Synthesis of Surface-Modified Iron Oxides for the Solvent-Free

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Synthesis of Surface Modified Iron Oxides for the Solvent Free Recovery of Bacterial Bioactive Compound, Prodigiosin and Its Algicidal Activity Villalan Arivizhivendhan, Manacharaju Mahesh, Boopathy Ramasamy, Karmegam Patchaimurugan, Ramasamy Regina Mary, Ganesan Sekaran, P Maharaja, and S Swarnalatha J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b03926 • Publication Date (Web): 03 Aug 2016 Downloaded from http://pubs.acs.org on August 5, 2016

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The Journal of Physical Chemistry

Synthesis of Surface Modified Iron Oxides for the Solvent Free Recovery of Bacterial Bioactive Compound, Prodigiosin and Its Algicidal Activity Arivizhivendhan K. V1, Mahesh M2, BoopathyR3, Patchaimurugan K2, Maharaja P2, Swarnalatha S2, Regina Mary R1*, Sekaran G2* 1

2

PG & Research Department of Zoology, Auxilium College, Vellore, India

Environmental Technology Division, Central Leather Research Institute (CSIR-CLRI), Chennai, India 3

Environment & Sustainability Department, Institute of Minerals and Materials Technology (CSIRIMMT), Bhubaneswar, Orissa, India

* Corresponding Author Dr. G. Sekaran, Tel.: +91-44-24911386 Fax: +91-44-24452941 Email:[email protected] * Corresponding Author R. Regina Mary Tel: 9952355724 E Mail: [email protected]

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Abstract Prodigiosin is a bioactive compound produced from several bacterial diversities. Currently, many technologies are being developed for the production of prodigiosin by fermentation processes. However, new challenges are being faced on the production of prodigiosin with recovery and purification steps owing to the labile nature of prodigiosin molecules and cost of purification steps. The conventional methods have limitations due to high cost, low reusability and health hazards. Hence, the present investigation was focused on the development of surface functionalized magnetic iron oxide ([Fe3O4]F) for the solvent free extraction of bioactive prodigiosin from the bacterial fermented medium. The Fe3O4 was functionalized with diethanolamine and characterized by FT-IR, DRS, TGA, SEM and confocal microscopy. The various process parameters such as contact time, temperature, pH and mass of Fe3O4 were optimized for the extraction of prodigiosin using functionalized Fe3O4. The instrumental analyses confirmed that the prodigiosin molecules were cross-linked with functional groups onto [Fe3O4]F through Vander Waals force of attraction. The prodigiosin extracted through Fe3O4 or [Fe3O4]F was separated from the fermentation medium by the applied external electromagnetic field and regenerated for successive reuse cycles. The purity of extracted prodigiosin was characterized by HPLC, FT-IR and UV-Visible spectroscopy. The iron oxidediethanolamine-prodigiosin cross linked ([Fe3O4]F-PG) composite matrix effectively deactivated the harmful fouling by

cyanaobacterial growth in water treatment plants. The present

investigation provides the possibility of solvent free extraction of bacterial bioactive prodigiosin from fermented medium using functionalized magnetic iron oxide.

Introduction 2 ACS Paragon Plus Environment

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Prodigiosin

is a red pigmented secondary metabolite, characterized by

molecular

formula, C20H25N3O; molecular weight, 323; dissociation constants pKa1, 7 and pKa2, 8 and cationic in nature.1,2 It has been widely used in pharmacological and industrial applications such as anticancer, immune suppressive, anti-inflammatory, antibacterial, antifungal, antiviral, antimalarial, mosquito larvicidal, insecticidal, anti-protozoa, anti-antemia, antioxidant, pH indicator and dyeing agent.3-14 Despite its vast applications, it has some limitations due to the high processing cost and huge energy consumption in the extraction step. Organic solvents such as chloroform, diethyl ether, petroleum ether etc are being used for the extraction of prodigiosin from fermented medium.15-20 The solvent extraction is considered to be disadvantageous due its toxic effect to deteriorate soil fertility and their volatile nature impair human health.21 Polymeric materials have been recommended for the extraction of prodigiosin from the fermented medium after certain chemical pretreatment such as alum, solvents and ultrafiltration.22-27 In addition, the organic molecules such as organic acids, peptides, proteins and nucleic acids from the fermented medium adsorbed onto polymeric materials.28,29 The adsorbed protein molecules by the polymeric materials affected the extraction efficiency and purity of prodigiosin. Overall the application of solvents and chemicals for the extraction of prodigiosin severely affect the environment besides purification process becomes cost intensive. Hence, it is important to develop an environmentally safe and cost- effective process for the extraction of prodigiosin from fermentation medium. Among these methods, extraction of prodigiosin by adsorption phenomena is one of the most investigated techniques for pigment removal from aqueous medium mainly due to its simplicity and high degree of effectiveness.30 Hence, the [Fe3O4]F was considered for the adsorption of prodigiosin from fermented medium for its high sorption capacity due to high reactive surfaces and large specific surface area and ease of regeneration property.31-33 The prodigiosin has greater aggregation tendency with heavy metal 3 ACS Paragon Plus Environment


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ions by strong electrostatic interactions between primary and secondary amine groups of the prodigiosin molecule. The addition of functionalized nanoparticles enables magnetic property, rendering these functionalized composites robust, and stimulate the active materials that could be employed in various biological and biomedical devices.34-35 We have demonstrated the functionalized Fe3O4 ([Fe3O4]F) using diethanolamine to enhance the functional groups on the surface of Fe3O4. The [Fe3O4]F was used for the extraction of prodigiosin from fermented medium. Moreover the algalicidal activity of prodigiosin impregnated [Fe3O4]F was demonstrated. Materials and methods Materials Ferrous sulphate (Fe SO4.7H2O), ammonia solution (25%), and diethanolamine were of analytical grade resourced from Merck chemicals (India). The microbiological constituents were purchased from HiMedia chemicals, India. Milli-Q water was used in all the experiments. Production of prodigiosin The bacterial strain employed in this study was isolated from proteinacious solid waste known as tannery fleshing (TF) discharged from leather industry and it was identified using 16s rRNA. A loop full of isolate was inoculated in nutrient broth and incubated at 37 0C for 24 h and named as mother culture. Mother culture (1% v/v) was transferred to the sterilized fermentation medium for the biosynthesis of prodigiosin. The fermentation medium containing TF 1% (w/v) was dispersed in M9 mineral medium of volume 100 mL.35 Then the fermented medium was incubated at 30 0C for 96 h. The influence of process parameters such as time, temperature, pH, concentration of tannery fleshing (TF) and glycine were optimized under batch culture method 4 ACS Paragon Plus Environment

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for the biosynthesis of prodigiosin. The optimum conditions for the production of prodigiosin were determined by central composite design using design expert 8.0 software. The fermented solution after biosynthesis of prodigiosin (PBFS) was directly considered for the extraction of prodigiosin by Fe3O4 or [Fe3O4]F. Preparation of Fe3O4 and [Fe3O4]F The [Fe3O4]F matrix was prepared by agitating ferrous sulphate with diethanolamine at alkaline condition.36 Ferrous sulphate, 1% (w/v) of volume 100 mL was taken in a 500 mL glass beaker and it was agitated at 600 rpm using magnetic stirrer until to get a homogeneous ferrous sulphate solution. The diethanolamine of volume 10 mL was added into ferrous sulphate solution under stirring at 100 rpm and continued for 15 min. Then, ammonia solution (25%) of volume 100 ml was added gently and the resultant solution was incubated at 80 oC for 30 min under stirring at 100 rpm. The solution color was changed from brownish to black, indicated the formation of [Fe3O4]F. Similarly, Fe3O4 was prepared by following the above steps without the addition of diethanolamine. The Fe3O4 and [Fe3O4]F were separated from the suspension by centrifugation at 5000 rpm for 20 min. The separated products were dried and stored at room temperature until to carry out further experiments. Extraction of prodigiosin from batch fermented solution The solvent free prodigiosin extraction from PBFS was carried out by extraction using Fe3O4 and [Fe3O4]F. The extraction of prodigiosin was carried out by taking 50 mL of PFB solution into 100 mL Erlenmeyer conical flask at 30 0C under agitation at 100 rpm. The Fe3O4 or [Fe3O4]F (20 g/L) was added into the PFB solution for the extraction of prodigiosin. The various process parameters such as contact time, temperature, pH and concentration of prodigiosin were



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studied for the prodigiosin extraction. The pH of PBFS was varied from 1 to 10 and temperature was varied from 20 to 50 ºC for the optimization process. The equilibrium conditions for the extraction of prodigiosin onto Fe3O4 or [Fe3O4]F were studied at different initial concentration of prodigiosin from 1 to 10 g/L. The used Fe3O4 or [Fe3O4]F materials were recovered from the experimental solution by the applied external electro magnets. The separated Fe3O4 or [Fe3O4]F was re-dispersed in methanol solution of volume 10 ml for desorption of prodigiosin. Then the Fe3O4 and [Fe3O4]F were thoroughly washed with hot distilled water (60-70 0C) under agitation at 200 rpm for 10 min. The prodigiosin extracted by Fe3O4 and [Fe3O4]F were named as Fe3O4–PG and [Fe3O4]F–PG respectively. The regenerated Fe3O4 and [Fe3O4]F was used in successive cycles. Characterization of matrices The Fe3O4, [Fe3O4]F, Fe3O4-PG and [Fe3O4]F–PG were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and thermo gravimetric analysis (TGA) for the surface characterization of the materials. XRD measurements were carried out using a PAN analytical X’Pert PRO-XRD instrument. Fourier transform- infrared red (FTIR) spectra were recorded using Perkin Elmer infrared spectrophotometer using KBr discs as reference, with average 32 scans for the scan range of 4000–400 cm−1 at a resolution of 4 cm−1. Thermo gravimetric analysis (TGA) was performed from 35 ºC to 800ºC at a heating rate of 5 ºC/min under nitrogen gas flow condition. The UV– DRS reflectance spectral data were recorded using Carry, varian-200 spectrophotometer. The extracted

prodigiosin

from

Fe3O4

or

[Fe3O4]F was

spectrophotometer , FT-IR and HPLC. Algicidal activity 6 ACS Paragon Plus Environment

characterized

by

UV-Visible

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The algicidal activity of Fe3O4-PG, or [Fe3O4]F–PG were studied against harmful cyanobacteria (Microcystis aeroginosa) commonly found in water collection systems. The M. aeroginosa was grown on BG 11 broth medium of volume 1 L for 5 days in photo-fermenter. After the growth of M. aeroginosa for 5 days, 1g of Fe3O4-PG or [Fe3O4]F–PG was dispersed into fermented solution separately and solution without addition of these products served as control. The fermenter was agitated at 200 rpm for 2 h in the presence of photo bulb at 100 W. After incubation, the sample was collected from the photo-fermenter and the M. aeroginosa morphology was evaluated under confocal microscopy and scanning electron microscopy (SEM) for the identification of algicidal activity. Results and discussions Biosynthesis of prodigiosin Central composite design (Response surface methodology using Design Expert software, Version 8.0) was employed to determine the significance and its optimal levels of the selected process parameters for the production of prodigiosin. In the present investigation, the parameters such as incubation time, temperature, pH, glycerol and TF concentration were optimized in fermenter. The agitation speed was kept as constant at 80 rpm throughout the experimental work. The change in broth color from light yellow to red indicated the production of prodigiosin.35 The TF was found to be significant role on prodigiosin production, the production of prodigiosin increased with increase in TF concentration. This may be due to the high protein content (5060%), sufficient volume of carbohydrate (10-20%) and moderate lipid content (5-10%) in the TF. The three dimensional surface graphs obtained from central composite design for the production of prodigiosin presented in Figure 1. The maximum yield of prodigiosin, 21.5 g was obtained at incubation time, 96 h; pH, 7; temperature, 30 ℃, glycerol, 5% and TF 3% (w/v). The 7 ACS Paragon Plus Environment


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statistical significance of equation was checked by f-test and ANOVA by second order polynomial model and the values are presented in table 1. The results are found to be both linear and quadratic terms were significant with 1% error. Therefore, the selected quadratic model matches with the experimental study carried out in the present investigation. The P-values were used as a tool to check the significance of each coefficient, which was necessary to understand the pattern of the mutual interactions of the best selected factors. The F-value of the selected model was found to be 9.77 which implies the model is significant. Further, the value of "Prob > F" was observed to be less than 0.0001 indicating the model terms are significant (Table 1). Preparation and characterization of matrices The prepared Fe3O4 and [Fe3O4]F were characterized by FT-IR as shown in Figure 2a. The FT-IR of Fe3O4 with peaks observed at 580 cm–1 and 647 cm–1 attributed to the presence of strong, Fe–O symmetric stretch in Fe3O4. The peak at 454 cm-1 is due to the asymmetric stretching of Fe–O. Broad absorption peak observed at 3413 cm–1 corresponding to the stretching vibration of O–H bonds in hydroxyl groups, may be due to the presence of residual moisture. The peaks observed at 1463 cm-1 and 1104 cm-1 are due to the primary and secondary amine structure of diethanolamine which was taken during synthesis of [Fe3O4]F. Two characteristic peaks observed at 2940 and 2870 cm-1 are due to the symmetric and asymmetric stretching of CH groups present in diethanolamine. The strong band at 3413 cm-1 confirmed the presence of stretching vibration of N-H group in the diethanolamine.37-39 The characteristic peaks at 580 cm-1 and 647 cm-1 indicates the presence of [Fe3O4]F. The surface morphology of Fe3O4, and [Fe3O4]F were captured using scanning electron microscopy and confocal microscopy. The Figure 2b shows the SEM image of Fe3O4 particles and they were found to be spherical in shape with smooth surface. The SEM image of [Fe3O4]F showed an uniform distribution of smooth surfaced 8 ACS Paragon Plus Environment

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particles on the peripheral of the spherical plumes, indicated the functionalization of diethanolamine (Figure 2c). Further Fe3O4 and [Fe3O4]F were characterized in diffuse reflectance spectroscopy (DRS) (Figure 2d). The calculated energy band gap values for the Fe3O4 and [Fe3O4]F were found to be 0.16 ev and 0.249 ev respectively. The energy band gap values suggest that the conductive Fe3O4 was changed into semiconductive after functionalization with diethanolamine. The thermal stability of Fe3O4 and [Fe3O4]F were evaluated by TGA and the results are presented in Figure 2e. Both Fe3O4 and [Fe3O4]F were found to be decomposed as the temperature increased from 100 to 600 °C, and the final residue of Fe3O4 and [Fe3O4]F was observed to be 91% and 95% respectively. The [Fe3O4]F was more stable than Fe3O4, may be due to functionalization of Fe3O4 which increased the stability by the presence of diethanolamine. Extraction of prodigiosin The prodigiosin was extracted from the fermented broth medium by solvent free method using magnetic [Fe3O4]F in batch fermenter (Figure 3). The extraction time was evaluated by varying the time from 1 min to 20 min while the other conditions were kept constant. The extraction of prodigiosin was increased with increase in extraction time and reached the maximum extraction time of 30 min. This phenomenon suggested that the adsorption was dynamic in equilibrium upto 15 min and remained the same even after a prolonged time of extraction (Figure 4a). The pH of the medium was also varied for the extraction of prodigiosin. Since the ionization of solution plays an important role in prodigiosin extraction by changing the ionic charge and stability of the prodigiosin in solution. Hence, the pH of the fermented solution was adjusted from 1.0 to 10.0 using 0.1 M HCl or 0.1M NaOH for the extraction of prodigiosin. The extraction of prodigiosin was significantly increased with decrease in solution pH from 7.0 to 1.0 (Figure 4b). This may be explained that the adsorption of prodigiosin onto [Fe3O4]F was favorable at acidic pH due to the negative charge of [Fe3O4]F particles and thus protonated 9 ACS Paragon Plus Environment


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prodigiosin may be adsorbed by ionic competition at acidic pH medium. While at solution pH higher than 7.0 the prodigiosin molecules are electrostatically repelled which tend to reduce the adsorption capacity. The adsorbent dose for the extraction of prodigiosin was evaluated under batch fermenter by dispersing known amount of [Fe3O4]F (10 to 100 g/L) in 1000 mL PBFS as shown in Figure 4c. The results showed that the increase in mass of [Fe3O4]F upto 50 mg completely adsorbed the prodigiosin from PBFS. The increase in mass of [Fe3O4]F increased the adsorption capacity owing to increase in surface area available on the prodigiosin molecules. However, the adsorption capacity was found to be remain the same even after the incremental increase in mass of [Fe3O4]F beyond 50 g. The extraction efficiency of prodigiosin by Fe3O4 and [Fe3O4]F were carried by varying the initial concentration of prodigiosin from 1 to 10 g/L. The prodigiosin was adsorbed by 3.1 and 5.12 g on the addition of 50 g of Fe3O4 and [Fe3O4]F respectively into PBFS (Figure 4d). The kinetic models (pseudo first and second order) were analyzed prodigiosin extraction by adsorption phenomena and presented in Table 2. log = log −

1 2.303

1 = + 2 Where qe and qt are the amount of prodigiosin adsorbed (mg g-1) at equilibrium and at time ‘t’, k1 (min-1) and k2 (g mg-1min-1) are the pseudo first and pseudo second order rate constants, respectively; The Langmuir adsorption isotherm model was fitted to explain the nature of the adsorption of prodigiosin molecules onto Fe3O4 or [Fe3O4]F. This isotherm is based on the assumption that maximum adsorption occurs when a saturated monolayer of prodigiosin 10 ACS Paragon Plus Environment

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molecules is present on the adsorbent surface, the energy of adsorption is constant, and there is no migration or interaction between the adsorbed molecules in the surface plane. The linear expression of the Langmuir isotherm model is defined by 1 = + 3 The values of and can be determined from the intercept and slope of the linear graph of 1/ against 1/. The parameter (mg/g) is the maximum adsorption capacity of prodigiosin per unit mass of Fe3O4 or [Fe3O4]F to form a complete monolayer on the surface. The essential characteristics of the Langmuir isotherm can be expressed in terms of a dimensionless constant separation factor (Table 3). The Freundlich isotherm explains that the extent of adsorption varies directly with concentration of prodigiosin. This empirical relationship helps to identify the multilayer adsorption of prodigiosin onto Fe3O4 or [Fe3O4]F. The linear model of the isotherm can be expressed logarithmically as in 1 log = log + log 4 ! The values of parameters # and ! (listed in Table 3) can be determined from the intercept and slope of the plot log against log , where KF is the Freundlich adsorption capacity constant and 1/! is related to the adsorption intensity constant that varies with the heterogeneity of the adsorbent surface. The Dubinin–Radushkevitch isotherm model, which is based on the Polanyi theory, is as expressed in the linear form as in Eq. (5)



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ln = ln % − 2&% '( ln )1 +

, = '( ln )1 +

.=

1

/2&%

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1 * 5

1 * 6

7

where R is the gas constant (8.314 J mol-1K-1) and T is the absolute temperature. Polanyi potential, is correlated to the constant ‘BD’ gives the mean free energy ‘E’ for adsorption of prodigiosin from bulk phase to solid surface of Fe3O4 or [Fe3O4]F. The Langmuir, Freundlich, Dubinin–Radushkevitch isotherm models were applied for the adsorption of prodigiosin onto Fe3O4 or [Fe3O4]F. The values of R2 and rate constant are presented in table 3. The best obeyed isotherm model fitted for the adsorption of prodigiosin onto Fe3O4 or [Fe3O4]F was identified based on the regression coefficient. Among the studied isotherm models Langmuir was the best obeyed for the adsorption of prodigiosin by Fe3O4 or [Fe3O4]F with R2, 0.99. This confirms that the adsorptive extraction of prodigiosin by Fe3O4 or [Fe3O4]F is through by monolayer formation. Molecular interaction between prodigiosin with [Fe3O4]F The FT-IR spectrum of [Fe3O4]F-PG showed a strong stretching vibration peak observed at 3424 cm-1 may be attributed to the presence of O-H and N-H functional groups (Figure 5a). Two broad stretching vibration peaks at 2934 cm-1 and 2852 cm-1 indicate the presence of C-H functional group of methylene groups in prodigiosin of [Fe3O4]F-PG. The stretching vibration peak observed at 2900 cm-1 may be attributed to the presence of methoxy group in the


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prodigiosin molecules.40 The aromatic ring in prodigiosin was identified by the peak observed at 1475 cm-1. The ethylene linkage (C6H5-O-CH3) this is methoxy linkage was confirmed by the presence of asymmetric band at 1271 cm-1 and symmetric band at 1035 cm-1. The presence of pyrrole ring in prodigiosin molecule was identified by the stretching vibration peak observed at 1107 cm-1 in Fe3O4-PG. This confirms the presence of prodigiosin in [Fe3O4]F after extraction . There is a report on the identification of B-pyrrole ring (phenyl, furan-2-yl) and A-pyrrole ring (thiophen-2-yl) of prodigiosin by FT-IR analysis.41,42 The SEM images of Fe3O4–PG and [Fe3O4]F–PG was were shown in Figure 5b & 5c. The results illustrated that the rough surface of spherical particles was smoothened after extraction of prodigiosin on Fe3O4 and [Fe3O4]F. Further, the prodigiosin adsorbed Fe3O4 and [Fe3O4]F particles were characterized by UV-DRS and its energy band gap were calculated and presented in Figure 5d. The calculated energy band gap values for Fe3O4-PG and [Fe3O4]F-PG were 0.25 ev and 0.29 ev respectively. The conductive nature of [Fe3O4]F was found to be changed to semiconductive nature due to the bonded prodigiosin molecules with [Fe3O4]F. The energy gap value was increased after bonding with prodigiosin molecules with [Fe3O4]F surface due to anchoring of d-orbital electron in Fe3O4, by coordinate linkage. The thermal stability of Fe3O4-PG and [Fe3O4]F-PG were evaluated by TGA and the results are presented in Figure 5e. Both Fe3O4-PG and [Fe3O4]F-PG were found to be decomposed as the temperature was increased from 100 to 600 °C, and the residue weight were 90% and 92% (w/w) respectively. There was an additional weight loss by about 2% and 3% for Fe3O4-PG and [Fe3O4]F-PG respectively, it may be attributed to decomposition of prodigiosin molecules bonded to Fe3O4-PG and [Fe3O4]F-PG. This confirmed the adsorption of prodigiosin molecules onto Fe3O4 or [Fe3O4]F. The XRD patterns of [Fe3O4]F and [Fe3O4]F-PG interaction were analyzed in Figure 6. The XRD patterns 2θ values at 30.1°, 35.5°, 43.3°, 53°, and 62.7° could be assigned to the characteristic 13 ACS Paragon Plus Environment


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peaks of [Fe3O4]F particles. The [Fe3O4]F-PG spectrum showed the broad peak in the range of 30.1°, 35.5°, 43.3°, 53°, and 62.7 correspondence to [Fe3O4]F and additionally slight hump in the range of 5° to 15°, it could be due to the adsorption of prodigiosin on [Fe3O4]F. Figure 7 shows confocal microscopic images of Fe3O4, [Fe3O4]F, Fe3O4–PG and [Fe3O4]F–PG. The Fe3O4–PG and [Fe3O4]F–PG were found to be aggregated because the hydrophobic nature of the prodigiosin molecules bound to the surface of the iron oxide particles and the [Fe3O4]F particles were densely packed

due to covalent overlapping between functional groups present on the matrix and

prodigiosin. The confocal microscopic image confirmed the more dense aggregation of prodigiosin on [Fe3O4]F (Figure 7d). The [Fe3O4]F was prepared by the addition of diethanolamine as a stabilizing agent and it also acted as active functional groups on the surface of Fe3O4 which was responsible for the prodigiosin extraction. The reason for the selection of diethanolamine as a functionalization reagent was to create amide functional groups on the surface of Fe3O4 for the effective extraction of hydrophobic prodigiosin from aqueous medium (Figure 8). It is reasonable to accept that the impenetrable network of Fe3O4−diethanolamine further precludes adsorption of other molecules onto the surface of [Fe3O4]F. The functional group of the diethanolamine consistently distributed on the surface of Fe3O4. Thus, proposed method of prodigiosin extraction reduced the solvent usage by about 95% than the conventional method of extraction (Figure 9). Recovery and reusability of Fe3O4 and [Fe3O4]F The recovery and reusability of the used Fe3O4 and [Fe3O4]F were evaluated and the results are presented in Figure 10a. The desorption of prodigiosin from Fe3O4-PG and [Fe3O4]FPG was carried out by using different polar and non-polar solvents such as acetone, ethanol, methanol, chloroform, diethyl ether, ethyl acetate, hexane, DMSO, dichloromethane and 14 ACS Paragon Plus Environment

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isopropanol as shown in Figure 10a. The desorption was carried out with 5 mL of solvent per gram of Fe3O4-PG or [Fe3O4]F-PG. The maximum desorption of prodigiosin was achieved with polar solvents such as acetone, ethanol, methanol, DMSO and isopropanol. Among the studied solvents acetone established the maximum elution than the other solvents. The regenerated Fe3O4 or [Fe3O4]F was washed with 10 mL of distilled water at 80 oC for 2 min in vortex mixture. The regenerated and washed Fe3O4 or [Fe3O4]F was collected and reused in successive cycles of prodigiosin extraction. This study revealed that the [Fe3O4]F could be regenerated and reused for many number of cycles after successful washing with hot water (Figure 10b). Characterization of prodigiosin extracted from Fe3O4 and [Fe3O4]F The recovered prodigiosin from Fe3O4 and [Fe3O4]F were characterized by high performance liquid chromatography as illustrated in Figure 11a. The prodigiosin extracted from both Fe3O4 and [Fe3O4]F showed a doublet peak at 4.6 min and 5.5 min of retention time, indicated the extracted compound

belongs to prodigiosin. However, the additional peak

observed in Fe3O4 extracted solution at 10.3 min and 10.9 min for may be due to the impurity from PBFS. The purity of the prodigiosin extracted from Fe3O4 and [Fe3O4]F were calculated to be 50% and 90%. The FT-IR spectra of extracted prodigiosin from Fe3O4 and [Fe3O4]F are presented in Figure 11b . The FT-IR spectrum of prodigiosin eluted from [Fe3O4]F shows strong stretching peaks observed at 1218 cm-1 and 2928 cm-1, may be attributed to the presence of aromatic (–CH) and methylene (CH2) stretching in prodigiosin molecules. Another stretching peak at 1630 cm-1 may be due to the presence of conjugated aromatic C=C bond in prodigiosin. The presence of N-H of the pyrrole ring in prodigiosin was confirmed by the stretching peak observed in the range of 3421 -3540 cm-1. The presence of C-C stretching was confirmed by the peak observed at 3295 cm-1. The strong peaks noticed at 2928 cm-1 and 2800 cm-1(C-O-CH3) 15 ACS Paragon Plus Environment


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Page 16 of 40

may be attributed to the presence of methyl groups in the prodigiosin molecules. The (–C-O-C-) –O-CH3 group attached in aromatic pyrrole ring in prodigiosin molecular structure was confirmed by strong band observed at 1133-1060 cm-1. The fingerprint region was characterized by the presence of medium peaks at 1734 cm-1 (C=O), 1452 cm-1 (C-O), 1133 cm-1 (C-N), 986 cm-1 & 817 cm-1 in prodigiosin molecule. Similarly, the prodigiosin extracted from Fe3O4 showed same characteristic additional peaks, this could be due to the presence of impurities from extracted solution. The purity of the extracted prodigiosin from Fe3O4 and [Fe3O4]F were evaluated by UV-visible spectrophotometer based on its characteristic peak intensity (Figure 11c). The extracted prodigiosin by [Fe3O4]F was characterized by peaks at λ532 nm in the visible region and at λ

290 nm

in the ultraviolet region. Similarly, the prodigiosin extracted by Fe3O4

showed a characteristic peak at λ

305 nm,

the positive shift in wavelength may be due to the

presence of impurity on extraction. Algicidal activity of Fe3O4-PG and [Fe3O4]F-PG The antifouling activity of Fe3O4-PG and [Fe3O4]F-PG were evaluated using scanning electron microscopy (SEM) and confocal microscopy (Figure 12). Figures 12a & 12d show that the virgin M. aeroginosa algal cells were found to be spherical in shape with smooth exterior and oval shaped cell structure. The Fe3O4 and [Fe3O4]F showed well defined structure of algal cells and confirmed that the Fe3O4 and [Fe3O4]F matrices alone not involved in the algal deactivation (Figure 12b & 12e). The Fe3O4-PG and [Fe3O4]F-PG exposed cells showed significant depression or distortion of cell structure (Figure 12b & 12e). The algal cells exposed to [Fe3O4]F-PG were found to have extended ruptured cell membrane with complete cell lysis than with Fe3O4-PG, indicated the cell toxicity due to the concentration of prodigiosin attached on to the matrices.43,44 The time dependent cell toxicity of [Fe3O4]F-PG was evaluated from SEM images as shown in 16 ACS Paragon Plus Environment

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Figure 13. The M. aeroginosa, control cells and Fe3O4-PG exposed cells for 1 hour showed clear oval shaped cell structure, indicated that Fe3O4-PG was not toxic to algal cells (Figure 13a & 13b). Hence, the exposure time of M. aeroginosa to Fe3O4-PG was increased to 3 h, cells showed slight damage on cell membrane (Figure 13c). The complete cell lysis was obtained after exposed to 6 h for Fe3O4-PG (Figure 13d). This indicates that [Fe3O4]F-PG showed higher toxicity to M. aeroginosa than Fe3O4-PG. The structural changes of cyanobacterial cells by [Fe3O4]F-PG was first initiated by rupture of plasma membrane leads to cell lysis as shown in Figure 13c & 13d. The prodigiosin with amphiphobic surface having both lipophilic and hydrophobic moieties could easily bind on to the surface of phospholipids bimolecular layer of the treated algae and ruptured the layers. The phenomenon was evidenced with SEM observation. The prominent membrane cell damage of alga cells was observed with the cells exposed to [Fe3O4]F-PG. This could reduce the adhesion zone in the soluble matrix of the enzymes required in the tricarboxylic acid cycle, where cellular respiratory activities were weakened and the aerobic glycometabolism was blocked. The functions of the organelles were completely damaged when the exposure time was prolonged and the cells were completely lysed at the end. This corroborates with the observation recorded by Ahn et al. (2003) that the surfactin (bioactive compounds) isolated from B. subtilis C1 was completely inhibited from the matured M. aeruginosa cells and the M. aeruginosa cells were mainly damaged due to destabilization of the cell membranes.45 Conclusion A simple and effective approach to extract the prodigiosin from fermented medium was developed using robust [Fe3O4]F. The Fe3O4 was functionalized using diethanolamine by coprecipitation technique. The diethanolamine was found to be acted as stabilizer to prevent the 17 ACS Paragon Plus Environment


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Page 18 of 40

aggregation of [Fe3O4]F and also acted as a cross-linking agent with prodigiosin molecules. The [Fe3O4]F was used for the extraction of prodigisin from the fermented medium and reused for several cycles after regeneration. Moreover, this approach can be extended to other metal oxides for the solvent free extraction of bioactive compounds from the microbial fermented medium. The antifouling activity of [Fe3O4]F–PG was found to be effective towards the removal of cyanobacterial cells. Hence, the proposed method for extraction of prodigiosin would be practically feasible and economically viable. Acknowledgement The authors thank the council of scientific and industrial research for the financial assistance to carry out the research work under the CSIR-STRAIT (CSC0201) and CSIR-SETCA (CSC0113) programmes. Reference (1)

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Ni, L.; Acharya, K.; Hao, X.; Li, S. Isolation and identification of an anti-algal

compound

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Page 25 of 40

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Tables Table 1. Analysis of variance for prodigiosin production using central composite design. Sum of

df

Mean

p-value

Sources

F Value Squares

Square

Prob > F

Model

1001.77

20

50.08

52.87

< 0.0001

A-Time

37.64

1

37.64

39.74

< 0.0001

B-pH

11.66

1

11.66

12.31

0.0015

C-Temperature

28.64

1

28.64

30.23

< 0.0001

D-Glycerol

1.01

1

1.01

1.06

0.31

E-TF

61.63

1

61.63

65.06

< 0.0001

Residual

27.46

29

0.94

Lack of Fit

27.46

22

1.24

Pure Error

0

7

0

Core Total

1029.24

49

Table 2. Kinetic rate of prodigiosin extraction by pseudo first order and pseudo second order First order Matrices

2

r

Fe3O4 [Fe3O4]F

Second order -1

K1(m )

2

r

K2(m-1)

0.91

0.08

0.94

8.71E-05

0.92

0.03

0.96

0.003



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 26 of 40

Table 3. Langmuir, Freundlich and Dubinin–Radushkevich isotherm constants for adsorption of prodigiosin by Fe3O4 and [Fe3O4]F.

Langmuir isotherm

Freundlich isotherm

Dubinin-Radushkevich isotherm

Matrices aL(L mg-1)

KL (L g-1)

QL(mg g-1)

r2

KF (L g-1)

n

r2

E

BD ( mg2 j-2)

QD (mg g-1)

r2

Fe3O4

0.01

5.43

292.39

0.99

68.05

5.45

0.33

0.68

1.02

348.12

0.95

[Fe3O4]F

0.1

31.47

314.46

0.99

49.28

4.27

0.41

0.59

1.19

367.31

0.95


Page 27 of 40

Prodigiosin

8.00

7.50

5.03393

7.20175

Prodigiosin

1.50

6.45854

7.88315

6.45854

7.82398

11.183

10.1287

11.1043 8.1774

9.30776

F:TF

B:pH

D:Glycerol

6.25

7.50

10.1287 9.15305

8

7.00

8

5.00

3.75

6.50

7.88315

60.00

78.00

96.00

42.00

A:Time 10.0461 11.0224 1.25

10.5343

8

0.75

10.0461 11.5106 11.0224

C:Temperature

D:Glycerol

60.00

78.00

24.00

8

30.00

11.5582 10.9082 27.50

8

5.00

11.0184

3.75

9.60822

10.2582

8.95822

9.60822

8.80493

9.91164 2.50 6.50

8.00

Prodigiosin

6.5915

7.69822

9.91164

7.00

7.50

8.00

6.00

6.50

B:pH

B:pH 1.50

96.00

6.25

6.00

7.50

78.00

Prodigiosin

7.50

25.00

7.00

60.00

A:Time

32.50

9.55798

6.50

42.00

8.80493

0.50 6.00

8

0.75

96.00

Prodigiosin

35.00

10.5343

1.00

10.3433

A:Time

Prodigiosin

1.50

9.5035 1.00

0.50 24.00

F:TF

42.00

8.66374

9.30776

6.45854 2.50 24.00

10.3433

1.25

10.7324

10.1287 6.00

6.78173

7.00

7.50

8.00

B:pH

Prodigiosin

7.50

Prodigiosin

7.50

7.88819

6.87807

7.91195

9.54339

8.99466

8.94582 9.9797

6.25

10.0636

6.25

11.1039

10.5837

10.1011

8

5.00

8

1.00

11.624

F:TF

1.25

F:TF

D:Glycerol

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


11.2076

3.75

0.75

8

5.00

11.0136

3.75

8.99466 9.54339

7.88819

9.9797

2.50

0.50 25.00

27.50

30.00

32.50

C:Temperature

35.00

8.94582

2.50

25.00

27.50

30.00

32.50

35.00

C:Temperature

0.50

0.75

1.00

1.25

1.50

D:Glycerol

Figure 1. Statistical optimization of variables such as time, temperature, pH, glycerol and TF concentration for the production of PG using RSM.



(b)

(a) % Transmittance

[Fe3O4]F

Fe3O4

(d)

3000

(c)

2000

-1 Wave number (cm )

1.0

Fe3O4

0.8

[Fe3O4]F

1000

(e) 100

Weight loss (%)

4000

Reflectance

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 28 of 40

0.6 0.4 0.2

Fe3O4 [Fe3O4]F

98 96 94 92 90

0.0

88

200 300 400 500 600 700 800

100 200 300 400 500 600 700 800

Wave length (nm)

0

Temperature (C )

Figure 2. The characterization of Fe3O4 and [Fe3O4]F matrices by (a) FT-IR spectroscopy, (b & c) SEM images, (d) DRS and (e) TGA


Page 29 of 40

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Figure 3. The schematic diagram of reactor for the extraction of PG under batch mode



(b)

60

Prodigiosin extraction (%)


(a) 70 50 40 30

Fe3O4 [Fe3O4]F

20 10

100

0

10

20

30

40

60 40 20

0

50

Prodigiosin (g/ 50 g of matrices)

(d)

100 80 60 Fe3O4 [Fe3O4]F

40 20 0 0

20

40

60

2

4

6

8

10

pH

Time (min)

(c)

Fe3O4 [Fe3O4]F

80

0


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

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80

100

Dosage of matrices (g/L)

6 Fe3O4 [Fe3O4]F

5

4

3

2

1 2

4

6

8

10

Dosage of prodigiosin (g/ L)

Figure 4. The optimization (a) time, (b) pH, (c) dosage of Fe3O4 and [Fe3O4]F and (d) concentration of prodigiosin for the extraction of PG in batch fermenter


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(a) % Transmittance

(b) [Fe3O4]F-PG

(c) Fe3O4-PG

(d) 1.0 0.8

3000

2000

1000

-1 Wave number (cm )

(e) 100

Fe3O4-PG

[Fe3O4]F-PG

Weight loss (%)

4000

Reflectance

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


0.6 0.4 0.2

Fe3O4-PG [Fe3O4]F-PG

98 96 94 92 90

0.0 200 300 400 500 600 700 800

88 100 200 300 400 500 600 700 800 0

Wave length (nm)

Temperature (C )

Figure 5. The characterization of [Fe3O4]F and [Fe3O4]F-PG matrices by (a) FT-IR spectroscopy, (b & c) SEM images, (d) DRS and (e) TGA



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Figure 6. XRD spectrum of [Fe3O4]F and [Fe3O4]F-PG


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(a)

(b)

(c)

(d)

Figure 7. Confocal microscopic images of the (a) Fe3O4, (b) [Fe3O4]F, (c) Fe3O4-PG and (d) [Fe3O4]F-PG.



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Figure 8. Diagrammatic representation of preparation of [Fe3O4]F and [Fe3O4]F-PG, and its molecular interaction with prodigiosin.


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Figure 9. Schematic diagram of prodigiosin extraction by [Fe3O4]F and compared with conventional method



100 80

Petrolium ether

Dichloromethan

0

Dichloromethan

Hexane

Chloroform

Benzene

Diethylether

Isopropanol

20

Methanol

40

Ethnol

60

Acetone

(a)

Prodigiosin desorption (%)

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

solvents

(b)

Figure 10. (a) Desorption of PG from [Fe3O4]F using different solvents and (b) Reusability efficiency of [Fe3O4]F for PG extraction


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(b) Transmittance (%)

(a)

Intensity

PG eluted from Fe3O4


PG eluted from [Fe3O4]F 0

4

8

12

16

PG eluted from[Fe3O4]F 4000

20

Retention Time (min)

3000

2000

1000

Wave length (cm-1)

1.2

(c)


1.0

PG eluted from [Fe3O4]F

0.8

Intensity

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


0.6 0.4 0.2 0.0 200

300

400

500

600

700

800

wave legth (cm-1)

Figure 11. Characterization of PG recovered from fermented medium using Fe3O4 and [Fe3O4]F by a) HPLC and b) FT-IR.



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(a)

(b)

(d)

(e)

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(c)

(f)

Figure 12. Algicidal effect of prodigiosin impregnated matrices using SEM and confocal microscopy. SEM images of cyanaobacteria before exposure a) control, b) after exposure to Fe3O4-PG and c) [Fe3O4]F-PG. Confocal microscopic images of cyanaobacteria before exposure d) control e) after exposure to Fe3O4-PG and f) [Fe3O4]F-PG


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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


(a)

(b)

(c)

(d)

Figure 13. The algicidal effect of Fe3O4-PG at different time exposure a) control, b) 1 h, c) 2 h and d) 3 h



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367x283mm (96 x 96 DPI)

ACS Paragon Plus Environment

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