Modeling and Analysis of Micellar and Microbubble Dynamics To

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Modeling and analysis of micellar and microbubble dynamics to derive new insights in molecular interactions impacting the packing behavior of surfactin for potential engineering implications Gunaseelan Dhanarajan, Partha Patra, Vivek Rangarajan, Ponisseril Somasundaran, and Ramkrishna Sen ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04428 • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 5, 2018

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Modeling and analysis of micellar and microbubble dynamics to derive new insights in molecular interactions impacting the packing behavior of a green surfactant for potential engineering implications Gunaseelan Dhanarajan1, Partha Patra*2, Vivek Rangarajan1, Ponisseril Somasundaran2 and Ramkrishna Sen*1 1

Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India Department of Earth and Environmental Engineering, Columbia University, New York, NY 10027, USA *email: [email protected]; [email protected] 2

ABSTRACT

The surfactin biosurfactants exhibit a unique form of molecular architecture - a heptapeptide lactone ring (LR) associates with a β-hydroxy fatty-acid tail. Their surface-active properties largely depend on the LR-charge and -conformation that differ with changes in pH conditions, and electrolyte-types/concentrations. We studied the surface-active property of a surfactin variant in terms of the molecular packing behavior at air/water interface, and considering the manner in which the charge and conformation of a LR differ with changes in respect to pH (6.5 9.5) and Ca2+ concentration (0 - 1 mM). The equilibrium surface tension values (γEq), micelle sizes (Rm) and the sizes of the surfactin-stabilized microbubbles (RMB) were determined to assess the packing behavior. In the absence of Ca2+ ions, the γEq vs. pH, Rm vs. pH and RMB vs. pH correlations were non-linear, which indicated that a marked reduction in the LR-charge occurs with increase in pH from 6.5 to 8, and marginally with increase in pH from 8 to 9.5. By using Circular Dichroism technique, we found that a LR undergoes conformational transition from β-sheet (pH 6.5) to β-turn (pH 8) as the ionized COOH group of a LR charge-neutralizes by forming an intramolecular hydrogen bond (HB), causing therefore a marked reduction in the LR-charge. The marginal reduction in LR-charge, with increase in pH from 8 to 9.5, occurs due to an increase in the HB strength; the LR conformation changes from β-turn to α-helix (pH 9.5). In the presence of Ca2+ ions, the LRs exhibited disordered β-sheet conformations as they formed LR-Ca-LR bridges. We developed a Double-Layered Aggregate (DLA) model to describe the packing behavior in the presence of Ca2+ ions. According to this model, all the LRs at the interface formed LR-Ca bridges as a critical Ca2+ concentration ( ) was attained, and the interfacial layer exhibited almost zero charge. At concentrations > , the surfactin molecules readily accumulated at this zero-charge interfacial layer and aggregated via LR-Ca bridging to form an additional layer. The γEq, Rm and RMB values, determined with respect to Ca2+ concentrations, validated the DLA model. KEYWORDS: Green surfactant; Molecular interactions; Micellar and microbubble dynamics; Modeling and analysis; Engineering implications per interfacial area: Γ) at the interface: ⁄ = −2.303 , where C is surfactant concentration, k is Boltzmann constant is the gas constant, and T is the absolute temperature. In the absence of electrolytes, the γ could deviate with small changes in pH in the range of 3 – 10 6, 8, 12-13 , primarily due the pH-dependency of the LR-charge 10, 14-20. The γ also differs depending on the random orientations probable for these molecules at a/w interface, which is because of their ball-type configurations where the β-hydroxy fatty-acid tail folds back to associate with the LR 9, 20. A broad range for Γ (0.9 - 1.16 µmoL m2 ) is determined in experiments and that using the Gibbs adsorption theorem and the Langmuir– Szyszkowski and Frumkin models 6, 10, 19-20. On the other hand, in molecular dynamics simulations (MDS) and, by probing neutron reflectivity technique, it has been evident that instead of a monolayer these molecules could pack at the interface in form of a multi-layered aggregate 16, 19-22. The surface-active property depends on the intermolecular forces ( ( among the surfactant molecules that pack/aggregate at an interface, where

INTRODUCTION The surfactin bio-surfactants, derived from several strains of Bacillus subtilis, exhibit unique molecular architecture: the head group as the heptapeptide (Glu-Leu-D-Leu-Val-Asp-D-Leu-Leu) lactone ring (LR) and the tail as the β-hydroxy fattyacid, constituting 13-17 alkyl groups 1. Because of their membrane-active, antibacterial and anti-cancer properties, these bio-surfactants are increasingly being considered for personal care and pharmaceutical applications2-5. However, their surface-active properties at air-water (a/w) and oilwater interfaces differ with changes in pH 6-7, and electrolyte-types and –concentrations 8-9. Therefore, an understanding of the interfacial behavior of these surfactants, under different conditions, is essential to consider them for a wide range of their potential engineering applications. The surface-active properties of the surfactin biosurfactants at the a/w interface are typically assessed in terms of their dynamic ( )10 and equilibrium surface tension ( )11 values. The depends on the molecular packing-density (molecules

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is given as: = ( ⁄ ; is the intermolecular distance. The magnitude of ( for a surfactin variant largely accounts for the electrostatic interactions among the molecules because the intertail hydrophobic interactions are comparatively much weaker. The net charge of LR therefore determines the ( for the packed layer of surfactin molecules at a/w interface. Considering that the COOH groups of the Glu and the Asp peptide-chromophores of a LR ionize at pH levels > pKa (5.8), the net charge of a LR is 2e- (e-: electron charge) 17. However, zetapotential values of mixed-micelles and liposomes, constituting surfactin molecules, indicate that the net charge typically differs from 2e-/LR at pH > 5.8 23-24. Penfold and coworkers inferred decrease in the net charge of LR as pH decreases from 9.5 to 5.59. Contrariwise, Heerklotz and others reported reduction in the net charge of LR with increase in pH from 7.4 to 8.5 23, 25-26. These contradictory results suggest that the net charge of a LR could differ due to factors other than pKa-dependent ionization of the COOH groups. The net charge of a LR differs as the RCOOH moiety of a LR forms an intramolecular hydrogen bond (HB) with a specific amine group (R indicates the number of carbon atoms of the Glu and the Asp peptide-chromophores that extend out of the peptide backbone of LR). The isomeric variants of surfactin, namely S1 and S2, could constitute one and two intramolecular HBs respectively. These HBs impart specific conformations to a LR; a LR typically exhibits stable β–sheet conformation at pH 7 and in the absence of electrolytes 17. The γ- and β-turn, and α-helix conformations of a LR have been identified by using the Nuclear Magnetic Resonance (NMR) and the Circular Dichroism (CD) techniques 15, 18. (Eleven amino acid moieties are typically required for a peptide sequence to describe its secondary structure.) The conformations of a LR, constituting only seven amino acid moieties, are interpreted in terms of the differences in the configurations of LR’s peptide backbone, where the –[N-C-C]- moieties of the peptide chromophores of a LR acquire specific bond- and dihedral-angles1, 18. The LR acquires specific conformations depending on the involvement of the RCOOH moiety in intramolecular HB formation. Alternately, the peptide backbone of a LR could be dis-configured as a RCOOH moiety forms a bond with an ion (electrolytes). The R-COOH moiety therefore exhibits specific orientations (θ) with respect to the peptide backbone, depending on the pH conditions and electrolyte-types and -concentrations 8, 15-16, 18 . We examined the manner in which the net charge of a LR differs as an R-COOH moiety acquires specific orientations, under different pH

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conditions and Ca2+ concentrations; we used the CD technique. Small angle neutron scattering (SANS)[10], neutron reflectometry27 and NMR techniques15, 18, 28, and MDS16 have been applied to study the packing behavior of surfactin molecules at a/w interface, and that in the presence of divalent ions. Accordingly, an aggregate of surfactin molecules form at the interface through bidentate-bridging of the LRs 29, which not only causes reduction in the net charge of LR but also induces inter-LR separation 30-31. The peptide backbone of LR dis-configures in an uncharacteristic manner as these bidentate bridges are short-lived and do not exhibit any particular bond-length/angle. We assessed the surface-active property of a surfactin variant (isolated from marine Bacillus megaterium) in terms of the molecular packing density at the a/w interface, in the pH range of 6.5 9.5 and in the Ca2+ concentration range of 0 - 1 mM. We determined the values to account for the packing density. However, considering the cases that surfactin molecules form a thick aggregate at the interface, spreading beyond the interfacial layer, such an aggregate could exhibit elastic behavior.32 Therefore, we prepared surfactin-stabilized microbubbles (MBs) where the sizes of the MBs were a measure of ‘E’. The modified Epstein and Plesset (EP) model33, proposed by Katiyar, relate the size of a MB ( !" ) to E as34: ( !" = $

% + ' ()

*+, *-

. − 10 ; % + ' 2 3)

0; % + ' 2 3)

*+, *-

*+, *-

− 1.4

− 1.4 ≤ 0

> 0 [1]

The % is according to the Γ at the a/w interface; R0 is the MB radius under stress-free state and !" being the actual radius of the MB. The ‘E’ was accounted here in a relative manner by comparing the sizes of the MBs that were prepared under different pH conditions and Ca2+ concentrations.

MATERIALS AND METHODS The surfactin variant used in this study was isolated from the culture of the marine Bacillus megaterium (from Andaman and Nicobar Islands, India) and by using a method described in our earlier publications 35-37. By using a reverse phase HPLC instrument (Agilent Technologies, CA, USA), equipped with a Zorbax C18 column and a diode array detector, the purity of the surfactin sample that we isolated here was determined to be > 95 % (wt.). The equilibrium surface tension (γEq) values under different pH conditions (6.5 - 9.5) and Ca2+ concentrations (0 - 1 mM) were determined using a tensiometer (S1). The conformations of the LR of this particular surfactin variant, under the aforementioned


units)38. The !" (average of the MB sizes) values at the pH levels of 6.5, 8 and 9.5 were respectively as 56, 32 and 31µ (Fig. 1 (a-d) and S2). As !" depends on γ ( ∆< = 2⁄ !" ), a non-linear relationship for the γ vs. pH correlation was established. Thus, the γ vs. pH correlation then validated that the packing density indeed differed with increase in pH. On the other hand, the MBs at pH 6.5 were large in their sizes and almost twice more in number (per unit suspension volume) than the small MBs at pH 9.5 (Fig. 2a and S2). A nonlinear relationship of the ‘number of MBs/suspension Surface tension values MB sizes, RMB

(a)

110

32

100 30 90 28

80

7

pH 8

70

9

MB size, R MB, Micron

conditions, were assessed by using the CD spectroscopy technique (S1). Gas (atmospheric air) MB suspensions were prepared by subjecting the surfactin solutions to ultra-sonication. The surfactin concentrations in the solutions were twice the critical micellar concentration (cmc); the cmc value determined for surfactin using a tensiometer was 2.5 × 10-5 M in the pH range of 6.5 – 8.5. One set of the MB suspensions were prepared without Ca2+ ions and at the pH levels of 6.5, 8 and 9.5; PBS (Phosphatebuffered saline) solutions were used. (The PBS solutions were devoid of the salts of divalent ions.) Another set of the MB suspensions were prepared with Tris buffer (pH 8), where desired amounts of CaCL2 was added to the surfactin solutions to attain Ca2+ concentrations of 0.25. 0.5 and 1 mM. The MB suspensions were prepared by activating a 20 kHz probe sonicator (Model: Q125; Make: Qsonica, Newtown, USA) for the surfactin solutions. The tip of a high intensity ultrasound probe was positioned at the air-solution interface and the sample was sonicated at 60% amplitude for 30 seconds. The suspensions were left standing for a few seconds, and until the MBs moved to the surface of the suspensions. The images of the MBs for a suspension were obtained using a camera and the MB size distribution profiles were developed (S1).

Surface Tension, mN/m

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(c) - pH 8

(b) - pH 6.5

(d) - pH 9.5

Fig. 1. The γEq vs. pH (red line) and the !" vs. pH (dashed line) correlations (a) are shown. The microscopic images are given for the MBs prepared at pH 6.5 (b), 8 (c), and 9.5 (d); bar indicates 50 µm.

RESULTS AND DISCUSSIONS pH-dependency of molecular packing behavior The equilibrium surface tension (γEq) values for the surfactin variant (isolated here) were determined as 32, 27 and 26 mN/m at the pH levels of 6.5, 8 and 9.5, respectively (Fig. 1a). The γEq reduced by 5 mN/m with a change in pH from 6.5 to 8, and marginally by ~1 mN/m for an equal change in pH from 8 to 9.5. Thus, the γEq vs. pH correlation was non-linear, which indicated that the packing density increased with increase in pH in a non-linear manner; the molecules packed more tightly at pH > 8 than at pH levels < 8. However, the γEq values differed within a narrow range (1 - 5 mN/m) to rely on this non-linear relationship of the γEq vs. pH correlation, and also on the ground that the γEq values could be confounded because of the elasticity (E) of the packed layer (Eqn. 1). Therefore, as an alternate technique to verify whether this γEq vs. pH relationship was indeed non-linear, we compared the sizes of surfactin-stabilized MBs that were prepared at pH 6.5, 7, 8 and 9.5. For the MBs that were prepared here through sonication approach, the MB size is expressed as: !" = 6378 ⁄9: , where γ is the specific heat ratio of the gas inside the bubble, p∞ is the ambient liquid pressure, ρ is the liquid density and ω is the angular frequency of ultrasound (all in SI

volume’ vs. pH correlation was established (S2). This relationship thus indicated that the total interfacial area stabilized by a surfactin molecule decreased with increase in pH in a non-linear manner, implying that the effective interfacial area occupied the surfactin molecules decreased with increase in pH. We thus verified that the packing density indeed increased with increase in pH in a non-linear manner. # of MBs in suspension

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pH

(a)

6.5 8 9.5

10000

5000

0 0

5

10

15

Time, min st

1

20

25

th

18

nd

22

(b) Fig. 2. The dissolution rate profiles for the MBs prepared under different pH conditions (a) are given. The microscopic images (b) are shown for the MBs that were prepared at pH 8 and taken in course of their



The R >? vs. pH correlation developed here was based on the R >? values determined after a few minutes of the formation of the MBs. However, MBs change their sizes through their dissolution processes that commence as soon as the MBs form. We developed the MB dissolution rate profiles (S1) for the MBs prepared at the pH levels of 6.5, 8 and 9.5. The dissolution rates were estimated as: ‘number of MBs/suspension volume’ vs. dissolution time (Fig. 2). The harmonic nature of the dissolution rate profiles for the MBs, prepared at different pH levels, verified that the MB sizes were according to the respective pH-dependent π and E values. dissolution, at 1st, 18th and 22nd minutes; bar: 25 µ.

electrostatic in nature for a surfactin variant, and depend largely on the LR-charge, because, the intertail hydrophobic interactions (δ and ) are least influenced with changes in pH conditions. Therefore, the effective area (I ) occupied by a LR in a micelle is an estimate of the intermolecular forces among the LRs, which is given as: I = 6α⁄, where α is the head group repulsion parameter. A measure of the I value under a particular pH condition can be easily obtained by determining the micelle size ( N ), where 2 N relates to I as: 4π N ∝ I ; N = micelle size. Therefore, the N vs. pH correlation that we developed (Fig. 3a) here was simply a magnified replication of the I vs. pH correlation (As the surfactin concentration was kept constant in this study, the value can be assumed same within the pH range of 6.5 -9.5). The N vs. pH profile exhibited a non-linear relationship (Fig. 3), where the N decreased significantly upon exceeding pH 8. (The trend for the N vs. pH correlation observed here corroborates with that reported by Heerklotz and others 9, 20, 24, 26.) Thus, the I vs. pH correlation was non-linear, where the I values for the molecules decreased significantly upon exceeding pH 8. Because I is a direct measure of the LR charge, we therefore concluded that the LR charge reduced significantly with increase in pH from 6.5 to that > 8 and marginally for further increase in pH (here, in the range of 8 to 9.5); the LRs packed loosely at pH < 8 than at pH >8.

100

RM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

90 80 6.5 7.0 7.5 8.0 8.5 9.0 9.5

pH

Fig. 3. The RM vs. pH correlation is shown.

Intermolecular forces for the LRs at a/w interface: Based on the γEq vs. pH and the !" vs. pH correlations, the packing density for the surfactin molecules at the a/w interface increased with increase in pH in the range of 6.5 - 9.5. We hypothesized that the molecular packing density increased because the LRs experienced less intermolecular repulsion forces with increase in pH. Considering that the packing of molecules at a/w interface and the ‘hydrophobic (core of micelles)-hydrophilic (water)’ interface of the micelles depends on the intermolecular forces, a measure of the intermolecular forces at the a/w interface can be obtained in terms of the sizes of the micelles these molecules form. The intermolecular interactions (chemical potential: μ%A, C ) that determine the magnitudes of these forces can be given for a spherical micelle as 39: ∆μ%A, C = ( − |E| ⁄F2G(

+ H(

+ I + HJ1 + K(

pH–dependency of LR conformation: We examined whether charge-reduction for a LR, with increase in pH from 6 to that > 8, was due to participation of the COOH group of the LR in pHdependent intra-molecular HB formation. If so, a LR was likely to acquire different conformations at the pH levels of 6.5 and 9.5. (The directions and strengths of intra-molecular HBs govern conformational states - secondary structures - of protein molecules.) To assess the conformations probable for a LR, the CD spectra were taken for the surfactin solutions having pH levels of 6.5, 8 and 9.5. Because the surfactin concentration in these solutions was twice the cmc value, the CD spectra were mostly for the LRs that resided at the hydrophobichydrophilic interface of the micelles; the LRs remained buried in the aqueous phase. This arrangement for the LRs in a micelle is similar to that for the LRs where they remain buried in the aqueous phase of the a/w interface. Considering a particular pH level, the CD spectrum taken for the micelles were then representative of the LRs at a/w interface. The CD spectrum taken for the micelles at pH 6.5 (Fig. 4a) exhibited a negative band at 219 nm

+ HLM [2]

where, is aggregation number, e is the proton charge, ε is the dielectric constant, δ is the separation distance between the hydrophobic surface and the location of charge on the head group, and is the radium of the hydrocarbon core (4πRs3/3 = υs, and υs is the volume of the hydrophobic tail of the surfactant molecule); K is the correlation parameter. The intermolecular interactions that determine the size of a micelle are mostly


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and a positive band at 196 nm, respectively indicating the b − b ∗ and d − b ∗ transitions for the peptide chromophores of LR. These bands were characteristics of the β-sheet conformation of LR40-41. The CD spectrum taken at pH 8 exhibited a positive band at 195 nm and the negative band 205 nm, demonstrating d − b ∗ and b − b ∗ transitions respectively. These bands were suggestive of β-turn type conformation of LR41. Notably however, a shallow band (225-245 nm) in this CD spectrum, taken at pH 8 (Fig. 4a), was for the negative red shifted d − b ∗ transition. Appearance 10

pH 6.5 pH 8 pH 9.5

Ellipticity (mdeg)

8 6

(a)

4 2

Wavelength (nm)

0 190 -2

200

210

220

230

240

250

-4 -6

VZ\]

(θ of RCOOH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(b)

VWXYW

x

VZY[

^. _

*

`

**

*** a. _

pH

* No HB ** Formation of an intramolecular HB *** Increase in HB strength

Figure 4. The CD spectra (a) taken for surfactin at pH 6.5 (red), 8 (blue) and pH 9.5 (green), indicate β-sheet, metastable state (β-turn and α-helix) and α-helix conformations of the LR, respectively. A graphical description is given for the changes (dashed line) in the orientations (θ) of the R-COOH moiety with respect to the peptide backbone of LR (b). Attainment of a critical θ value (e ) for the R-COOH moiety at pH 8 (***) led to the formation of an intramolecular HB.

of this shallow band meant that the LRs could also acquire α-helix conformation at pH 8, and alongside their β-turn type conformation. Considering that this CD spectrum (at pH 8) was only for the LRs, the spectral features in this spectrum then indicated that both the β-turn and α-helix conformations were probable for a LR at pH 8. These dual conformations

5

for a LR can be explained as LR switches its conformation from β-turn to α-helix, and vice versa. This inference of LR existing as equilibrium mixture of these two conformational states at pH ~ 8 was supported by the fact that the dual spectral features were absent in the CD spectrum taken at pH 9.5. The positive band at 190 nm and the negative bands at 209 nm and 221.5 nm (Fig. 4a) in the CD spectrum taken at pH 9.5 were characteristics of the α-helix conformation of LR, where signature spectral features for β-turn were indeed absent. The positive band at 190 nm was indicative of the exciton coupling of the b − b ∗ transition, and for the perpendicularly polarized positive ( b − b ∗ ) transition. The negative bands at 209 nm and 221.5 nm were respectively for the parallel-polarized b − b ∗ transitions and the redshifted d − b ∗ transitions. 18, 42. The ability of a LR to undergo pHdependent transition in its conformation from β-sheet at the pH 6.5 to ‘β-turn and α-helix mixed state’ at pH 8, and then to α-helix at the pH 9.5 revealed that seven -[N-C-C]- moieties of the LR twisted across their central carbons in a specific manner with increase in the pH level from 6.5 to 9.5. The LR backbone could twist to acquire such a stable α-helix configurational state with aid of one or two intramolecular HB(s) 43-45. The conformational transition from β-sheet to α-helix therefore meant that the R-COOH moiety of LR participated in HB formation as the pH changed from 6.5 to 9.5, where the out-of-‘LR plane’ configuration of a R-COOH moiety at pH 6.5 changed to in-‘LR-plane’ hydrogenbonded configuration at pH 9.5 (Fig. 4b). Because of the attachment of the R-COOH moieties to the central carbons of the -[N-C-C]- groups of the Glu and Asp peptide chromophores, these changes in the configurations of the R-COOH moieties led to twisting of the -[N-C-C]- groups across their central carbons. The changes in the b − b ∗ to d − b ∗ transitions for the -[N-C-C]- entity, with a change in pH from 6.5 to 9.5, substantiated that a [-N-C-C-] group indeed twisted across its central carbon as the pH level was increased from 6.5 to 9.5. Apart from these out-of-‘LR-plane’ and the in-‘LR-plane’ configurations, the R-COOH moieties also exhibited intermediate states (Fig. 4b). These intermediate configurations were justified in terms of the characteristic d − b ∗ and b − b ∗ transitions observed for the LRs in the CD spectrum taken at pH 8. Considering that θ was maximum (out-of-‘LR plane’) at pH 6.5 and minimum (in-‘LR plane’) at pH 9.5, the θ value then decreased with increase in pH in the range of 6.5-9.5 (Fig. 4b). Formation of a HB bond with increase in pH from 6.5 to 8 meant that a critical θ (e value for a R-COOH moiety was attained at

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The Ca2+ ions those accumulate at the interface then facilitate Ca2+-bridging of the LRs of the molecules at the interfacial layer. There exists therefore a critical Ca2+ concentration ( ), where all the LRs (at the interface) form bidentate-bridges with Ca2+ ions; the LRs undergo charge-neutralization. The o% for the interface is therefore almost zero (Fig. 5a) at . (A model view of the molecules that form the zeropotential 1st layer at the a/w interface is shown (Fig. 5b) for the surfactin-stabilized MBs prepared at .) The zero o% interface is ideal for the amphiphilic surfactin molecules to partition from the aqueous phase (far away from the interface) to that near to the interface in large proportions 58. On the other hand, whether the o% is zero or negative, the Ca2+ ions invariably accumulate in the DL in large proportions as the Ca2+ concentration increases. The Ca2+ content in the DL is mostly governed by the pHdependency of the solubility levels of CaCO3 (atm CO2 in the aqueous phase) 57. Thus, at Ca2+ > , 2+ the Ca ions and the surfactin molecules accumulate at the interfacial layer (ideally a monolayer) in large amounts. This scenario is ideal for bidentate-bridging of the surfactin molecules with the Ca2+ ions for the formation a large aggregate (gel) structure 59-60, which spreads beyond (Fig. 5c) the interfacial layer 30-31, 52, 58, 61 . Thus, the DLA model can be described as that the surfactant molecules that populate at a/w interface at Ca2+ > , segregate into two regions (Fig. 5c): the interfacial layer (1st layer) and an extra layer (2nd layer) that extent from the 1st layer. It is notable that the net electrostatic potential for the 2nd layer is almost zero as well, because the LRs undergo charge-neutralization due to bidentate bridging. Therefore, the volume of the 2nd layer would increase with availability of Ca2+ ions in large amounts to bridge the LRs of the surfactin molecules. Ideally, the electrostatic interactions between these two layers can be considered negligible because the 2nd layer could only form at the zero o% 1st layer. We validated this DLA model in the subsequent sections.

around pH 8. Further decrease in the θ value with increase in pH then associated with a corresponding reduction in the HB length - the distance between the oxygen of a COOH group and the hydrogen atom of the amine group - H*f ghhi..…k l mk*n - (n and m denote positions of the peptides in a LR)46. The HB strength therefore increased as the R-COOH moieties inclined more (θ < e ) towards the LR-plane 47. In summary, the LR charge reduced significantly upon exceeding pH 8 because of the participation of the COOH group(s) of LR in intramolecular HB formation. With further increase in pH at pH levels > 8, the effective charge of a LR reduced only according to the HB strength. 48-49. Thus, the LRs undergo reduction in their net charge or chargeneutralization, depending on the HB strength of the intramolecular HB formed by the COOH groups of the LRs.49-50 Packing behavior of surfactin molecules at a/w interface in the presence of Ca2+ ions: The surfactin molecules could readily form a thick aggregate structure at the a/w interface in the presence of Ca2+ ions. The physico-chemical characteristics of this aggregate structure is complex 9, 19-20, 22, 51. As for example, the molecules arrange in a relatively ordered manner at the interface, and randomly in the aggregate structure that spread beyond the interfacial layer 16, 22, 52. To assess the packing behavior in such an aggregate structure, we developed a DoubleLayered Aggregate (DLA) model to describe the arrangements probable for the molecules in an aggregate. We developed this model by considering the typical phenomenological steps that lead to the formation of such an aggregate structure 53. We viewed (Fig. 5a) the a/w interface as an electrical double layer (EDL) 54. The distribution of ions in the EDL, with increase in the Ca2+ content, was accounted in terms of the Poisson-Boltzmann theory 51, 55 . For aggregation of surfactin molecules at the interface, we accounted for the surface potential (o% ) of the a/w interface that determines the extent of partitioning of the Ca2+ ions and the surfactin molecules from the aqueous phase to that at the interface. The o% for the a/w interface is either negative or zero across the pH range of 6.5-9.5 and, in the absence of Ca2+ ions, because the LRs of the surfactin molecules that adsorb at the interface exhibit either negative or zero charge (as shown in the earlier sections). The EDL models then propose that: as the Ca2+ ions are incorporated into the aqueous phase, a significant proportion of them invariably accumulate in the Debye Layer (DL) 56-57, and that determined according to the PoissonBoltzmann distribution of counterions at interfaces.

Molecular packing in micelles formed in the presence of Ca2+ ions: Considering a monolayer of surfactin molecules at either the a/w interface or that for micelles, the EDL models propose either negative or zero o% for both the cases, and across the pH range of 6.5 - 9.5. Therefore, the EDL for the monolayer of surfactin molecules at the hydrophobic-hydrophilic interface of micelles mimics that of the a/w interface. The phenomenological steps described in the DLA model hence are applicable to assess the aggregation behavior of the surfactin molecules at the


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hydrophobic-hydrophilic interface of micelles, and that with respect to increase in Ca2+ concentrations.

(a)

EDL representation of a/w interface 1st

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Layer

o% w 0

2nd Layer

2nd

Layer

o% − dEtuvE

1st Layer

conc.

Air

(b)

2+ Ca2+ Ca = = C cric . (c)

Ca2+>

).

Fig. 5. The packing behavior of surfactin molecules at a/w interface, in the presence of Ca2+ ions, is described in terms of the Double Layered Aggregate (DLA) model. The surface potential (electrostatic) profile (a) with respect to Ca2+ concentrations is shown for the a/w interface that is viewed as an Electrical Double Layer (EDL). The cartoons (b and c) are shown to illustrate molecular-organization and -packing in the interfacial aggregate that form at Ca2+concentrations below (b) and above (c) the level. At (b), the LRs at the interface are bridged by Ca2+ ions and form the 1st layer. A large nd volume of the 2 layer that form at Ca2+ > (c), spreads beyond the 1st layer; a green layer shown here (c) signify weak intermolecular interactions for the molecules associating these layers.

We studied the aggregation behavior of the molecules at the micellar monolayer by developing the Rm vs. ‘Ca2+ content’ correlation. The average Rm at pH 8 was 100 nms (S3, Fig. 6a) in the absence of Ca2+ ions and 110 nm in the presence of Ca2+ ions (0.25 mM). The increase in Rm by 10 nms indicated bidentate-bridging-induced increase in inter-LR distances.30 From the viewpoint of the DLA model and, as shown in Fig. 6a (dashed vertical line), if we consider that the was around 0.3 - 0.4 mM, the 2+ micelles at Ca content ≤ 0.4 mM constituted only a monolayer of surfactin molecules - the 1st layer. On the other hand, as Ca2+ content exceeded 0.4 mM, the Rm values (pH 8) were 400 and 1000 nms at 0.5 and 1 mM Ca2+, respectively (Fig. 6a); the Rm increased significantly. (As reported in literature, the Rm increases significantly upon exceeding a critical level of Ca2+ content 25, 31.) We interpreted that the 2nd layer was formed at Ca2+ content > 0.4 mM, where surfactants aggregated at the 1st layer to cause an increase in the Rm by one fold. Thus, validating the DLA model, the 2nd layer was formed beyond the 1st layer, where the volume of the 2nd layer increased with increase in Ca2+ content.

LRs in the micelles were expected to be bridged according to the DLA model. We acquired CD spectra for the micelles, prepared under different Ca2+ concentrations, to assess the bridging-induced changes in the conformations of a LR. For the micelles prepared at Ca2+ concentration of 0.1 and 0.25 mM, the CD spectra (Fig. 6b) exhibited a positive band at 195 nm and a shallow and broad band across the range of 200-225 nm. These bands were representative of either the native (without electrolytes) β-turn (at pH 8) or the disordered forms of β-sheet conformations. The β-turn conformations represented those LRs that were free of bidentatebridging. As we acquired the CD spectrum for the micelles prepared at Ca2+ concentration of 0.5 mM, a negative band at 195 nm was prominent (Fig. 6b). This negative band was mostly indicative of the nonnative (upon bridging) forms of β-sheet conformations of the LRs. The native form of β-turn conformations for the LRs were not observed at 0.5 mM Ca2+ (Ca2+ concentrations > (0.4 mM)) because it was most likely that all the LRs formed LR-Ca bridges with Ca2+ ions. These LR-Ca bridges typically exhibit short life span, and lack any particular bond length/angle. Therefore, the observation of disordered β-sheet conformations for the LRs, as evident in the CD spectrum at 0.5 mM Ca2+, explained that the LR-Ca bond(s) caused twisting the peptide backbone of LR uncharacteristically, and because the R-COOH moieties oriented away from the LR-plane. Absence of the broad band across the 225-245 nm in the CD spectra taken form the micelles prepared at 0.75 and

LR conformations in presence of Ca2+ ions: We assessed the conformations of the LRs in the micelles that were formed at pH 8 and at different Ca2+ concentrations. The LRs in the micelles were expected to form bidentate bridges depending on the availability Ca2+ ions62-63. The proportion of bridgedLRs were likely to be higher with increase in Ca2+ content in the Ca2+ concentration range of 0 - 0.4 mM. On the other hand, at Ca2+ > 0.4 mM, all the



1 mM Ca2+ > further confirmed that the LRs exhibited only non-native forms of disordered β-sheet conformations. On the other hand, this CD spectrum exhibited a broad positive band across the 195 - 205 nm range and a broad negative band across 205-240 nm range. Significant broadening of both these bands meant that they were the convoluted forms of the diverse ranges of b − b ∗ and d − b ∗ transitions probable for the LR’s peptide chromophores. Thus, as the R-COOH moieties formed bidentate bridges, they exhibited a diverse range of out-of-‘LR-plane’ orientations and caused the -[N-C-C]- backbones of the LRs to be twisted across their central carbons uncharacteristically and to varying degrees. (Such changes in conformations of LRs from native-todisordered forms has been reported to occur at a level 16, 25, 31, 52, 64.)

we have seen in the last two sections, an increase in leads to an increase the Ca2+ content at that > nd in the volume of the 2 layer. Therefore, it can be concluded that the γEq changed marginally irrespective of the increase in the volume of the 2nd layer. Negligible effect of 2nd layer on γEq substantiated that weak electrostatic interactions associated the 1st and 2nd layers. The γEq vs. ‘Ca2+ content’ correlation was verified by comparing the sizes of the MBs that were prepared at different concentrations of Ca2+ ions. The size of a MB provide a measure of γEq, and in terms of that the MB size depend on the surface pressure. Fig. S3 showed that the MB size at the Ca2+ content of 0.25 mM and at pH 8 was on an average 70 µ, which was significantly higher than that in the absence (56 µ) of Ca2+ ions at pH 8. However, with respect to an increase in the Ca2+ content at concentrations > 0.3 mM and, at

33 32

600 31 400

30

200

29

0

28 0.0

0.2

0.4 2+

Ca

0.6

0.8

75

1.0

content, mM Ca2+ concentration (mM) 0.1 0.25 0.5 0.75 1

10 8 6 4

(b)

-2

190

200

210

220

230

240

33

65

32

60

31

55 50

30

45

29

Avg. RMB Surface tension

40 35 0.0

0.2

0.4

0.6

0.8

28 27

1.0

Ca2+ concentration, mM

Fig. 7. The RMB vs. Ca2+ content and γEq vs. Ca2+ content correlations are shown.

2 0

34

70


γ

Average MB size,µm

800

Rm

34

Surface tension, mN/M

Micelle sizes, Rm, nms

(a)

1000

Ellipticity (mdeg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 20

250

pH 8, the MB sizes differed marginally, at most by 23 µ (Fig. 7 and S4). First, we examined whether the MB sizes that we determined after a few minutes of their generation were representative of their π and E values, and considering their lifetimes. As we determined the MB dissolution rate profiles to be harmonic in nature, the size comparison was according to the π and E values for the MBs. However, it was important to note that the MB dissolution rates were higher (Fig. S4) with increase in Ca2+ content > 0.3 mM (Ca2+ content > ). Thus, according to Epstein and Plesset (EP) model33, proposed by Katiyar, the elasticity of the of the 2nd layer at the a/w interface of the MBs caused an increase the dissolution rate. In fact, the dissolution rate increased with increase in Ca2+ content, > , implying that the dissolution rate increased with increase in the volume of 2nd layer. Thus, the elasticity (E) of the 2nd layer was higher with increase in its volume (Ca2+ content) and enhanced the gas

Wavelength (nm)

-4 -6 -8

Figure. 6 The N vs. Ca2+ conc., and the γ vs. Ca2+ conc. correlations are shown (a). The CD spectra (b) of the LRs at different Ca2+concentrations are given.

Dependency of γEq on Ca2+ st concentrations: In view of that the 1 layer is formed at 0.25 mM Ca2+ (< ), bidentate bridging should cause inter-LR separation at the interface, reducing the Γ value. Accordingly, the γEq was determined to be higher as 32 mN/m (Fig. 6a) at 0.25 mM Ca2+ (pH 8), as opposed to lower γEq value (27 mN/m) in the absence of Ca2+ ions (Fig. 1a). However, considering the Ca2+ concentration (> ) range of 0.5 - 1 mM, the γEq differed marginally with increase in Ca2+ content (Fig. 6a). As


Page 9 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


diffusion rate for the MBs (S4). These observations thus brought further clarity to our view of the DLA model by revealing that though the elasticity of the 2nd layer was measurable, its effect on influencing the π/γEq was marginal. We accounted for the elasticity contributions (Ec) of each of the two layers of the interfacial aggregate to the γEq values. The Ec ('( ⁄ % − 1) values for the two layers were estimated in a relative manner, and in terms of the differences in the sizes of the MBs (Eqn. 2) that were prepared in the absence and presence of Ca2+ ions (0 - 1 mM). The γEq value at pH 8 (Fig. 7) and at 0.25 mM Ca2+ was same (31.6 mN/m) as that in the absence of Ca2+ ions at pH 6 (Fig. 1). Although the γEq values were determined to be the same, the sizes (avg. 70 of the MBs with Ca2+ ions (0.25 mM were larger than that (avg. 56 ) in the absence of Ca2+ ions (Fig. 7 and S4). As the 1st layer could only be formed at 0.25 mM Ca2+, the Ec of this layer was accounted as causing an increase in the π, which led to an increase in the average size for the MBs from 56 to 70 ; 56 is an arbitrary reference point where Ec was considered negligible (reference line (pH 8) in Fig. 7). (The elasticity effects mostly ascribe to the flexible nature of the LR-Ca-LR bridges with lifetime of about 140 nanoseconds63, 65.) On the other hand, with increase in Ca2+ content at that > 0.25 mM, both the γEq values and the sizes of the MB changed negligibly with respect to increase in Ca2+ content, which meant that the Ec from the 2nd layer was negligible. Therefore, the π/γEq (assessed in a relative manner) changed negligibly even though the volume of the 2nd layer increased with increase in Ca2+ content. In spite of the fact that the elasticity (E) of the 2nd layer was measurable, negligible effect of this elasticity on π/γEq validated the DLA model - both the layers associated with each other via weak electrostatic interactions.

that the marked reduction in the net charge of a LR occurred due to the formation of an intramolecular HB, and that with increase in the pH from 6.5 to 8. Formation of this HB was evident as a LR was found to acquire pH-dependent conformations: β-sheet conformation at pH 6.5, metastable β-turn at pH 8, and α-helix form at pH 9.5. These conformations for a LR meant that the R-COOH moiety of a LR exhibited out-of-‘LR-plane’ configuration at pH 6.5, and in-‘LR-plane’ hydrogen-bonded configuration at pH 9.5. Besides these two configurations, the metastable β-turn conformation at pH 8 was indicative of the intermediate configurations probable for the R-COOH moiety. Thus, an R-COOH moiety oriented (θ) with respect to the peptide backbone of a LR in a pH-dependent manner, orienting more towards the LR-plane with increase in pH. Considering θ being maximum and minimum respectively for ‘out of -LR plane’ and in-‘LR plane’ configurations, the θ decreased to a critical value e - with increase in pH from 6.5 to 8, and led to the formation of an intramolecular HB. The θ value then decreased further to that less than e as the pH was increased from 8 to 9.5. As a result, the distance between the COOH group of an R-COOH moiety and the hydrogen of the amine group - H*f ghhi..…k lmk*n (n and m are the positions of the peptides in LR) – decreased, causing a corresponding increase in the HB strength. Thus, the pH-dependency of the net charge of LR owe to formation of an intra-molecular HB with an increase in pH from 6.5 to 8 and strengthening of the HB with further increase in pH (here, in the range of 8 to 9.5). The LRs of the surfactin molecules at the a/w interface participated in LR-Ca-LR bridging in the presence of Ca2+ ions. These bridges caused charge-neutralization of the LRs, and that depending on the availability of Ca2+ ions for the LRs. However, as these LR-Ca bridges do not exhibit any characteristic bond-length or -angle, they caused twisting of the –[N-C-C]- groups of a LR across their central carbons in a non-uniform manner. The CD spectra taken for the LRs evidently demonstrated their disordered forms of β-sheet conformations. On the other hand, the packing density increased with increase in Ca2+ content in a non-linear manner, where the packing density increased by one fold upon exceeding a critical Ca2+ concentration ( ). In spite of this significant increase in the packing density at Ca2+ content > , the γEq values and the sizes of the surfactin-stabilized MBs differed marginally. In order to explain this anomaly, we developed a Double-Layered-Aggregate (DLA) model to describe the organization and aggregation behavior of the surfactin molecules at a/w interface. According to this DLA model, the interface – 1st

CONCLUDING REMARKS We assessed the surface-active property of a surfactin variant (derived from the marine Bacillus megaterium) in terms of the packing behavior of the molecules at a/w interface, in the pH range of 6.5 9.5, and Ca2+ concentration range of 0 - 1 mM. In the absence of divalent ions, the molecules packed more tightly at the a/w interface with increase in pH from 6.5 to 8, than for an increase in pH from 8 to 9.5; thus, revealing the nonlinear relationship for the ‘packing density’ vs. pH correlation. This non-linear relationship indicated that there was a marked reduction in the net charge of LR with increase in pH from 6.5 to 8, and marginally with further increase in pH from 8 to 9.5. Based on CD assessment of the LR conformation, we found



layer – acquired zero-potential (electrostatic) at the level, where all the LRs at the interface were charge-neutralized through bidentate-bridging. As this 1st layer exhibited almost zero-potential, the surfactin molecules partitioned to this layer from the aqueous phase in large proportions at Ca2+ content > . Therefore, at Ca2+ content > , a large nd aggregate structure - 2 layer – of these partitionedsurfactants could easily form through bidentatebridging, spreading beyond the 1st layer. This view of the DLA model was supported by the fact that the micelle sizes increased marginally with increase in Ca2+ content at that < -1st layer, and, by one 2+ fold with increase in Ca content at that > . nd Because the 2 layer could only form at a zeropotential 1st layer, these layers associated with each other via weak intermolecular electrostatic interactions. Thus, with increase in Ca2+ content at that > , the γEq and the sizes of the surfactinstabilized MBs differed marginally even though the volume of the 2nd layer increases significantly.

2. Mnif, I.; Ghribi, D., Lipopeptides Biosurfactants: Mean Classes and New Insights for Industrial, Biomedical, and Environmental Applications. Biopolymers 2015, 104 (3), 129-147. DOI 10.1002/bip.22630. 3. Rodrigues, L.; Banat, I. M.; Teixeira, J.; Oliveira, R., Biosurfactants: potential applications in medicine. Journal of Antimicrobial Chemotherapy 2006, 57 (4), 609618. DOI: 10.1093/jac/dkl024. 4. Kanlayavattanakul, M.; Lourith, N., Lipopeptides in cosmetics. Int J Cosmet Sci 2010, 32 (1), 1-8. DOI: 10.1111/j.1468-2494.2009.00543.x. 5. Lintner, K.; Peschard, O., Biologically active peptides: from a laboratory bench curiosity to a functional skin care product. Int J Cosmet Sci 2000, 22 (3), 207-18. DOI: 10.1046/j.1467-2494.2000.00010.x. 6. Magetdana, R.; Ptak, M., Interfacial Properties of Surfactin. J Colloid Interf Sci 1992, 153 (1), 285-291. DOI: 10.1016/0021-9797(92)90319-H. 7. Morikawa, M.; Hirata, Y.; Imanaka, T., A study on the structure-function relationship of lipopeptide biosurfactants. Bba-Mol Cell Biol L 2000, 1488 (3), 211218. DOI: 10.1016/S1388-1981(00)00124-4. 8. Magetdana, R.; Ptak, M., Interactions of Surfactin with Membrane Models. Biophys J 1995, 68 (5), 1937-1943. DOI: 10.1016/S0006-3495(95)80370-X. 9. Shen, H. H.; Lin, T. W.; Thomas, R. K.; Taylor, D. J. F.; Penfold, J., Surfactin Structures at Interfaces and in Solution: The Effect of pH and Cations. J Phys Chem B 2011, 115 (15), 4427-4435. DOI: 10.1021/jp109360h. 10. Eastoe, J.; Dalton, J. S., Dynamic surface tension and adsorption mechanisms of surfactants at the air-water interface. Adv Colloid Interfac 2000, 85 (2-3), 103-144. DOI: 10.1016/S0001-8686(99)00017-2. 11. Razafindralambo, H.; Thonart, P.; Paquox, M., Dynamic and equilibrium surface tensions of surfactin aqueous solutions. J Surfactants Deterg 2004, 7 (1), 41-46. DOI: 10.1007/s11743-004-0286-x. 12. Iglesias-Fernandez, J.; Darre, L.; Kohlmeyer, A.; Thomas, R. K.; Shen, H. H.; Domene, C., Surfactin at the Water/Air Interface and in Solution. Langmuir 2015, 31 (40), 11097-11104. DOI: 10.1021/acs.langmuir.5b02305. 13. Amirkhanian, J. D.; Merritt, T. A., The Influence of Ph on Surface-Properties of Lung Surfactants. Lung 1995, 173 (4), 243-254. DOI: 10.1007/bf00181876. 14. Fan, H. Y.; Nazari, M.; Raval, G.; Khan, Z.; Patel, H.; Heerldotz, H., Utilizing zeta potential measurements to study the effective charge, membrane partitioning, and membrane permeation of the lipopeptide surfactin. Bba-Biomembranes 2014, 1838 (9), 2306-2312. DOI: 10.1016/j.bbamem.2014.02.018. 15. Ferre, G.; Besson, F.; Buchet, R., Conformational studies of the cyclic L,D-lipopeptide surfactin by Fourier transform infrared spectroscopy. Spectrochim Acta A 1997, 53 (4), 623-635. DOI: 10.1016/S1386-1425(96)01787-8. 16. Gallet, X.; Deleu, M.; Razafindralambo, H.; Jacques, P.; Thonart, P.; Paquot, M.; Brasseur, R., Computer simulation of surfactin conformation at a hydrophobic/hydrophilic interface. Langmuir 1999, 15 (7), 2409-2413. DOI: 10.1021/La980954r. 17. Ishigami, Y.; Osman, M.; Nakahara, H.; Sano, Y.; Ishiguro, R.; Matsumoto, M., Significance of BetaSheet Formation for Micellization and Surface-Adsorption

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: AUTHOR INFORMATION Corresponding Authors * Ramkrishna Sen Email: [email protected] Phone: +91-3222-283752. Fax: +91-3222-278707 *Partha Patra E-mail: [email protected] ACKNOWLEDGEMENTS The authors thankfully acknowledge the financial support received from the Department of Biotechnology, Government of India, for the project grant (No.: BT/PR6909/PBD/26/391/2013, 21/03/2014).The authors acknowledge the support received for this research work from the National Science Foundation Industry/University Collaborative Research Center of Particulate and Surfactant Systems (IIP 1362078). FINANCIAL CONFLICT There are no financial conflicts to report. REFERNCES 1. Bonmatin, J. M.; Genest, M.; Labbe, H.; Ptak, M., Solution 3-Dimensional Structure of Surfactin - a Cyclic Lipopeptide Studied by H-1-Nmr, Distance Geometry, and Molecular-Dynamics. Biopolymers 1994, 34 (7), 975-986. DOI 10.1002/bip.360340716.


Page 10 of 20

Page 11 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of Surfactin. Colloid Surface B 1995, 4 (6), 341-348. DOI: 10.1016/0927-7765(94)01183-6. 18. Nicolas, J. P., Molecular dynamics simulation of surfactin molecules at the water-hexane interface. Biophys J 2003, 85 (3), 1377-1391. DOI: 10.1016/S00063495(03)74571-8. 19. Onaizi, S. A.; Nasser, M. S.; Al-Lagtah, N. M. A., Benchmarking the Self-Assembly of Surfactin Biosurfactant at the Liquid-Air Interface to those of Synthetic Surfactants. J Surfactants Deterg 2016, 19 (3), 645-652. DOI: 10.1007/s11743-016-1796-9. 20. Shen, H. H.; Thomas, R. K.; Chen, C. Y.; Darton, R. C.; Baker, S. C.; Penfold, J., Aggregation of the Naturally Occurring Lipopeptide, Surfactin, at Interfaces and in Solution: An Unusual Type of Surfactant? Langmuir 2009, 25 (7), 4211-4218. DOI: 10.1021/la802913x. 21. Ko, S. O.; Schlautman, M. A., Partitioning of hydrophobic organic compounds to sorbed surfactants. 2. Model development/predictions for surfactant-enhanced remediation applications. Environ Sci Technol 1998, 32 (18), 2776-2781. DOI: 10.1021/Es9710767. 22. Song, C. S.; Ye, R. Q.; Mu, B. Z., Molecular behavior of a microbial lipopeptide monolayer at the airwater interface. Colloid Surface A 2007, 302 (1-3), 82-87. DOI: 10.1016/j.colsurfa.2007.01.055. 23. Fan, H. Y.; Nazari, M.; Raval, G.; Khan, Z.; Patel, H.; Heerklotz, H., Utilizing zeta potential measurements to study the effective charge, membrane partitioning, and membrane permeation of the lipopeptide surfactin. Biochimica et Biophysica Acta (BBA) Biomembranes 2014, 1838 (9), 2306-2312. DOI: 10.1016/j.bbamem.2014.02.018. 24. Heerklotz, H.; Wieprecht, T.; Seelig, J., Membrane perturbation by the lipopeptide surfactin and detergents as studied by deuterium. J Phys Chem B 2004, 108 (15), 4909-4915. DOI: 10.1021/jp0371938. 25. Knoblich, A.; Matsumoto, M.; Ishiguro, R.; Murata, K.; Fujiyoshi, Y.; Ishigami, Y.; Osman, M., Electron Cryo-Microscopic Studies on Micellar Shape and Size of Surfactin, an Anionic Lipopeptide. Colloid Surface B 1995, 5 (1-2), 43-48. DOI: 10.1016/09277765(95)01207-Y. 26. Heerklotz, H.; Seelig, J., Detergent-like action of the antibiotic peptide surfactin on lipid membranes. Biophys J 2001, 81 (3), 1547-1554. DOI: 10.1016/S00063495(01)75808-0. 27. Li, Z. X.; Dong, C. C.; Thomas, R. K., Neutron reflectivity studies of the surface excess of gemini surfactants at the air-water interface. Langmuir 1999, 15 (13), 4392-4396. DOI: 10.1021/La981551u. 28. Bonmatin, J. M.; Genest, M.; Petit, M. C.; Gincel, E.; Simorre, J. P.; Cornet, B.; Gallet, X.; Caille, A.; Labbe, H.; Vovelle, F.; Ptak, M., Progress in Multidimensional Nmr Investigations of Peptide and Protein 3-D Structures in Solution - from Structure to Functional-Aspects. Biochimie 1992, 74 (9-10), 825-836. DOI: 10.1016/0300-9084(92)90065-M. 29. Ruano, M. L. F.; García-Verdugo, I.; Miguel, E.; Pérez-Gil, J.; Casals, C., Self-Aggregation of Surfactant Protein A. Biochemistry 2000, 39 (21), 6529-6537. DOI: 10.1021/bi000188z.

30. Gang, H.; Liu, J.; Mu, B., Binding structure and kinetics of surfactin monolayer formed at the air/water interface to counterions: A molecular dynamics simulation study. Biochimica et Biophysica Acta (BBA) Biomembranes 2015, 1848 (10, Part A), 1955-1962. DOI: 10.1016/j.bbamem.2015.05.016. 31. Li, Y.; Zou, A. H.; Ye, R. Q.; Mu, B. Z., Counterion-induced changes to the micellization of surfactin-C16 aqueous solution. J Phys Chem B 2009, 113 (46), 15272-15277. DOI: 10.1021/jp9062862. 32. Noskov, B. A.; Loglio, G.; Miller, R., Dilational surface visco-elasticity of polyelectrolyte/surfactant solutions: Formation of heterogeneous adsorption layers. Adv Colloid Interfac 2011, 168 (1-2), 179-197. DOI: 10.1016/j.cis.2011.02.010. 33. Epstein, P. S.; Plesset, M. S., On the Stability of Gas Bubbles in Liquid-Gas Solutions. J Chem Phys 1950, 18 (11), 1505-1509. DOI: Doi 10.1063/1.1747520. 34. Katiyar, A.; Sarkar, K.; Jain, P., Effects of encapsulation elasticity on the stability of an encapsulated microbubble. J Colloid Interf Sci 2009, 336 (2), 519-525. DOI: 10.1016/j.jcis.2009.05.019. 35. Dhanarajan, G.; Mandal, M.; Sen, R., A combined artificial neural network modeling-particle swarm optimization strategy for improved production of marine bacterial lipopeptide from food waste. Biochem Eng J 2014, 84, 59-65. DOI: 10.1016/j.bej.2014.01.002. 36. Dhanarajan, G.; Rangarajan, V.; Sen, R., Dual gradient macroporous resin column chromatography for concurrent separation and purification of three families of marine bacterial lipopeptides from cell free broth. Sep Purif Technol 2015, 143, 72-79. DOI: 10.1016/j.seppur.2015.01.025. 37. Dhanarajan, G.; Rangarajan, V.; Sridhar, P. R.; Sen, R., Development and Scale-up of an Efficient and Green Process for HPLC Purification of Antimicrobial Homologues of Commercially Important Microbial Lipopeptides. Acs Sustain Chem Eng 2016, 4 (12), 66386646. DOI: 10.1021/acssuschemeng.6b01498. 38. Young, F. R., Cavitation. McGraw-Hill: 1989. 39. Florenzano, F. H.; Dias, L. G., Critical micelle concentration and average aggregation number estimate of zwitterionic amphiphiles: Salt effect. Langmuir 1997, 13 (21), 5756-5758. DOI: 10.1021/La970176n. 40. Greenfield, N. J., Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 2006, 1 (6), 2876-2890. DOI: 10.1038/nprot.2006.202. 41. Lopes, J. L. S.; Miles, A. J.; Whitmore, L.; Wallace, B. A., Distinct circular dichroism spectroscopic signatures of polyproline II and unordered secondary structures: Applications in secondary structure analyses. Protein Sci 2014, 23 (12), 1765-1772. DOI: 10.1002/pro.2558. 42. Wieprecht, T.; Apostolov, O.; Beyermann, M.; Seelig, J., Thermodynamics of the alpha-helix-coil transition of amphipathic peptides in a membrane environment: Implications for the peptide-membrane binding equilibrium. J Mol Biol 1999, 294 (3), 785-794. DOI: 10.1006/jmbi.1999.3268. 43. Micsonai, A.; Wien, F.; Kernya, L.; Lee, Y. H.; Goto, Y.; Refregiers, M.; Kardos, J., Accurate secondary structure prediction and fold recognition for circular



dichroism spectroscopy. P Natl Acad Sci USA 2015, 112 (24), E3095-E3103. DOI: 10.1073/pnas.1500851112. 44. Tapiolas, D. M.; Roman, M.; Fenical, W.; Stout, T. J.; Clardy, J., Octalactin-a and Octalactin-B - Cytotoxic 8-Membered-Ring Lactones from a Marine Bacterium, Streptomyces Sp. J Am Chem Soc 1991, 113 (12), 46824683. DOI: 10.1021/ja00012a048. 45. Rosengren, K. J.; Daly, N. L.; Plan, M. R.; Waine, C.; Craik, D. J., Twists, knots, and rings in proteins - Structural definition of the cyclotide framework. J Biol Chem 2003, 278 (10), 8606-8616. DOI: 10.1074/jbc.M211147200. 46. Hubbard, R. E.; Kamran Haider, M., Hydrogen Bonds in Proteins: Role and Strength. In eLS, John Wiley & Sons, Ltd: 2001. DOI: 10.1002/9780470015902.a0003011.pub2. 47. Zhao, G.-J.; Han, K.-L., Role of Intramolecular and Intermolecular Hydrogen Bonding in Both Singlet and Triplet Excited States of Aminofluorenones on Internal Conversion, Intersystem Crossing, and Twisted Intramolecular Charge Transfer. The Journal of Physical Chemistry A 2009, 113 (52), 14329-14335. DOI: 10.1021/jp903200x. 48. Efimov, A. V.; Brazhnikov, E. V., Relationship between intramolecular hydrogen bonding and solvent accessibility of side-chain donors and acceptors in proteins. Febs Lett 2003, 554 (3), 389-393. DOI: 10.1016/S00145793(03)01189-X. 49. Shan, S. O.; Herschlag, D., The change in hydrogen bond strength accompanying charge rearrangement: Implications for enzymatic catalysis. P Natl Acad Sci USA 1996, 93 (25), 14474-14479. DOI: 10.1073/pnas.93.25.14474. 50. Gilli, P.; Pretto, L.; Bertolasi, V.; Gilli, G., Predicting Hydrogen-Bond Strengths from Acid-Base Molecular Properties. The pK(a) Slide Rule: Toward the Solution of a Long-Lasting Problem. Accounts Chem Res 2009, 42 (1), 33-44. DOI: 10.1021/ar800001k. 51. Nag, K.; Perez-Gil, J.; Ruano, M. L. F.; Worthman, L. A. D.; Stewart, J.; Casals, C.; Keough, K. M. W., Phase transitions in films of lung surfactant at the airwater interface. Biophys J 1998, 74 (6), 2983-2995. DOI: 10.1016/S0006-3495(98)78005-1. 52. Vass, E.; Besson, F.; Majer, Z.; Volpon, L.; Hollosi, M., Ca2+-induced changes of surfactin conformation: A FTIR and circular dichroism study. Biochem Bioph Res Co 2001, 282 (1), 361-367. DOI: 10.1006/bbrc.2001.4469. 53. Walters, R. W.; Jenq, R. R.; Hall, S. B., Distinct steps in the adsorption of pulmonary surfactant to an airliquid interface. Biophys J 2000, 78 (1), 257-266. DOI: 10.1016/S0006-3495(00)76589-1. 54. Takahashi, M., xi potential of microbubbles in aqueous solutions: Electrical properties of the gas-water interface. J Phys Chem B 2005, 109 (46), 21858-21864. DOI: 10.1021/jp0445270.

55. Bu, W.; Vaknin, D.; Travesset, A., Monovalent counterion distributions at highly charged water interfaces: Proton-transfer and Poisson-Boltzmann theory. Phys Rev E 2005, 72 (6). DOI: 10.1103/PhysRevE.72.060501. 56. Braunschweig, B.; Schulze-Zachau, F.; Nagel, E.; Engelhardt, K.; Stoyanov, S.; Gochev, G.; Khristov, K.; Mileva, E.; Exerowa, D.; Miller, R.; Peukert, W., Specific effects of Ca2+ ions and molecular structure of betalactoglobulin interfacial layers that drive macroscopic foam stability. Soft Matter 2016, 12 (27), 5995-6004. DOI: 10.1039/c6sm00636a. 57. Moulin, P.; Roques, H., Zeta potential measurement of calcium carbonate. J Colloid Interf Sci 2003, 261 (1), 115-126. DOI: 10.1016/S00219797(03)00057-2. 58. Jungwirth, P.; Tobias, D. J., Specific ion effects at the air/water interface. Chem Rev 2006, 106 (4), 12591281. DOI: 10.1021/cr0403741. 59. Monteux, C.; Williams, C. E.; Meunier, J.; Anthony, O.; Bergeron, V., Adsorption of oppositely charged polyelectrolyte/surfactant complexes at the air/water interface: Formation of interfacial gels. Langmuir 2004, 20 (1), 57-63. DOI: 10.1021/la0347861. 60. Taneva, S. G.; Keough, K. M. W., Calcium-Ions and Interactions of Pulmonary Surfactant Proteins Sp-B and Sp-C with Phospholipids in Spread Monolayers at the Air-Water-Interface. Bba-Biomembranes 1995, 1236 (1), 185-195. DOI: 10.1016/0005-2736(95)00046-6. 61. Saleem, M.; Meyer, M. C.; Breitenstein, D.; Galla, H. J., Calcium Ions as "Miscibility Switch": Colocalization of Surfactant Protein B with Anionic Lipids under Absolute Calcium Free Conditions. Biophys J 2009, 97 (2), 500-508. DOI: 10.1016/j.bpj.2009.05.011. 62. Francois, J. M.; Sedarous, S. S.; Gerday, C., Ca2+-induced conformational shift of the COOH-domain of eel skeletal muscle troponin C in the presence of physiological concentrations of Mg2+. J Muscle Res Cell M 1997, 18 (3), 323-334. DOI: 10.1023/A:1018622109391. 63. Bala, T.; Prasad, B. L. V.; Sastry, M.; Kahaly, M. U.; Waghmare, U. V., Interaction of Different Metal Ions with Carboxylic Acid Group: A Quantitative Study. The Journal of Physical Chemistry A 2007, 111 (28), 61836190. DOI: 10.1021/jp067906x. 64. Arutchelvi, J.; Sangeetha, J.; Philip, J.; Doble, M., Self-assembly of surfactin in aqueous solution: Role of divalent counterions. Colloids and Surfaces B: Biointerfaces 2014, 116, 396-402. DOI: 10.1016/j.colsurfb.2013.12.034. 65. Cote, A. S.; Freeman, C. L.; Darkins, R.; Duffy, D. M., Structure and Orientation of MDBA Self-Assembled Monolayers and Their Interaction with Calcite: A Molecular Dynamics Study. J Phys Chem C 2013, 117 (14), 7148-7153. DOI: 10.1021/jp4006235.


Page 12 of 20

Page 13 of 20

In absence of Ca2+ ions

In presence of Ca2+ ions ions

𝑎𝑒 LR β-sheet

Loosely packed LRs

D𝐿𝑒𝑢

pH: 8, Ca2+ = Ca2+cric L𝐺𝑙𝑢

L𝐿𝑒𝑢

Single layer at interface 2nd Layer Water

𝐶𝐻

α-helix

Mixed β-sheet

L𝐴𝑠𝑝

L𝐿𝑒𝑢

𝐶𝐻2

𝑎𝑒

β-sheet to α-helix transition

D𝐿𝑒𝑢

𝐿 − 𝑉𝑎𝑙

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ሺ𝐶𝐻2 ሻ9

1st Layer

Disordered β-sheet

𝐶𝐻 𝐶𝐻3

𝐶𝐻3 Tightly packed LRs

Air Air Water

pH: 8, Ca2+ > Ca2+cric Double layer at interface

The results from this study have chemistry and engineering implications of surfactin molecules in sustainable synthesis of micro-emulsions and micro-bubbles.



Figure 1

(a)

Surface tension values (b) - pH 6.5 MB sizes, RMB

110

32

100 30 90 28

(c) - pH 8

80

7

pH 8

9

70

MB size, RMB, Micron


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(d) - pH 9.5

Fig. 1. The γEq vs. pH (red line) and the 𝑅𝑀𝐵 vs. pH (dashed line) correlations (a) are shown. The microscopic images are given for the MBs prepared at pH 6.5 (b), 8 (c), and 9.5 (d); bar indicates 50 µm.


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Page 15 of 20

FIGURE 2

(a)

# of MBs in suspension

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pH 6.5 8 9.5

10000

5000 (b)

0 0

5

10

15

20

25

Time, min 1st

18th

22nd

(b) Fig. 2. The dissolution rate profiles for the MBs prepared under different pH conditions (a) are given. The microscopic images (b) are shown for the MBs that were prepared at pH 8 and taken in course of their dissolution, at 1 st, 18th and 22nd minutes; bar: 25 µ.



Figure 3

100

RM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

90 80 6.5 7.0 7.5 8.0 8.5 9.0 9.5

pH Fig. 3. The Rm (micelle size) vs. pH correlation is shown.


Page 16 of 20

Page 17 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




Figure 5 (a) EDL representation of a/w interface 1st Layer

𝐶𝑎2+ 𝑐𝑟𝑖𝑐

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 20

2nd Layer

2nd

1st Layer

Layer

𝜓0 ≈ 0

𝜓0 − 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒

𝐶𝑎2+ conc.

Air

(b)

2+2+=C = Ccric cric (c) CaCa

Ca2+>Ccric

Fig. 5. The packing behavior of surfactin molecules at a/w interface, in the presence of Ca2+ ions, is described in terms of the Double Layered Aggregate (DLA) model. The surface potential (electrostatic) profile (a) with respect to Ca2+ concentrations is shown for the a/w interface that is viewed as an Electrical Double Layer (EDL). The cartoons (b and c) are shown to illustrate molecular-organization and -packing in the interfacial aggregate that form at 2+ 2+ Ca2+concentrations below (b) and above (c) the 𝐶𝑎𝑐𝑟𝑖𝑐 level. At 𝐶𝑎𝑐𝑟𝑖𝑐 (b), the LRs at the interface are bridged by Ca2+ ions and form the 1st layer. A large volume of the 2nd layer that 2+ form at Ca2+ > 𝐶𝑎𝑐𝑟𝑖𝑐 (c), spreads beyond the 1st layer; a green layer shown here (c) signify weak intermolecular interactions for the molecules associating these layers.


Page 19 of 20

Figure 6

1000 800

Rm

34

g

33 32

600 31 400

30

(b)

200

29

0

Surface tension, mN/M

Micelle sizes, Rm, nms

(a)

28 0.0

0.2

0.4

0.6

0.8

1.0

Ca2+ content, mM Ca2+ concentration (mM) 0.1 0.25 0.5 0.75 1

10 8

Ellipticity (mdeg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6 4 2 0 -2

190

200

210

220

230

240

250

Wavelength (nm)

-4 -6 -8

Figure. 6 The 𝑅𝑚 vs. Ca2+ conc., and the γ vs. Ca2+ conc. correlations are shown (a). The CD spectra (b) of the LRs at different Ca2+concentrations are given.



FIGURE 7

75 33

65

32

60

31

55 50

30

45

29

Avg. RMB Surface tension

40 35 0.0

0.2

0.4

0.6

0.8

28


34

70

Average MB size,mm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 20

27

1.0

Ca2+ concentration, mM Fig. 7. The RMB vs. Ca2+ content and γEq vs. Ca2+ content correlations are shown.


Modeling and Analysis of Micellar and Microbubble Dynamics To

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