ

Δ


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


34






Δ

Δ

Δ

Δ


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


35





36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


36






Δ

Δ

Δ

Δ


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


37




Δ

Δ

Δ

Δ


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


38






Δ

Δ


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


39




Δ

Δ


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


40





41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


41





Δ

Δ


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


42




Δ

Δ


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


43





44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


44






Δ

Δ

Δ

Δ


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


45





46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


46






Δ

Δ


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


47





Δ

Δ


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


48





49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


49






Δ

Δ

Δ

Δ


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


50






51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


51



50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


50


Surfactant

NaCl (wt)























51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


51

45 Conclusions







52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


52

5 References





















53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


53




















54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


54




















55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


55




















56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


56





















57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References


57










2 Introduction

21 Surfactants



222 Biosurfactants









31 Materials

32 Methods





323 Conductivity


4 Results





45 Conclusions

5 References

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