Waste Biomass Valor (2011) 2:7786 DOI 10.

1007/s12649-010-9052-4

ORIGINAL PAPER

Eco-friendly Technologies Based on Banana Peel Use for the Decolourization of the Dyeing Process Wastewater
Carolyn Palma Elsa Contreras Johana Urra Mara Jesus Martnez

Received: 12 March 2010 / Accepted: 26 October 2010 / Published online: 12 November 2010 Springer Science+Business Media B.V. 2010

Abstract This study analyzed some alternatives to the valorization of agricultural residues considering its use in the treatment of colored efuents. The acidbase behavior of the banana peel surface was thus determined in order to establish the feasibility of its use as a bioadsorbent for dyes. The adsorption capacity of Acid Black 1 was evaluated, through the equilibrium isotherm and the kinetics of this uptake process was also analyzed. Additionally, banana peel was used as substrate-support to evaluate the growth of Inonotus sp SP2, Stereum hirsutum RU 104 and Pleurotus eryngii IJFM 169 and their ligninolytic enzymes production. The decolourization ability of strain fungi was moreover screened. The concentration of functional basic groups in the banana peel surface was determined in 5.5 mmol g-1 as six and a half times higher than acid groups, while the lowest value of the maximum adsorption capacity of Acid Black 1 was 250 mg g-1. The adsorption kinetics of this dye was suitably represented by a pseudo second order model, obtaining correlation coefcients greater than 0.98. Additionally, the banana peel was demonstrated to be a source of carbon available for growth of the fungi studied. Reducing sugars supplied for banana peel were abruptly consumed up to the 5th day by S. hirsutum and Inonotus sp, while a slower consumption was observed in the case of P. eryngii. Manganese Peroxidase was produced by the three fungal strains, Inonotus sp. additionally produces Laccase and Aryl-alcohol oxidase.
C. Palma (&) E. Contreras J. Urra Chemical Engineering Department, Universidad de Santiago de Chile, Alameda 3363, Estacion Central, Santiago, Chile e-mail: carolyn.palma@usach.cl M. J. Martnez Centro de Investigaciones Biologicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain

Screening assays showed that all of the dyes were decolorized, resulting in efciencies between 50 and 99% by the three strains, with the exception of Basic Violet 4. Acid Black 1 was decolorized efciently by Inonotus sp and S. hirsutum. In conclusion, banana peel is a promising material for development of an integral bioremediation strategy for wastewater containing hazardous compounds. Keywords Agricultural waste Valorization White rot fungi Banana peel Dye

Introduction A signicant amount of lignocellulosic biomass is generated every year during cultivation, harvesting, processing and consumption of agricultural products, such as straw, stover, stalks, seeds, bagasse, peels, among others [1]. There are opportunities for addingvalue to these lignocellulosic residues using them as raw material to produce biosorbents and low-cost adsorbents [2], support-substrate for the production of biomass, enzymes and metabolites [3, 4], or feedstock for producing biofuels and biochemicals [5]. Additionally, using these residues to obtain valueadded products can contribute to their removal from the environment, avoiding their handling as solid waste [6]. Fungi, the major recyclers of carbon, are decomposersorganisms capable to hydrolyze complex organic compounds, such as agricultural waste. At present many lamentous fungi are utilized as producers of enzymes of industrial interest using same lignocellulosic residues as a source of carbon and energy. The use of the agricultural residues as a nutrient source for microorganisms of biotechnological interest is an alternative which continues to be explored. White rot fungi (WRF) have been extensively studied for their ability to produce

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extracellular ligninolytic enzymes usually during the secondary metabolism, which are involved in the degradation of lignin and other recalcitrant aromatic compounds [7, 8]. The white rot fungi are able to produce various kinds of ligninolytic enzymes such as laccase and peroxidase in liquid or solid-state fermentation cultures [9, 10], but their secretion depends on the type of fungi, the strain and the culture conditions. In all cases, a good carbon/nitrogen ratio is necessary to make fungi grow, and glucose is the more usual carbon source when using a dened medium. The search for alternative sources of nutrients, such as agricultural residues, has a double advantage: they add value to this waste while lowering the costs of producing enzymes. Another interesting feature of lignocellulosic residues is the physicalchemical properties of the functional groups available on their surface. These groups are responsible for the adsorption capacity of some specic solutes through ionic interactions. Naturasorbents have been obtained from agricultural waste, such as corn cobs, coconut shell, sugar cane bagasse and fruit peel like orange and banana [2]. Banana peel is a solid waste with high carbohydrate content, around 60% of dry matter. It is thus possible that it supports fungal growth [3]. The production of bananas and plantains in the world exceeded 94 million tons by 2008, with Africa, Latin America and the Caribbean being the major exporters [11]. At the time of harvest, a banana plant is estimated to have a weight of 100 kg, of which 15 kg correspond to leaves, 50 kg to pseudo-stalks, 33 kg to fruits and 2 kg to rachis [12]. At harvest, the percentage of rejected products due to size, contamination, handling, transport and storage is estimated at about 20% [13]. The proportion of the banana which is wasted as peel is 1820%. The disposal, minimization and valorization of this organic waste have become a major task. In Central America for example, the wastes from both, banana farming and commercial packaging, are normally disposed in large open-air dumps located in the same plantations, where the decomposition process is very slow due to high bre contents. Additionally, bacterial degradation is affected by the presence of chemicals in the disposal area. Poor disposal and lack of an appropriate treatment for these bio-degradable wastes also causes the proliferation of pathogenic organisms. At the same time, the lixiviated liquid causes a negatively impact over soil and groundwater [14]. The immediate use of this waste has been for animal feed but there are periods of restricted use, such as when natural forage is plentiful. Banana residues are highly fermentable due to the high starch content while still green (about 72% dry basis). Then during ripening, the presence of simple sugars (saccharose, glucose and fructose) facilitates silage [15]. The bers extracted from banana leaves and stem have been used in the production of particle boards for construction of social housing [16, 17]. From the

environmental standpoint, the banana peel has been used as bioadsorbent of soluble contaminants, such as dyes [2], metals [18, 19], and phenolic compounds [20]. In addition, its use in the production of pectin [21, 22] and ethanol [23, 24], as well as for production of biomass and metabolites of biotechnological interest [3, 4, 25], has also been reported. The release of dyes into industrial wastewater causes serious environmental problems, because their chemical structure gives them a persistent and recalcitrant nature. The discharge into natural waterways may have an inhibitory effect on photosynthesis affecting aquatic ecosystem. Besides, dye molecules are broken down during the anaerobic processes occurring in the sediment, generating toxic amines that may also pose a serious environmental problem. According to this background, it is interesting to explore the feasibility of colored wastewater bioremediation by a ligninolytic enzyme system of white rot fungi using banana peel as substrate-support. This substrate contains biopolymers, such as starch, cellulose and lignin, whose functional groups may have a signicant afnity for the adsorption of dyes. Therefore, it is important to determine which extension belongs to the process involved: bioadsorption and biodecolorization. The aim of this study was to analyze some alternative valorization of agricultural residues, considering its use in the treatment of colored efuents. The physicalchemical properties of the banana peel surface were determined in order to establish the feasibility of its use as dyes bioadsorbent. Additionally, banana peel was evaluated as substrate-support for the growing Inonotus sp SP2, Stereum hirsutum RU 104 and Plerotus eryngii IJFM 169, and the ligninolytic enzymes secreted by these fungi during the biodecolorizing process were evaluated. Finally the ability of decolorization/degradation of various types of dyes by these fungal strains was investigated.

Materials and Methods Agricultural Waste Banana peel (Mussa ssp) was obtained from fruit (CHIQUITA) purchased at a local market. The banana fruits were selected according to their ripening classication based on the Color Index corresponding to stage 6 or entirely Yellow [26]. The banana peel (BP) was dried in an oven for 1 week, reaching the equilibrium moisture content of around 10.5% (wt). Then the solid waste was crushed and screened, selecting the [-18 ? 60] mesh cut which corresponds to particles larger than 0.25 mm and smaller than 1 mm. The sample was then separated using different opening of the superior sieve, obtaining three cuts which are denominated 0.25, 0.5 and 1 mm.

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Dry matter, ash and nitrogen contents of BP were determined by a Proximate Analysis and the elemental composition (C, H and O) was obtained by Ultimate Analysis. Furthermore, the correlation between the contents of BP in the culture medium and the concentration of the available reducing sugars were established. Microorganisms Inonotus sp SP2 and Stereum hirsutum RU 104, (isolated in the Araucana region of southern Chile by the group of researchers at the Laboratory of Environmental Biotechnology, University of La Frontera) and Pleurotus eryngii IJFM 169 (provided by Dr. Maria Jesus Martinez from CIB-CSIC, Madrid, Spain). The fungi were maintained on malt extract agar plates at 4C. Dyes The dyes used were as shown in Table 1. All of them were of analytical grade and from SigmaAldrich Co. Physical Characterization of Banana Peel Density Density is evaluated with the mercury intrusion technique using the Porosimeter 2000 instrumental equipment (CARLO ERBA INSTRUMENTS). Particle size Distribution

PhysicalChemical Characterization of Banana Peel Surface Surface Acidity and Basicity Surface acidity and basicity were determined by potentiometric titration of the sodium hydroxide or chloride acid excess, after contacting 0.5 g of BP with 50 mL of NaOH 0.01 M (acidity) or 50 mL of 0.1 M HCl (basicity) until the equilibrium was reached. An aliquot of 20 mL ltrate obtained from contact assay (24 h) was then analyzed by titration with 0.01 M HCl (acidity) or 0.1 M NaOH (basicity) [27]. Point Zero Charge (pHPZC) Point zero charge (pHPZC) a set of BP suspensions were prepared adding 10, 7.5, 5, 2.5 and 1.25 mL of 0.1 M HCl to 1 g of BP. Similarly, BP suspensions were prepared with the same volumes of 0.1 M NaOH. Then, in all cases 5 mL of KCl 0.1 M were added and the volume was completed with distilled water until 100 mL. A sample with KCl 0.1 M and distilled water was also included. The asks were agitated for 1 h and then, pH values (pH1) were recorded. Subsequently 5 mL of KCl 1 M were added to each sample, maintaining the agitation an extra 1 h. Then the nal pH of each sample (pH2) was measured. The pH solution value, which makes the surface charge of BP is equal to zero, was determined by the method informed by Navas and Carrasquero [28]. Bioadsorption of Dyes In Banana Peel Determination of Contacting Time

Particle size Distribution is determined by laser diffraction using the Mastersizer X equipment (MALVERN INSTRUMENTS, MSX1). In this analysis a wet method was used. The sample was dispersed in a support solution, which is not soluble and does not alter its physical and chemical properties. Specic Surface Specic Surface is determined by nitrogen gas adsorption according to Brunauer, Emmett and Teller (BET) isotherm method. In comparison, Methylene Blue (Basic Blue 9) adsorption using the Langmuir isotherm was also obtained. Samples with an initial concentration between 50 and 600 mg L-1 of dye and a dose of 2 g of BP per liter of solution were used. These samples were continuously stirred in a thermostated shaker at 20C for 24 h. Finally the residual dye concentration in the solution was determined by visible spectrophotometry at 665 nm (Helios Gamma UVvis spectrophotometer).

Determination of contacting time 50, 200 and 500 mg L-1 of AB1 dye solution was contacted with 2 g L-1 dose of BP. The dynamic process was identied through the evolution of residual concentration of dye in solution for different contact times. For this, the BP was separated by ltration and the dye concentration of ltrate was determined by absorbance measurements in a UVVis spectrophotometer (Spectronic Helios Gamma model) at maximum absorbance wavelength. Adsorption Dyes Isotherms 2 g L-1 of BP was contacted with different concentration of AB1 dye solution (50500 mg L-1) in the thermostatted and agitated system (20 2C, 150 rpm) during 2 days. The samples were ltrated in a vacuum system and the dye concentration in the solution was determined by the absorbance measures at maximum wavelength in a Helios Gamma model UVVis spectrophotometer.

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80 Table 1 Characteristics of dyes

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Dye Acid black 1 (AB1)

Chromophore Diazo

k (nm) 618.5

Structure

Acid red 27 (AR27)

Azo

522.5

Basic blue 41 (BB41)

Azo

617.0

Basic blue 24 (BB24)

Thiazine

633.0

Basic orange 2 (BO2)

Azo

454.0

Basic violet 4 (BV4)

Triphenylmethane

595.0

Reactive black 5 (RB5)

Diazo

597.5

Reactive blue 19 (RB19)

Anthraquinone

592.5

Reactive orange 16 (RO16)

Azo

493.0

Growth, Decolorization Ability and Ligninolytic Enzyme Production of Fungi Previous studies indicate that strains of Inonotus sp SP2 and Stereum hirsutum RU 104 grow in media with 10 g L-1 glucose; in contrast Pleutorus eryngii requires twice the concentration of glucose. Experiments were designed based on this information. In the assays using BP, the equivalent concentration of BP was added.

Carbon Assimilation Carbon assimilation a pre-inoculate was prepared in a Fernsbatch (2 L) which contained 1012 plugs (5 mm in diameter) as an inoculate with 100 mL of sterilized glucose-peptone medium (5 g L-1 peptone, 2 g L-1 Malt Extract, 1 g L-1 KH2PO4, 0.5 g L-1 MgSO4 7H2O). The cultures were incubated at 28C for 58 days. Then, the mycelium was homogenized and

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inoculated in a 10% (v/v) proportion in Erlenmeyer asks (250 mL) which contained 81 mL of BP-peptone medium. In this medium the glucose was replaced by 36.16 and 60.56 g L-1 BP, for Inonotus sp SP2, Stereum hirsutum RU 104 and Pleutorus eryngii, accordingly. The cultures (in triplicate) were incubated at 28C in a shaker, at 150 rpm. Reducing sugars were determined by the dinitrosalicylic acid method, using D-glucose as a standard [29]. Decolorization Screening

tartrate buffer (pH 5) in the presence of 0.1 mM H2O2 [31]. Lignin peroxidase (LiP) activity was determined with 2 mM veratryl alcohol in 0.1 M sodium tartrate at pH 3 and 0.1 mM H2O2 [32]. Aryl-alcohol oxidase (AAO) was determined by the development of veratraldehyde from 5 mM veratryl alcohol 0.1 M sodium phosphate, pH 6 [33].

Results and Disscusion Characterization of Banana Peel This test was carried out in order to establish the degrading capacity of the recently isolated strains Inonotus sp and S. hirsutum. The assay was conducted in an Erlenmeyer (100 mL) ask covered with cotton and gauze stoppers, with 1.5 mL homogenized mycelium and 13.5 mL of glucose-peptone medium supplemented with 50 and 100 mg L-1 of dyes. The medium was autoclaved. Biotic and abiotic controls were carried out in parallel. The biotic controls were realized with mycelium and culture medium, and abiotic controls consisted of water and culture medium supplemented with dye. All cultures (in triplicate) were incubated for 15 days at 28C in an air atmosphere with 100% relative humidity. Enzyme Activities Laccase activity was measured using 5 mM 2,6-dimethoxyphenol (DMP) in 100 mM sodium citrate buffer (pH 5.0; e469 = 27,500 M-1 cm-1, referred to DMP concentration) [30]. Mn oxidizing peroxidase activity (named as MnP) was estimated by the development of Mn?3-tartrate complex (e238 = 6,500 M-1 cm-1) during the oxidation of 0.1 mM MnSO4 in 100 mM sodium
Fig. 1 SEM analysis of banana peel. a inside surface b outside surface

The pore size distribution of BP shows that the 40% of void volume has pore radius lower than 1 l, the remaining 60% corresponds to macropores, which leads to denominate this biomaterial as mesoporous. Consequently its density is 1.45 g L-1. The surface structure of these particles was observed with scanning electron microscopy (SEM). The electron micrographs revealed that the particles are of irregular shape and its surface exhibits a micro-rough texture, which can promote the adherence of the mycelium in a similar way to that found in natural habitats (Fig. 1). The particle size distribution of cut-off so-called 0.25 mm shows that the mode and median of the frequency distribution curve are 72.50 and 50.36 lm, respectively. These results agree with the mean particle size corresponding to 57.49 lm. The specic surface determined by the BET method has a very low value of 5 m2 g-1, probably due to de operational complexity for degassing samples lignocellulosic [34]. Other authors have reported values of specic surface for BP between 1323.5 m2 g-1 using the same method [18, 35]. On the other hand, estimation of the same property by the methylene blue adsorption method yielded

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82 Table 2 Chemical composition of banana peel Method Carbon Hydrogen Oxygen Total solid Nitrogen Ash Chloride Sulfur Phosphorus Ultimate analysis Ultimate analysis Ultimate analysis AOAC 17 AOAC 992.23 MOD combust AOAC 18 AOAC 16 AOAC 990.08C, EPA 2007 AOAC 990.08C, EPA 2007 41.1% (Dry wt.) 5.32% (Dry wt.)
0.1 0.05 0 0 -0.05 2 4

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0.25 mm 6

0.5 mm 8 10

1 mm 12

pH2- pH1

33.4% (Dry wt.) 15.7% (wt) 1.27% (wt) 17.3% (wt) 1.47% (wt) 855 mg kg-1 1,749 mg kg-1

-0.1 -0.15 -0.2 -0.25 -0.3 -0.35 -0.4

pH Suspension

510.2 m2 g-1, value which has the same order of magnitude that obtained for other bioadsorbent of low-cost such as bamboo cane, olive stones, peanut shells, peat moss (350550 m2 g-1) [36, 37]. The carbon and nitrogen contents in BP were 41.1 and 1.27 (wt)%, respectively (Table 2). These results make BP an alternative substrate well-suited for carrying out the enzyme production processes [4]. BP also presents significant oxygen contents (33.4 (wt) %) from carbohydrates and ber, which are in the range of those report in the literature are 59 and 32 (wt) %, respectively [3, 37]. This high content of polymers, such as cellulose and hemi-cellulose, suggest that BP could be a promising bioadsorbent of synthetic organic contaminants, such as dyes [38]. The mechanism governing the adsorption of the synthetic organic compounds into lignocellulosic waste is difcult to predict, since their diverse origin prevents us from knowing the chemical structure and the functional groups existing in the particle surface. The physicochemical characterization of the bioadsorbent surface, in particular the acidbase behavior of functional groups, might play a crucial role in ionic interactions occurring during the process, and thus it is fundamental to postulate the mechanisms involved, such as complexation reactions, ionic exchange and electrostatic interactions, among others. For example, the selective uptake of basic dyes on starchbased polymer was achieved by ionic exchange mechanism involving the acid carboxylic groups [39]. The deprotonation of these functional groups contributed to the adsorption occurring by electrostatic interactions between the COO- and cationic groups of basic dyes [40]. During the adsorption of the dyes, there were interactions between the ionic groups of the dye and the opposite polarity of the adsorbent sites. The higher the available number of sites capable of polarization and the stronger the interaction with the dye molecules, the greater the adsorption capacity of the biosorbent [41]. All bioadsorbents are characterized by having two main regions with a buffering capacity in the pH region between 35 and 810. According to the

Fig. 2 Point zero charge of banana peel

literature, the rst zone could correspond to carboxylic groups and the second, to hydroxyl groups [42]. The knowledge of pHpzc allows us to hypothesize on the ionization of functional groups and their interaction with ionic species in solution. The zero-charge point of BP corresponds to solution pH value of 2.41 (Fig. 2). This result is of the same order of magnitude as that reported value for peat moss which is 3.1, so it indicates that the concentration of basic sites is greater than that of acid sites and therefore BP can be classied as a bioadsorbent with a basic character. The concentrations of adsorption sites of acidic type and basic type corroborate this result. Indeed, the concentration of basic functional groups is six and a half times higher than acids, i.e. 5.50 and 0.84 mmol g-1, respectively. Moreover, the basicity is ve times higher than the values informed for peat moss and activated carbon (F-400), while the acidity is in the same order of magnitude. If the pH of a solution is higher than the value of pHpzc, the surface of the biosorbent has a negative net charge since the acid groups are de-protonated and could preferably interact with cationic species. In solutions with a lower pH than pHpzc, the net charge of solid surface is positive since the basic groups have the ability to share electrons, i.e., they are proton acceptors, and could do with those negatively charged. According to these results, BP could be a low-cost bioadsorbent to uptake acid dyes from industrial wastewater. Adsorption of Acid Black 1 dye According to the contact times studied, it was established that the equilibrium between AB1 and BP is reached at 30 h, period in which removal was achieved up to 80% for operating conditions corresponding to particle size identied as 0.25 mm and 500 mg L-1 of dye. The system was operating unbuffered, so that the process was conducted at the pH given by the dye solution (6.5). At this pH value, the charge of the BP surface is slightly negative (Fig. 2) and therefore

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there will be medium-scale repulsive forces with AB1 dye. The kinetic of the dye removal process under the studied conditions was accurately interpreted by a pseudo second order model [43] shown in the following Eq. 1. dqt k2 qe q2 dt 1

Although there are many models proposed in the literature to describe the equilibrium biosorption, the most used are the Langmuir and Freundlich isotherms, which are shown in Eqs. 3 and 4. qe kL qm Ce 1 kL Ce 3

where the integrated form, considering the following boundary conditions, is presented in Eq. 2: t = 0 =[ qt = 0 and t = t =[ qt = qt t qt 1 : 2 t k2 q2 qe
e

qm: maximum adsorption capacity (complete monolayer) (mg g-1) which correspond to the ratio between parameters kL and aL, kL: afnity between sorbate and adsorbent (L g-1), aL: parameter associated with the energy of adsorption (L mg-1) qe kF Ce
1 n

k2 is the kinetic rate constant (g mg-1 min-1) qe is the maximum capacity of adsorption (mg g-1) and, k2 q2 = h e represents the initial rate of adsorption (mg g-1 min-1) Table 3 provides information on model parameters obtained for each particle size. The effect of particle size was observed for initial dye concentrations above 200 mg L-1, for example, the maximum capacity of adsorption decreased approximately 40% for the largest particle size (1 mm). Moreover, for the particles of smaller size (0.25 and 0.5 mm) the maximum capacity of adsorption increased when increasing the initial concentration of dye, tripling its value. An effect of initial dye concentration in the kinetic rate constant was observed for all particle size studied. The highest value of kinetic rate constant was obtained for the lower concentration. The result indicated that this concentration is insufcient to saturate the outer surface of the particle and therefore the adsorption process is governed by the diffusion of the dye from the bulk solution to the solidliquid interface. If the system is operating with the higher concentrations, the driving force is greater and therefore the sites of the outer surface are saturated. The mechanism which controls the process is under these conditions, the intraparticle diffusion through of the mesopores. The combined effect of both phenomena causes the lower value of the kinetic rate constant to be obtained at 250 mg L-1 of dye. No signicant effect was observed from these variables in the initial rate of dye uptake process with the exception of smaller particle size.
Table 3 Effect of the operations conditions in the kinetics parameters of AB1 adsorption onto BP dp (mm) 0.25 C (mg L-1) 50 200 500 0.5 50 200 500 1 50 200 500

kF: equilibrium constant (mg g-1 (L mg-1)1/n, n: parameter associated with the afnity between sorbate and adsorbent) Figure 3 presents the equilibrium concentration of adsorption of AB1 dye on BP and tted curves for Langmuir and Freundlich models at different particle sizes. The isotherms parameters are given in Table 4. The experimental equilibrium data can be t adequately by both models (correlation coefcients [0.99). The values of maximum adsorption capacity of AB1 onto banana peel, which depends on the particle size, are in the range 250620 mg g-1; whereas, in other bioadsorbents such as magellanic moss peat [44] and cells fodder yeast (Kluyveromyces fragilis) magnetically modied [45], the maximum adsorption capacity of AB1 reported, is in the range of 25.0 and 29.8 mg g-1, respectively. The maximum adsorption capacity increases 20% when the particle diameter decreases by half, while the effect is not signicant for kL constant. Carbon Assimilation by WRF Several lignocellulosic wastes, such as agricultural wastes and by-products of this sector, have been used for enzyme production. The main reason is the carbohydrate content as well as the presence of sources of N and other inducers on
k2 (g mg-1 min-1)*105 9.77 1.53 3.58 14.63 5.36 5.04 13.46 6.73 8.27 h (mg g-1 min-1)*103 2.17 0.96 2.28 3.29 3.08 3.13 3.05 2.50 3.06 r2 0.99 0.98 0.97 0.99 0.98 0.98 0.99 0.99 0.99

qe (mg g-1) 22.27 62.50 63.69 22.47 57.47 62.11 22.68 37.17 37.07

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Concentration Relative (C/Co)

1 mm 250 200

0.5 mm

0.25 mm

Langmuir

Freundlich

1.2 Pleurotus 1 0.8 0.6 0.4 0.2 0 0 2 4 6 8 10 Stereum Inonotus

qe (mg g -1)

150 100 50 0 0 20 40 60 80 100 120

Time (d)
Fig. 4 Banana peels carbon assimilation by WRF Table 5 Phenoloxidases enzymes production by WRF

Ce (mg L-1)
Fig. 3 Adsorption isotherms of AB1 in banana peel Table 4 Parameters of the adsorption equilibrium isotherms models of acid black 1 Langmuir model dp (mm) 0.25 0.5 1 qm (mg g-1) 322. 14 256.44 623.56 kL (L g-1) 8.57 7.42 9.04 r
2

Enzyme

Freundlich model kF (mg g-1) (mg L-1)-n 16.78 17.24 13.18 n 0.61 0.54 0.78 r2 0.99 0.99 0.99 MnP Lacasse AAO

Pleurotus eryngii IJFM 169 4

Inonotus sp. SP2 4 4 4

Stereum hirsutum RU 104 4

0.99 0.99 0.94

Table 6 Dyes screening Pleurotus eryngii IJFM 169 Decolorization (%) AB1 AR27 BB24 BB41 BO2 BV4 RB19 RB5 RO16 81.68 2.11 70.06 29.48 ND 63.33 4.71 70.05 5.83 8.73 1.80 45.05 1.95 96.96 0.68 78.90 1.59 Dye Inonotus sp. SP2 96.88 0.45 96.52 0.52 86.79 2.41 97.3 1.42 56.17 5.73 ND 97.69 2.13 98.83 0.56 99.32 0.98 Stereum hirsutum RU 104 95.74 0.63 63.3 5.55 96.75 3.58 47.46 5.73 ND 93.33 9.33 98.13 0.01 86.64 5.38

the soluble fractions of these residues. As a result, these biomaterials have become an excellent substitute for synthetic sources of C and N. The origin and composition of these residues is critical for the type and amount of enzymes that could produce white fungus putrefaction. The production of extracellular oxidative enzymes occurs during secondary metabolism, usually in media in which one of the sources (C, N or S) is the limiting factor of growth. However, this phenomenon does not seem to be general for all fungi [46]. Figure 4 shows that the fungal strains assayed consumed the carbon source provided by BP. Reducing sugar was abruptly consumed up to the 5th day for S. hirsutum and Inonotus sp. cultures. Slower consumption was observed in cultures of P. eryngii, keeping from day 5 a residual concentration of about 40%. While a banana ripens, increases the contents of soluble sugar in the peel and consequently, the starch contents will be lower. In order to induce the growth of WRF using BP as a source of nutrients, high maturity fruits were selected (Color Index = 6) [26]. Ligninolitic enzyme activities were analyzed in the supernatants obtained after biomass separations with the sole purpose of detecting enzyme expression. Table 5, identies the ligninolitic enzymes produced by each fungal strain studied. MnP activity was detected in all fungal strains. LiP was not detected in any culture. Laccase and AAO were detected only in the Inonotus sp culture.

Numerous studies report the application of degradation processes on a large variety of efuents and recalcitrant compounds through the action of white-rot fungi by means of a highly oxidative, nonspecic, extracellular ligninolytic enzymatic system. In order to evaluate the degrading capacity of the phenoloxidase enzyme produced by WRF, dyes with an azoic structure were preferably selected (Table 6). Azo dyes are synthetic organic compounds most used in textile processing dyeing, paper printing and manufacture of foods and pharmaceutical drugs, among others. This type of dyes, which is characterized by the presence of at least one azo bond besides having aromatic rings, corresponds to the most marketed dyestuff. They represent 70%

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85 2. Gupta, V.K.: Suhas: Application of low-cost adsorbents for dye removala review. J. Environ. Manag. 90 8, 23132342 (2009) 3. Essien, J.P., Akpan, E.J., Essien, E.P.: Studies on mould growth and biomass production using waste banana peel. Bioresour. Technol. 96, 14511456 (2005) 4. Osma, J.F., Toca Herrera, J.L., Rodrguez Couto, S.: Banana skin: A novel waste for laccase production by trametes pubescens under solid-state conditions. Application to synthetic dye decolouration. Dyes Pigments 75(1), 3237 (2007) 5. Chen, S., Zhang, X., Singh, D., Yu, H., Yang, X.: Biological pretreatment of lignocellulosics: potential, progress and challenges. Biofuels 1(1), 177199 (2010) 6. Mtui, G.Y.S.: Review: Recent advances in pretreatment of lignocellulosic waste and production of value added products. African. J. Biotechnol. 8, 8, 13981415 7. Schoemaker, H.E., Tuor, U., Muheim, A., Schmidt, H.W.H., Leisola, M.S.A.: White-rot degradation of lignin and xenobiotics. In: Betts, W.B. (ed.) Biodegradation: Natural and synthetic materials, pp. 157174. Springer-Verlag, London (1991) 8. Rodrguez, E., Nuero, O., Guillen, F., Martnez, A.T., Martnez, M.J.: Degradation of phenolic and non-phenolic aromatic pollutants by four Pleurotus species: The role of laccase and versatile peroxidase. Soil Biol. Biochem. 36, 909916 (2004) 9. Couto, S.R., Sanroman, M.A.: Application of solid-state fermentation to ligninolytic enzyme production. Biochem Eng. J. 22, 211219 (2005) 10. Martnez, A.T., Speranza, M., Ruiz-Duenas, F.J., Ferreira, P., Camarero, S., Guillen, F., Martnez, M.J., Gutierrez, A., del Ro, J.C.: Biodegradation of lignocellulosics: Microbiological, chemical and enzymatic aspects of fungal attack to lignin. Int. Microbiol. 8, 195204 (2005) 11. FAO (Food and Agriculture Organization of the United Nations).: Faostat Statistics Database (last updated December 2009), Agriculture, Rome, Italy. (2008) 12. Bao, M., Delgado, S., Garca, M., Torres, M.: Aprovechamiento de residuos de plataneras. I. produccion en islas canarias, sus caractersticas y alternativas de utilizacion. Rev. Agroquim. Tecnol. Aliment. 27, 2430 (1987) 13. Zhang, P., Whistler, R.L., Bemiller, J.N., Hamaker, B.R.: Banana starch: Production, physicochemical properties, and digestibilitya review. Carbohydr. Polym. 59, 443458 (2005) 14. Astorga, Y.: The environmental impact of the banana industry: A case study of costa rica. First International Banana Conference, Brussels, Belgium (1998) 15. Dividich, J.L., Ceoffory, F., Canope, I., Chenost, M.: Using waste bananas as animal feed. World Anim. Rev. 20 20, 2230 (1976) 16. Contreras, W., de Contreras, M.E., Contreras, Y.: Determinacion de las propiedades de resistencia de los tableros aglomerados de partculas, fabricados con vastago de platano y adhesivo fenol formaldehdo (R10/R13%). Tecnologa y construccion, 24 3, 1525 (2008) 17. Biagiotti, J., Puglia, D., Kenny, J.M.: A review on natural brebased composites-Part I, structure, processing and properties of vegetable bres. J. Nat. Fibers 1(2), 3768 (2004) 18. Memon, J.R., Memon, S.Q., Bhanger, M.I., Memon, G.Z., ElTurki, A., Allen, G.C.: Characterization of banana peel by scanning electron microscopy and FT-IR spectroscopy and its use for cadmium removal. Colloids Surf. B 66, 260265 (2008) 19. Memon, J.R., Memon, S.Q., Bhanger, M.I., El-Turki, A., Hallam, K.R., Allen, G.C.: Banana peel: a green and economical sorbent for the selective removal of Cr(VI) from industrial wastewater. Colloids Surf. B 70, 232237 (2009) 20. Achak, M., Hadi, A., Ouazzani, N., Sayadi, S., Mandi, L.: Low cost biosorbent banana peel for the removal of phenolic compounds from olive mill wastewater: Kinetic and equilibrium studies. J. Hazard. Mat. 166(1), 117125 (2009)

of the world-wide market of dyes and 510% of this amount is discharged in industrial efuents, thus being a major environmental concern [47]. Decolorization experiments were carried out by using S. hirsutum, Inonotus sp and P. eryngii. The fungi were incubated under static conditions transformed at 28C with 100 mg L-1 of dye during 15 days. As shown in Table 6, all those dyes whose azoic group is joined to benzenenaphthalene rings or naphthalene-naphthalene rings, such as AB1, AR27, RB5 and RO16, were highly decolorized (mean value above 90%). On the contrary, dyes with a triphenylmethane structure, such as BV4, were not degraded by P. eryngii and Inonotus sp. Only S. hirsutum decolorized BV4, but scarcely (8.7%). Biodegradation of triphenylmethane dyes has been studied by bacteria, actinomycetes, yeasts, and fungi. In the specic case of WRF, degradation by laccases has only been reported [48]. Currently the degradation pattern of the dyes by the fungi is being studied as well as the development of an efuents treatment system based in their metabolism. Based on the above presented results, a combined two stage strategy of coloured wastewater treatment is proposed. In the rst stage, the dye is adsorbed by the banana peel. In the second stage, a process of bioremediation of the solid phase by means of a treatment with ligninolytic enzymes is carried out. The enzyme production can be performed using the same banana peel as carbon source.

Conclusion In conclusion, it is possible to use banana peel as promising material for the development of a global bioremediation strategy for wastewater containing hazardous compounds, such as dyes. Its high adsorption capacity of Acid Black 1 (250 mg g-1) as well as the high decolourizing efciency of the ligninolytic enzymes produced by Inonotus sp SP2 (97%) and Stereum hirsutum RU 104 (82%) allow to postulate the use of BP to implement a hybrid strategy of remediation based on the combination of physicochemicalbiological treatments.
Acknowledgments We wish to thank Maria Cristina Diez (University of La Frontera, Chile) for providing the Inonotus sp. SP2 and Stereum hirsutum RU 104. This research has been partially funded by Fondecyt Project 1090098 and a collaboration project between Spain and Chile (CSIC-USACh 2008-CL0030).

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