Accepted Manuscript Production of rhamnolipid surfactant and its application in bioscouring of cotton fabric Zulfiqar Ali Raza, Aisha Rehman, Muhammad Tahir Hussain, Rashid Masood, Anwar ul Haq, Muhammad Tahir Saddique, Amjed Javid, Niaz Ahmad PII: DOI: Reference:
S0008-6215(14)00104-9 http://dx.doi.org/10.1016/j.carres.2014.03.009 CAR 6704
To appear in:
Carbohydrate Research
Received Date: Revised Date: Accepted Date:
27 December 2013 10 March 2014 12 March 2014
Please cite this article as: Raza, Z.A., Rehman, A., Hussain, M.T., Masood, R., Haq, A.u., Saddique, M.T., Javid, A., Ahmad, N., Production of rhamnolipid surfactant and its application in bioscouring of cotton fabric, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/j.carres.2014.03.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Production of rhamnolipid surfactant and its application in bioscouring of cotton fabric Zulfiqar Ali Raza*,, Aisha Rehman, Muhammad Tahir Hussain, Rashid Masood, Anwar ul Haq, Muhammad Tahir Saddique, Amjed Javid, and Niaz Ahmad Chemistry Research Laboratory, National Textile University, Faisabad-37610, Pakistan Abstract In the present study, a biosurfactant was synthesized by using a bacterial strain of Pseudomonas aeruginosa in minimal media provided with n-heptadecane as sole carbon source under shakeflask conditions. The biosurfactant was isolated (by acid precipitation, solvent extraction and rotary evaporation), purified (by column chromatography and TLC), identified (by FAB-MS, FTIR and 1D-1H NMR) and chemo-physical characterized (by tensiometry). Two principal rhamnolipid congeners were identified as dirhamnolipid RRC10C10 and monorhamnolipid RC10C10 with a CMC of 50 mg/L. The biosurfactant, hence produced, was applied in sole and in combination with pectinase in scouring of cotton fabric in contrast to conventional scouring agents of NaOH and anionic surfactant SDS. The scoured cotton fabric was investigated for its weight loss, residual oil and grease, wettability, whiteness and tensile strength. The results were compared both for conventional and biological approaches. The scouring with biosurfactant plus pectinase was equivalent to or better in efficiency than conventional alkaline scouring. The former process is additionally environmental friendly and bio-compatible. Scanning electron microscopy of cotton fabric showed that the alkaline scouring deteriorates the fabric texture whereas bioscouring with biosurfactant plus pectinase gently removes hydrophobic impurities from the cotton fabric. Keywords: Bioscouring, Pectinase, Rhamnolipids, Surfactants, Textile Running title: Rhamnolipid for bioscouring of cotton fabric * Correspondence: Tel: +92 41 9230081, Fax: +92 41 9230098, E-mail: [email protected] (ZA Raza) 1. Introduction The cotton fiber is a single biological cell with a multilayer structure. These layers are structurally and chemically different, and contain approximately 10% by weight of noncellulosic substances such as lipids, waxes, pectins, minerals, proteins/nitrogenous substances, 1
non-cellulosic polysaccharides, miscellaneous substances such as pigments, hemicelluloses, reducing sugars, and other unidentified compounds.1-3 Pectins are a family of complex polysaccharides that contain 1,4-linked a-D-galactosyluronic acid residues.4 The hydrophobic nature of waxes and pectins, is responsible for the non-wetting behavior of native cotton and impedes uniform and efficient dyeing and finishing commonly performed in aqueous systems; hence must be removed from griege fabric before further processing.5-7 Conventionally, scouring is done by treating the fabric with sodium hydroxide and/or detergents for the removal of waxes, pectins dirt, and so on. The alkaline scouring although is effective and cheap yet is costly because of consumption of large quantities of energy, water and auxiliary agents. The potential for the environmental contamination and depletion of natural resources is also serious. Moreover, strict pH, high temperature (90-100°C) and high concentrations of NaOH (e.g., 15g/L) requirements for alkaline scouring make the cotton fibers susceptible to oxidative damage.7-9 Moreover, fabric treated with harsh chemicals is unsafe for human health. So, alternative approaches must be considered to make the scouring process environment friendly and cost effective. One option is to shift from chemical scouring to enzymatic scouring. The idea is that pectin acts as a sort of cement or matrix that stabilizes the primary cell wall of the cotton fibers. During incubation, the enzymes degrade pectin, thereby destabilizing the structure in the outer layers. The weakened outer layers could then be removed in a subsequent washing. In this context, a number of studies have been undertaken to use the enzymes for cotton scouring.10-12 Successful applications of bioscouring with cellulase, pectinase and protease in different combinations have been reported elsewhere.13 Apart from enzymes, bio/surfactants might be employed for scouring process either alone or in combination with enzymes.14 Biosurfactants had been produced by various strains15 and shown diverse applications in surface physiology.16 The rhamnolipids are extra/intra-cellular secondary metabolites of certain native and recombinant bacteria, yeast and fungi, as they grow on different carbon sources.17,18 Being amphiphilic molecules, rhamnolipids tend to partition preferentially at the interface between the phases of different degrees of polarity and hydrogen bonding (such as aqueous/solid or air/solid interfaces). The formation of such an ordered molecular film at the interface lowers the surface and interfacial tensions and is responsible for the unique properties 2
of surfactant molecules. This phenomenon helps dispersion of hydrophobic substances in aqueous media. The biosurfactants have advantage over their chemical counterparts because of higher biodegradability, surface activity, emulsifying properties and biological activity, and lower toxicity and critical micelle concentration (CMC).18-21 Up till now, no study has been reported on the use of rhamnolipid surfactants for bioscouring of cotton fabric. The present study aimed to modify existing conventional process chemicals with renewable and sustainable agents/biochemicals; hence to minimize the environmental issues related to textile pretreatments. This was done by the production and purification of biosurfactant under shake flask conditions and its identification and characterization. The biosurfactant was, then, investigated as bio-scouring agent in the presence and absence of pectinase in contrast to conventional scouring agents in the scouring of cotton fabric. The bio/scoured cotton fabric was characterized for various chemo-physical and textile properties. 2. Experimental 2.1. Materials Following analytical grade chemicals were purchased from respective made: hydrochloric acid (Riedel-de Haen), sodium hydroxide (Merck); methanol and sodium dodecyl sulfate (SDS) Sigma Aldrich), silica gel (60 F254 ) - coated aluminum sheets (20 × 20 cm), silica gel (230-400 mesh) and sulfuric acid (Merck); acetic acid, ethanol and alpha naphthol (BDH); chloroform (Fisher); L-rhamnose (Hopkin and Williams); orcinol (Fluka), and methanol (Riedel-de Haen). The scourzyme L (a pectinase) was donated by Novozymes. All the chemicals were used without any pre-treatment. All the solutions were prepared in distilled water. Griege 100% cotton plain weave fabric was purchased from Ahmed Fine Textile Mills Ltd., Multan, Pakistan. 2.2. Production of biosurfactant The microorganism used for biosurfactant production was EBN-8, a gamma ray-induced mutant of Pseudomonas aeruginosa.22 An iron limited minimal medium was used comprising (g/L): KH2PO4 (0.7); Na2HPO4 (0.9); NaNO3 (2.0); MgSO4.7H2O (0.4); CaCl2.2H2O (0.1) and FeSO4.7H2O (0.001) supplemented with (10 g /L) n-heptadecane as sole carbon source and incubated at 37°C and 100 rpm in 250 ml Erlenmeyer flask on an orbital shaker. 2.3. Biosurfactant extraction and purification 3
After a certain time interval, the culture flasks were removed from the orbital shaker and culture media were centrifuged at 7,740×g and 4°C for 15 min to remove the cell biomass, hence to get cell free culture broth (CFCB). The crude biosurfactant was extracted from the CFCB by acid precipitation followed by solvent extraction, according to Zhang and Miller.23 For the removal of neutral and/or phospholipids, and other possible contaminating molecules from rhamnolipids, the crude biosurfactant was passed through a preparative chromatographic column according to a modified method.18 The rhamnolipids, hence collected, were freeze dried, weighed and stored for further study. 2.4. Mass spectrometry of biosurfactant A stock of column purified rhamnolipid fraction was prepared in glycerol. The chemical analysis was performed on a double focusing JMS-HX110, JEOL mass spectrometer in negative ion mode, using a gun voltage of 1 kV, accelerating voltage of 10 kV and emission current of 10 mA. Xe gas was used under the pressure in the range of 10-5 - 10-7 Torr as a primary source of ionization. The scanning molecular mass range was 100-800 Da. 2.5. Thin layer chromatography (TLC) The column-purified rhamnolipids were dissolved in chloroform (300 mg/L) and 10 µl of it were applied on a silica gel-coated aluminum sheet. The thin layer chromatograms were developed in the chloroform/methanol/acetic acid (65/25/4, v/v) system and visualized using the Molish reagent.24 The retardation factors (Rf) of the rhamnose lipid purple spots were determined. The TLC-purified rhamnolipids, with separate fractions on the basis of Rf, were saved for Fourier transform infrared (FT-IR) and one dimensional proton nuclear magnetic resonance (1-D 1H NMR) analyses. 2.6. FT-IR analysis The IR spectra of TLC-purified rhamnolipids were recorded on an FT-IR spectrometer (8201 PC, Shimadzu, Germany) in the spectral region 4000-400 cm-1 at a resolution of 2 cm-1, using a 0.23mm liquid cell of KBr. 2.7. NMR analysis 4
The TLC-purified rhamnolipids were re-dissolved in deuterated chloroform (CDCl3) and analyzed with an NMR machine (Avance 400 MHz, Bruker, Germany) for 1-D 1H NMR analysis. 2.8. Tensiometric characterization of biosurfactant Surface tension and interfacial tension of the rhamnolipids aqueous solutions against nhexadecane under working conditions were measured by using a Theta lite Optical Tensiometer (Biolin, Finland). CMC, the concentration at which the surface tension of the media suddenly increases, was determined from the break point of the curve of surface tension vs. rhamnolipids concentration. 2.9. Enzymatic desizing of cotton fabric Enzymatic desizing of the griege cotton fabric was done by using 2 g/L amylase at 95°C for 40 min at lab scale jigger machine. The efficiency of desizing process was assessed according to Tagawa rating using a standard iodine solution. The desized fabric was washed in distilled water to remove starch and other degraded impurities. 2.10. Bio/scouring of cotton fabric Separate alkaline and SDS scourings were done at 95°C and pH 9-11while bioscourings at 65°C and pH 8 for 40 min of incubation on a lab scale launder-o-meter with fabric-to-liquor ratio of 1:30 (w/v) under designed experimental conditions (Table 1). The treated cotton fabric samples were air dried and then brought to moisture equilibrium under conditions of relative humidity (65±2%) and temperature (25±1°C) for 24 h. The input parameters were scouring agent types (i.e., NaOH, SDS, pectinase and biosurfactant), their combinations and concentrations. The output parameters were weight loss (g/g), residual oil and grease (ROG) (g/g), whiteness (CIE), wettability (s) and tensile strength (KgF). 2.11. Weight loss measurement The weight loss of cotton fabric was recorded as dried sample weight change. The drying was done at 105°C for 1 h. The sample was weighed after cooling in a closed weighing bottle placed in a desiccator. The weight loss is reported as grams of weight loss per gram of fabric (g/g). 2.12. Residual oil and grease measurement 5
The ROG in the fabric sample before and after bio/scouring was determined according to a modified method of Kokub et al.25 The fabric sample was dipped thrice in petroleum ether in ratio 1:20 (w/v) under shaking conditions at room temperature for 1 h. The organic phase was separated and evaporated to dryness under vacuum using a rotary evaporator at 30°C and then oven-dried at 60°C to a constant mass. 2.13. Whiteness measurement The whiteness measurement was done by using a spectrophotometer (Color Eye 7000A, GretagMacbeth) according to AATCC 110-2011 test method.26 2.14. Wettability measurement The wettability of cotton fabric was characterized by the wetting time (in s) and evaluated by means of the drop test of AATCC 79-2010.26 The time between the contact of a water drop, carefully deposited on the fabric surface, and its disappearance into the fabric matrix was recorded as the fabric-wetting time. 2.15. Tensile strength measurement The tensile strength was determined according to ASTM D 5035 standard method. Fabric sample of dimension 6×4 inches was cut and the measurements were taken along warp- and weft-wise using tensile strength tester (Model KG-300) under the standard conditions of humidity and temperature. All above measurements were performed in triplicate. 2.16. Scanning electron microscopy (SEM) The effect of various scouring approaches on the surface morphologies of cotton fabric was examined by using a scanning electron microscope (JSM-5910, JEOL, Japan), operating at an acceleration voltage of 10 kV. Each sample was coated with sputtered gold for 40 s at 15 mA prior to the observation. 3. Results and discussion 3.1. Production of biosurfactant The culture media were incubated for seven consecutive days till the entire carbon source utilized and the CFCB achieved a constant concentration of rhamnolipid surfactant. After 24 h of lag phase, the bacterial growth entered the exponential phase. After that the bacterial growth 6
entered the stationary phase. On the other hand, rhamnolipid production started at 48 h of incubation and lasted till the end of incubation period (7 d). 3.2. Identification of rhamnolipids The rhamnolipids hence produced were identified by using a negative mode FAB-MS analysis followed by spectroscopic characterization. Several types of rhamnolipid congeners, of molecular masses in the range m/z 504-678, were observed (Figure 1). The proton abstraction of rhamnolipid molecules yielded [M-H]- pseudomolecular anions at m/z 503 (RC10C10), 621 (RRC10C8/RRC8C10), 649 (RRC10C10) and 677 (RRC12C10/RRC10C12), with the relative abundances 35, 6, 42 and 7, respectively. According to Deziel et al.27 most of the ions above m/z 447 are rhamnolipid pseudomolecular [M - H]- ions and the most ions between m/z 163 and 503 are fragment ions produced by cleavage. The mass spectrum demonstrates that the principal congeners in the rhamnolipids mix were, respectively, dirhamnolipid RRC10C10 (L-rhamnosyl-Lrhamnosyl-β-hyroxydecanoyl-β-hyroxydecanoate) and monorhamnolipid RC10C10 (L-rhamnosylβ-hyroxydecanoyl-β-hyroxydecanoate). Again, the TLC of rhamnolipids showed that it contained two main groups of a monorhamnolipids (with Rf = 0.73) and dirhamnolipids (with Rf = 0.52), dirhamnolipids being predominant species. The FTIR spectra of rhamnolipids were recorded in the spectral region of 4,000-400 cm-1 (IR spectra are not shown). Several C-H stretching bands of -CH2- and -CH3 groups were observed in the region 3,000-2,700 cm-1. The carbonyl stretching peak was observed at 1,635 cm-1, which is characteristic of ester compounds. The ester carbonyl group was also confirmed from the peak at 1,013 cm-1, which corresponds to C-O stretching vibration. The lack of characteristic bands for organic acids that usually appear at 3,500-2,700 cm-1, 1,635-1,650 cm-1 and 1,013-1,018 cm-1 indicates the presence of an ester compound. The present of rhamnolipids in glycolipidic biosurfactants was confirmed by the 1-D 1H NMR. The data of 1H shifts of the absorption frequencies are shown in Table 2. The NMR results indicate that the purified biosurfactant comprises two principal rhamnolipid homologs, i.e. RC10C10 and RRC10C10, supporting FAB-MS results. 3.3. Chemo-physical properties of rhamnolipids
7
Because of their diverse molecular structures, rhamnolipids exerted excellent surface activities especially in lowering the surface and interfacial tensions, and exhibiting emulsifying behavior. They could reduce the surface tension of distilled water from 72.0 to ≤ 30.0 mN/m and the interfacial tension vs. n-hexadecane from 40.0 to 0.6 mN/m at 25oC. The CMC of the rhamnolipids extract was 50.0 mg/L. At higher concentrations, rhamnolipids molecules travelled into the air/liquid or oil/water interfaces to saturate it, resulting in significant reduction in surface and interfacial tensions, respectively. The abrupt changes in surface activity at CMC could be employed in various textile processes related to laundry and so on. 3.4. Pre-treatment of cotton fabric The desized cotton fabric was somehow hydrophobic and showed wettability of >40 s. It was then treated with hot distilled water at 95°C for 40 min. This improved wettability of fabric to ~ 10 s, but still lower than acceptable limit. This justified that scouring treatment was essential to further improve the wettability of cotton fabric. The ROG contents in desized fabric and hot water treated fabric were, respectively, 0.022 and 0.018 g/g. The other specifications of desized cotton fabric are shown in Table 3. 3.5. Alkaline scouring of cotton fabric There observed a pronounced weight loss in the treated fabric on increasing the NaOH concentration during the alkaline scouring (Figure 2a). This loss in weight might be due pectin degradation as well as the removal of oil, grease, and so on. The scouring process under goes saponification of fatty substances, the resultant soaps then act as emulsifier and detergent hence to extend the removal of non-cellulosic impurities from the fabric surface. Another parameter to determine the efficiency of scouring process was the measurement of ROG in the treated fabric. Lesser ROG contents in the treated fabric meant better scouring and vice versa. There observed gradual improvement in scouring efficiency up to 30 g NaOH/L; above this concentration, ROG value remained almost constant (Figure 2a). As dirt, dust and lint play a role for rendering color; these impurities were also removed during scouring process hence the resultant fabrics were found with improved whiteness. Figure 2b shows the effect of alkaline scouring on the tensile strength (both warp- and weft-wise) and wettability of treated fabric. It could be seen that tensile strength decreased gradually with increase in alkali concentration. The water wettability of
8
treated fabric was improved with increase in alkali concentration which depicts better removal of hydrophobic impurities like pectins, waxes etc.3,28 3.6. SDS scouring of cotton fabric SDS being anionic surfactant acts both as an emulsifier and a detergent. It picks hydrophobic substances from fabric surface and emulsify them and to suspend water insoluble impurities in the aqueous media; hence to decrease the weight of resultant fabric with improved whiteness. Figure 3a depicts the effect of SDS on weight loss, ROG and whiteness. It could be seen that weight loss increased with increase in SDS concentration until it reach at 500 mg/L. After that, there observed a sharp decrease in weight loss at 1000 mg SDS/L. This unusual behavior is attributed to aggregation of SDS above its CMC, which made it ineffective for scouring as a result of higher ROG and lower whiteness. There observed that the whiteness was not significantly improved during SDS scouring as compared to alkaline scouring. Figure 3b shows that tensile strength of treated fabric was not affected much during SDS scouring. The wettability of treated overall improved on increasing SDS concentration, however, below its CMC. 3.7. Bioscouring of cotton fabric There observed a noticeable enhancement in the wettability, weight loss and whiteness, meanwhile ROG and tensile strength of the treated fabric decreased, by increasing the pectinase doses. A combined effect of SDS plus pectinase can be seen in Figure 4a. The weight loss of treated fabric increased with increase in SDS concentration. This effect was more pronounced at a concentration of 4 g pectinase/L. In the case of ROG, a decrease trend in curves was observed for all the three tested concentrations of pectinase on increasing SDS concentration. These results could be explained in terms of enzymatic hydrolysis of the impurities like pectic substances and waxy materials from raw cotton fabric when pectinase was used. Such hydrolysis yields soluble products and is accompanied by a certain loss of tensile strength which is proportional to the amount of weight loss.29 On the other hand, SDS emulsified the degraded and departed impurities and showed detergency so that all the impurities of the fabric removed.2 The degradation and removal of pectins could make cotton fibers achieve higher hydrophilic properties without fiber deterioration.30 The whiteness was not affected much by concentration of both SDS and pectinase: which shows coloured impurities were not effectively removed by SDS plus pectinase. Figure 4b shows the 9
effect combined concentration of SDS plus pectinase on both wrap- and weft-wise tensile strength and the wettability of the fabric. The tensile strength was not much reduced on increasing SDS and pectinase concentrations. However, the wettability increased with increase in amounts of SDS and pectinase. The biosurfactant produced by using P. aeruginosa EBN-8 grown on n-heptadecane was applied as bioscouring agent and the process was investigated for intended output parameters. On increasing biosurfactant concentration, there observed slight weight loss and decrease in ROG content in the treated fabric (Figure 5a). This is because of the oil removal from the cotton fabric.2 A slight increase in wettability and decrease in tensile strength along both warp- and weft-wise was also noticed on increasing biosurfactant dose (Figure 5b). These results are significant as compared to that of SDS on the basis of amount of scouring agents used. The combined effect of biosurfactant plus pectinase on weight loss, ROG and whiteness are shown in Figure 6a. There observed that both weight loss and whiteness increased by increasing concentration of pectinase from 4 to 6 g/L whereas ROG contents were decreased to some extent. Figure 6b shows that tensile strength of fabric was almost unaffected with different combinations of biosurfactant plus pectinase. However, a sharp increase in wettability was observed on increasing the concentration of pectinase from 4 g to 5g/L while further increase in concentration showed no apparent effect. 3.8. Comparison of output parameters The weight loss of cotton fabric during bio/scouring is attributed to removal of pectins, oil, grease, dirt, dust and lint. The samples scoured with combinations of pectinase plus SDS/biosurfactant showed better removal of unwanted materials. Figure 7a shows the comparison for weight loss of all the treated samples and control. On increasing the enzyme concentration, there observed increased weight loss. The samples scoured with pectinase (4 g/L) plus SDS (400 mg/L) showed the highest weight loss value of 0.04 g/g, whereas the samples treated with pectinase (4 g/L) plus SDS (300 mg/L), and pectinase (4-6 g/g) plus biosurfactant (100 mg/L) showed reasonable weight losses in the range 0.028-0.032 g/g. A significant weight loss during alkaline scouring has also been reported elsewhere.10,12,31 The comparison of ROG contents of all the scoured samples is shown Figure 7b. The camples treated with alkali and combination of pectinase plus SDS/biosurfactant showed ROG in the 10
lowest range. A sample having low ROG values means less oil and grease have been left after the treatment, hence the sample is effectively scoured. The ROG values in samples in the highest range were observed for control, water treated and that treated with 1000 mg SDS/L. The whiteness index of fabric is associated with removal of cotton hydrophobic (pectin, waxes and lipids) and colored impurities from fabric. Figure 8a shows the comparison of whiteness indices of all treated samples. The samples scoured with alkali showed whiteness in the highest range of 44.16 to 49.9 CIE. The whiteness of treated fabrics increased with increasing NaOH concentration. The lowering of whiteness in the case of SDS/biosurfactant treatments might be due to result of the interfering effect of the residual starch. Nevertheless, on increasing pectinase dose there observed increment in whiteness index up to 30.81 CIE. The wettability was a function of both NaOH concentration and pectinase dose. Both in the case of alkaline and pectinase scourings, the water wettability was
S0008-6215(14)00104-9 http://dx.doi.org/10.1016/j.carres.2014.03.009 CAR 6704
To appear in:
Carbohydrate Research
Received Date: Revised Date: Accepted Date:
27 December 2013 10 March 2014 12 March 2014
Please cite this article as: Raza, Z.A., Rehman, A., Hussain, M.T., Masood, R., Haq, A.u., Saddique, M.T., Javid, A., Ahmad, N., Production of rhamnolipid surfactant and its application in bioscouring of cotton fabric, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/j.carres.2014.03.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Production of rhamnolipid surfactant and its application in bioscouring of cotton fabric Zulfiqar Ali Raza*,, Aisha Rehman, Muhammad Tahir Hussain, Rashid Masood, Anwar ul Haq, Muhammad Tahir Saddique, Amjed Javid, and Niaz Ahmad Chemistry Research Laboratory, National Textile University, Faisabad-37610, Pakistan Abstract In the present study, a biosurfactant was synthesized by using a bacterial strain of Pseudomonas aeruginosa in minimal media provided with n-heptadecane as sole carbon source under shakeflask conditions. The biosurfactant was isolated (by acid precipitation, solvent extraction and rotary evaporation), purified (by column chromatography and TLC), identified (by FAB-MS, FTIR and 1D-1H NMR) and chemo-physical characterized (by tensiometry). Two principal rhamnolipid congeners were identified as dirhamnolipid RRC10C10 and monorhamnolipid RC10C10 with a CMC of 50 mg/L. The biosurfactant, hence produced, was applied in sole and in combination with pectinase in scouring of cotton fabric in contrast to conventional scouring agents of NaOH and anionic surfactant SDS. The scoured cotton fabric was investigated for its weight loss, residual oil and grease, wettability, whiteness and tensile strength. The results were compared both for conventional and biological approaches. The scouring with biosurfactant plus pectinase was equivalent to or better in efficiency than conventional alkaline scouring. The former process is additionally environmental friendly and bio-compatible. Scanning electron microscopy of cotton fabric showed that the alkaline scouring deteriorates the fabric texture whereas bioscouring with biosurfactant plus pectinase gently removes hydrophobic impurities from the cotton fabric. Keywords: Bioscouring, Pectinase, Rhamnolipids, Surfactants, Textile Running title: Rhamnolipid for bioscouring of cotton fabric * Correspondence: Tel: +92 41 9230081, Fax: +92 41 9230098, E-mail: [email protected] (ZA Raza) 1. Introduction The cotton fiber is a single biological cell with a multilayer structure. These layers are structurally and chemically different, and contain approximately 10% by weight of noncellulosic substances such as lipids, waxes, pectins, minerals, proteins/nitrogenous substances, 1
non-cellulosic polysaccharides, miscellaneous substances such as pigments, hemicelluloses, reducing sugars, and other unidentified compounds.1-3 Pectins are a family of complex polysaccharides that contain 1,4-linked a-D-galactosyluronic acid residues.4 The hydrophobic nature of waxes and pectins, is responsible for the non-wetting behavior of native cotton and impedes uniform and efficient dyeing and finishing commonly performed in aqueous systems; hence must be removed from griege fabric before further processing.5-7 Conventionally, scouring is done by treating the fabric with sodium hydroxide and/or detergents for the removal of waxes, pectins dirt, and so on. The alkaline scouring although is effective and cheap yet is costly because of consumption of large quantities of energy, water and auxiliary agents. The potential for the environmental contamination and depletion of natural resources is also serious. Moreover, strict pH, high temperature (90-100°C) and high concentrations of NaOH (e.g., 15g/L) requirements for alkaline scouring make the cotton fibers susceptible to oxidative damage.7-9 Moreover, fabric treated with harsh chemicals is unsafe for human health. So, alternative approaches must be considered to make the scouring process environment friendly and cost effective. One option is to shift from chemical scouring to enzymatic scouring. The idea is that pectin acts as a sort of cement or matrix that stabilizes the primary cell wall of the cotton fibers. During incubation, the enzymes degrade pectin, thereby destabilizing the structure in the outer layers. The weakened outer layers could then be removed in a subsequent washing. In this context, a number of studies have been undertaken to use the enzymes for cotton scouring.10-12 Successful applications of bioscouring with cellulase, pectinase and protease in different combinations have been reported elsewhere.13 Apart from enzymes, bio/surfactants might be employed for scouring process either alone or in combination with enzymes.14 Biosurfactants had been produced by various strains15 and shown diverse applications in surface physiology.16 The rhamnolipids are extra/intra-cellular secondary metabolites of certain native and recombinant bacteria, yeast and fungi, as they grow on different carbon sources.17,18 Being amphiphilic molecules, rhamnolipids tend to partition preferentially at the interface between the phases of different degrees of polarity and hydrogen bonding (such as aqueous/solid or air/solid interfaces). The formation of such an ordered molecular film at the interface lowers the surface and interfacial tensions and is responsible for the unique properties 2
of surfactant molecules. This phenomenon helps dispersion of hydrophobic substances in aqueous media. The biosurfactants have advantage over their chemical counterparts because of higher biodegradability, surface activity, emulsifying properties and biological activity, and lower toxicity and critical micelle concentration (CMC).18-21 Up till now, no study has been reported on the use of rhamnolipid surfactants for bioscouring of cotton fabric. The present study aimed to modify existing conventional process chemicals with renewable and sustainable agents/biochemicals; hence to minimize the environmental issues related to textile pretreatments. This was done by the production and purification of biosurfactant under shake flask conditions and its identification and characterization. The biosurfactant was, then, investigated as bio-scouring agent in the presence and absence of pectinase in contrast to conventional scouring agents in the scouring of cotton fabric. The bio/scoured cotton fabric was characterized for various chemo-physical and textile properties. 2. Experimental 2.1. Materials Following analytical grade chemicals were purchased from respective made: hydrochloric acid (Riedel-de Haen), sodium hydroxide (Merck); methanol and sodium dodecyl sulfate (SDS) Sigma Aldrich), silica gel (60 F254 ) - coated aluminum sheets (20 × 20 cm), silica gel (230-400 mesh) and sulfuric acid (Merck); acetic acid, ethanol and alpha naphthol (BDH); chloroform (Fisher); L-rhamnose (Hopkin and Williams); orcinol (Fluka), and methanol (Riedel-de Haen). The scourzyme L (a pectinase) was donated by Novozymes. All the chemicals were used without any pre-treatment. All the solutions were prepared in distilled water. Griege 100% cotton plain weave fabric was purchased from Ahmed Fine Textile Mills Ltd., Multan, Pakistan. 2.2. Production of biosurfactant The microorganism used for biosurfactant production was EBN-8, a gamma ray-induced mutant of Pseudomonas aeruginosa.22 An iron limited minimal medium was used comprising (g/L): KH2PO4 (0.7); Na2HPO4 (0.9); NaNO3 (2.0); MgSO4.7H2O (0.4); CaCl2.2H2O (0.1) and FeSO4.7H2O (0.001) supplemented with (10 g /L) n-heptadecane as sole carbon source and incubated at 37°C and 100 rpm in 250 ml Erlenmeyer flask on an orbital shaker. 2.3. Biosurfactant extraction and purification 3
After a certain time interval, the culture flasks were removed from the orbital shaker and culture media were centrifuged at 7,740×g and 4°C for 15 min to remove the cell biomass, hence to get cell free culture broth (CFCB). The crude biosurfactant was extracted from the CFCB by acid precipitation followed by solvent extraction, according to Zhang and Miller.23 For the removal of neutral and/or phospholipids, and other possible contaminating molecules from rhamnolipids, the crude biosurfactant was passed through a preparative chromatographic column according to a modified method.18 The rhamnolipids, hence collected, were freeze dried, weighed and stored for further study. 2.4. Mass spectrometry of biosurfactant A stock of column purified rhamnolipid fraction was prepared in glycerol. The chemical analysis was performed on a double focusing JMS-HX110, JEOL mass spectrometer in negative ion mode, using a gun voltage of 1 kV, accelerating voltage of 10 kV and emission current of 10 mA. Xe gas was used under the pressure in the range of 10-5 - 10-7 Torr as a primary source of ionization. The scanning molecular mass range was 100-800 Da. 2.5. Thin layer chromatography (TLC) The column-purified rhamnolipids were dissolved in chloroform (300 mg/L) and 10 µl of it were applied on a silica gel-coated aluminum sheet. The thin layer chromatograms were developed in the chloroform/methanol/acetic acid (65/25/4, v/v) system and visualized using the Molish reagent.24 The retardation factors (Rf) of the rhamnose lipid purple spots were determined. The TLC-purified rhamnolipids, with separate fractions on the basis of Rf, were saved for Fourier transform infrared (FT-IR) and one dimensional proton nuclear magnetic resonance (1-D 1H NMR) analyses. 2.6. FT-IR analysis The IR spectra of TLC-purified rhamnolipids were recorded on an FT-IR spectrometer (8201 PC, Shimadzu, Germany) in the spectral region 4000-400 cm-1 at a resolution of 2 cm-1, using a 0.23mm liquid cell of KBr. 2.7. NMR analysis 4
The TLC-purified rhamnolipids were re-dissolved in deuterated chloroform (CDCl3) and analyzed with an NMR machine (Avance 400 MHz, Bruker, Germany) for 1-D 1H NMR analysis. 2.8. Tensiometric characterization of biosurfactant Surface tension and interfacial tension of the rhamnolipids aqueous solutions against nhexadecane under working conditions were measured by using a Theta lite Optical Tensiometer (Biolin, Finland). CMC, the concentration at which the surface tension of the media suddenly increases, was determined from the break point of the curve of surface tension vs. rhamnolipids concentration. 2.9. Enzymatic desizing of cotton fabric Enzymatic desizing of the griege cotton fabric was done by using 2 g/L amylase at 95°C for 40 min at lab scale jigger machine. The efficiency of desizing process was assessed according to Tagawa rating using a standard iodine solution. The desized fabric was washed in distilled water to remove starch and other degraded impurities. 2.10. Bio/scouring of cotton fabric Separate alkaline and SDS scourings were done at 95°C and pH 9-11while bioscourings at 65°C and pH 8 for 40 min of incubation on a lab scale launder-o-meter with fabric-to-liquor ratio of 1:30 (w/v) under designed experimental conditions (Table 1). The treated cotton fabric samples were air dried and then brought to moisture equilibrium under conditions of relative humidity (65±2%) and temperature (25±1°C) for 24 h. The input parameters were scouring agent types (i.e., NaOH, SDS, pectinase and biosurfactant), their combinations and concentrations. The output parameters were weight loss (g/g), residual oil and grease (ROG) (g/g), whiteness (CIE), wettability (s) and tensile strength (KgF). 2.11. Weight loss measurement The weight loss of cotton fabric was recorded as dried sample weight change. The drying was done at 105°C for 1 h. The sample was weighed after cooling in a closed weighing bottle placed in a desiccator. The weight loss is reported as grams of weight loss per gram of fabric (g/g). 2.12. Residual oil and grease measurement 5
The ROG in the fabric sample before and after bio/scouring was determined according to a modified method of Kokub et al.25 The fabric sample was dipped thrice in petroleum ether in ratio 1:20 (w/v) under shaking conditions at room temperature for 1 h. The organic phase was separated and evaporated to dryness under vacuum using a rotary evaporator at 30°C and then oven-dried at 60°C to a constant mass. 2.13. Whiteness measurement The whiteness measurement was done by using a spectrophotometer (Color Eye 7000A, GretagMacbeth) according to AATCC 110-2011 test method.26 2.14. Wettability measurement The wettability of cotton fabric was characterized by the wetting time (in s) and evaluated by means of the drop test of AATCC 79-2010.26 The time between the contact of a water drop, carefully deposited on the fabric surface, and its disappearance into the fabric matrix was recorded as the fabric-wetting time. 2.15. Tensile strength measurement The tensile strength was determined according to ASTM D 5035 standard method. Fabric sample of dimension 6×4 inches was cut and the measurements were taken along warp- and weft-wise using tensile strength tester (Model KG-300) under the standard conditions of humidity and temperature. All above measurements were performed in triplicate. 2.16. Scanning electron microscopy (SEM) The effect of various scouring approaches on the surface morphologies of cotton fabric was examined by using a scanning electron microscope (JSM-5910, JEOL, Japan), operating at an acceleration voltage of 10 kV. Each sample was coated with sputtered gold for 40 s at 15 mA prior to the observation. 3. Results and discussion 3.1. Production of biosurfactant The culture media were incubated for seven consecutive days till the entire carbon source utilized and the CFCB achieved a constant concentration of rhamnolipid surfactant. After 24 h of lag phase, the bacterial growth entered the exponential phase. After that the bacterial growth 6
entered the stationary phase. On the other hand, rhamnolipid production started at 48 h of incubation and lasted till the end of incubation period (7 d). 3.2. Identification of rhamnolipids The rhamnolipids hence produced were identified by using a negative mode FAB-MS analysis followed by spectroscopic characterization. Several types of rhamnolipid congeners, of molecular masses in the range m/z 504-678, were observed (Figure 1). The proton abstraction of rhamnolipid molecules yielded [M-H]- pseudomolecular anions at m/z 503 (RC10C10), 621 (RRC10C8/RRC8C10), 649 (RRC10C10) and 677 (RRC12C10/RRC10C12), with the relative abundances 35, 6, 42 and 7, respectively. According to Deziel et al.27 most of the ions above m/z 447 are rhamnolipid pseudomolecular [M - H]- ions and the most ions between m/z 163 and 503 are fragment ions produced by cleavage. The mass spectrum demonstrates that the principal congeners in the rhamnolipids mix were, respectively, dirhamnolipid RRC10C10 (L-rhamnosyl-Lrhamnosyl-β-hyroxydecanoyl-β-hyroxydecanoate) and monorhamnolipid RC10C10 (L-rhamnosylβ-hyroxydecanoyl-β-hyroxydecanoate). Again, the TLC of rhamnolipids showed that it contained two main groups of a monorhamnolipids (with Rf = 0.73) and dirhamnolipids (with Rf = 0.52), dirhamnolipids being predominant species. The FTIR spectra of rhamnolipids were recorded in the spectral region of 4,000-400 cm-1 (IR spectra are not shown). Several C-H stretching bands of -CH2- and -CH3 groups were observed in the region 3,000-2,700 cm-1. The carbonyl stretching peak was observed at 1,635 cm-1, which is characteristic of ester compounds. The ester carbonyl group was also confirmed from the peak at 1,013 cm-1, which corresponds to C-O stretching vibration. The lack of characteristic bands for organic acids that usually appear at 3,500-2,700 cm-1, 1,635-1,650 cm-1 and 1,013-1,018 cm-1 indicates the presence of an ester compound. The present of rhamnolipids in glycolipidic biosurfactants was confirmed by the 1-D 1H NMR. The data of 1H shifts of the absorption frequencies are shown in Table 2. The NMR results indicate that the purified biosurfactant comprises two principal rhamnolipid homologs, i.e. RC10C10 and RRC10C10, supporting FAB-MS results. 3.3. Chemo-physical properties of rhamnolipids
7
Because of their diverse molecular structures, rhamnolipids exerted excellent surface activities especially in lowering the surface and interfacial tensions, and exhibiting emulsifying behavior. They could reduce the surface tension of distilled water from 72.0 to ≤ 30.0 mN/m and the interfacial tension vs. n-hexadecane from 40.0 to 0.6 mN/m at 25oC. The CMC of the rhamnolipids extract was 50.0 mg/L. At higher concentrations, rhamnolipids molecules travelled into the air/liquid or oil/water interfaces to saturate it, resulting in significant reduction in surface and interfacial tensions, respectively. The abrupt changes in surface activity at CMC could be employed in various textile processes related to laundry and so on. 3.4. Pre-treatment of cotton fabric The desized cotton fabric was somehow hydrophobic and showed wettability of >40 s. It was then treated with hot distilled water at 95°C for 40 min. This improved wettability of fabric to ~ 10 s, but still lower than acceptable limit. This justified that scouring treatment was essential to further improve the wettability of cotton fabric. The ROG contents in desized fabric and hot water treated fabric were, respectively, 0.022 and 0.018 g/g. The other specifications of desized cotton fabric are shown in Table 3. 3.5. Alkaline scouring of cotton fabric There observed a pronounced weight loss in the treated fabric on increasing the NaOH concentration during the alkaline scouring (Figure 2a). This loss in weight might be due pectin degradation as well as the removal of oil, grease, and so on. The scouring process under goes saponification of fatty substances, the resultant soaps then act as emulsifier and detergent hence to extend the removal of non-cellulosic impurities from the fabric surface. Another parameter to determine the efficiency of scouring process was the measurement of ROG in the treated fabric. Lesser ROG contents in the treated fabric meant better scouring and vice versa. There observed gradual improvement in scouring efficiency up to 30 g NaOH/L; above this concentration, ROG value remained almost constant (Figure 2a). As dirt, dust and lint play a role for rendering color; these impurities were also removed during scouring process hence the resultant fabrics were found with improved whiteness. Figure 2b shows the effect of alkaline scouring on the tensile strength (both warp- and weft-wise) and wettability of treated fabric. It could be seen that tensile strength decreased gradually with increase in alkali concentration. The water wettability of
8
treated fabric was improved with increase in alkali concentration which depicts better removal of hydrophobic impurities like pectins, waxes etc.3,28 3.6. SDS scouring of cotton fabric SDS being anionic surfactant acts both as an emulsifier and a detergent. It picks hydrophobic substances from fabric surface and emulsify them and to suspend water insoluble impurities in the aqueous media; hence to decrease the weight of resultant fabric with improved whiteness. Figure 3a depicts the effect of SDS on weight loss, ROG and whiteness. It could be seen that weight loss increased with increase in SDS concentration until it reach at 500 mg/L. After that, there observed a sharp decrease in weight loss at 1000 mg SDS/L. This unusual behavior is attributed to aggregation of SDS above its CMC, which made it ineffective for scouring as a result of higher ROG and lower whiteness. There observed that the whiteness was not significantly improved during SDS scouring as compared to alkaline scouring. Figure 3b shows that tensile strength of treated fabric was not affected much during SDS scouring. The wettability of treated overall improved on increasing SDS concentration, however, below its CMC. 3.7. Bioscouring of cotton fabric There observed a noticeable enhancement in the wettability, weight loss and whiteness, meanwhile ROG and tensile strength of the treated fabric decreased, by increasing the pectinase doses. A combined effect of SDS plus pectinase can be seen in Figure 4a. The weight loss of treated fabric increased with increase in SDS concentration. This effect was more pronounced at a concentration of 4 g pectinase/L. In the case of ROG, a decrease trend in curves was observed for all the three tested concentrations of pectinase on increasing SDS concentration. These results could be explained in terms of enzymatic hydrolysis of the impurities like pectic substances and waxy materials from raw cotton fabric when pectinase was used. Such hydrolysis yields soluble products and is accompanied by a certain loss of tensile strength which is proportional to the amount of weight loss.29 On the other hand, SDS emulsified the degraded and departed impurities and showed detergency so that all the impurities of the fabric removed.2 The degradation and removal of pectins could make cotton fibers achieve higher hydrophilic properties without fiber deterioration.30 The whiteness was not affected much by concentration of both SDS and pectinase: which shows coloured impurities were not effectively removed by SDS plus pectinase. Figure 4b shows the 9
effect combined concentration of SDS plus pectinase on both wrap- and weft-wise tensile strength and the wettability of the fabric. The tensile strength was not much reduced on increasing SDS and pectinase concentrations. However, the wettability increased with increase in amounts of SDS and pectinase. The biosurfactant produced by using P. aeruginosa EBN-8 grown on n-heptadecane was applied as bioscouring agent and the process was investigated for intended output parameters. On increasing biosurfactant concentration, there observed slight weight loss and decrease in ROG content in the treated fabric (Figure 5a). This is because of the oil removal from the cotton fabric.2 A slight increase in wettability and decrease in tensile strength along both warp- and weft-wise was also noticed on increasing biosurfactant dose (Figure 5b). These results are significant as compared to that of SDS on the basis of amount of scouring agents used. The combined effect of biosurfactant plus pectinase on weight loss, ROG and whiteness are shown in Figure 6a. There observed that both weight loss and whiteness increased by increasing concentration of pectinase from 4 to 6 g/L whereas ROG contents were decreased to some extent. Figure 6b shows that tensile strength of fabric was almost unaffected with different combinations of biosurfactant plus pectinase. However, a sharp increase in wettability was observed on increasing the concentration of pectinase from 4 g to 5g/L while further increase in concentration showed no apparent effect. 3.8. Comparison of output parameters The weight loss of cotton fabric during bio/scouring is attributed to removal of pectins, oil, grease, dirt, dust and lint. The samples scoured with combinations of pectinase plus SDS/biosurfactant showed better removal of unwanted materials. Figure 7a shows the comparison for weight loss of all the treated samples and control. On increasing the enzyme concentration, there observed increased weight loss. The samples scoured with pectinase (4 g/L) plus SDS (400 mg/L) showed the highest weight loss value of 0.04 g/g, whereas the samples treated with pectinase (4 g/L) plus SDS (300 mg/L), and pectinase (4-6 g/g) plus biosurfactant (100 mg/L) showed reasonable weight losses in the range 0.028-0.032 g/g. A significant weight loss during alkaline scouring has also been reported elsewhere.10,12,31 The comparison of ROG contents of all the scoured samples is shown Figure 7b. The camples treated with alkali and combination of pectinase plus SDS/biosurfactant showed ROG in the 10
lowest range. A sample having low ROG values means less oil and grease have been left after the treatment, hence the sample is effectively scoured. The ROG values in samples in the highest range were observed for control, water treated and that treated with 1000 mg SDS/L. The whiteness index of fabric is associated with removal of cotton hydrophobic (pectin, waxes and lipids) and colored impurities from fabric. Figure 8a shows the comparison of whiteness indices of all treated samples. The samples scoured with alkali showed whiteness in the highest range of 44.16 to 49.9 CIE. The whiteness of treated fabrics increased with increasing NaOH concentration. The lowering of whiteness in the case of SDS/biosurfactant treatments might be due to result of the interfering effect of the residual starch. Nevertheless, on increasing pectinase dose there observed increment in whiteness index up to 30.81 CIE. The wettability was a function of both NaOH concentration and pectinase dose. Both in the case of alkaline and pectinase scourings, the water wettability was
