SOLUTIONS BY RHIZOPUS STOLONIFER BIOMASS
Y. ZEROUAL1,2, B. S. KIM1, C. S. KIM3,∗, M. BLAGHEN2and K. M. LEE1
1Laboratory of Enzyme Technology, College of Natural Science, Chonbuk National University, Chonju 561–756, Korea;2Laboratory of Microbiology, Biotechnology and Environment, Faculty of
Sciences, University Hassan II, Casablanca, Morocco;3Division of Electronics and Information Engineering, College of Engineering, Chonbuk National University, Chonju 561-756, Korea
(∗author for correspondence, e-mail: kmlee@chonbuk.ac.kr)
(Received 29 August 2005; accepted 12 February 2006)
Abstract. The removal of bromophenol blue dye (BPB), from aqueous solutions, by biosorption on a non-living biomass of Rhizopus stolonifer was investigated in a batch system. Pretreatment of the biomass with NaOH was found to be the most effective means to enhance the biosorption of BPB. The fungal biomass exhibited the highest dye sorption capacity at pH 2 and the uptake process followed the pseudo-second order reaction model. The equilibrium sorption capacity of the biomass increased as the initial dye concentration increased, and the maximum uptake value was estimated at 1111 mg/g according to Langmuir adsorption isotherm. The adsorbed dye was easily desorbed from a fungal biomass with 0.1 M NaOH solution and the regenerated biomass could be reused for other biosorption essays with similar performances.
Keywords: biosorption, bromophenol blue, Rhizopus stolonifer
1. Introduction
Dyeing industry effluents constitute one of the most problematic wastewaters to be treated not only due to the toxicity of the dyes but also due to its visibility even at small concentrations (Banat et al., 1996; Robinson et al., 2001; Fu et al., 2001).
Consequently considerable research efforts have been devoted to optimizing color removal from effluents.
The main techniques, which have been utilized for the treatment of dye wastewa- ter, include chemical coagulation/flocculation, ozonation, oxidation, ion exchange, irradiation, precipitation and adsorption (Lin et al., 1994a; Lin et al., 1994b; Lin et al., 1996). These methods have been found to be limited, since they often involve high capital and operational costs and may also be associated with the generation of secondary wastes, which present treatment problems.
Adsorption technology is generally considered to be the most promising option for quickly lowering the concentration of dissolved dyes in an effluent (Tsai et al., 2004). In this regard, activated carbon has been evaluated extensively for the removal of color resulting from different classes of dyes. However, due to its high price it is not used on a great scale (El-Geundi, 1991; Choy et al., 1999; Al-Degs et al., 2000).
Water, Air, and Soil Pollution (2006) 177: 135–146
DOI: 10.1007/s11270-006-9112-3 C Springer 2006
This has led many workers to search for the use of cheap and efficient alternative materials, such as bagasse pith, peat, and rice husk (Juang et al., 1997; Ramakrishna and Viraraghvan, 1997; Morais et al., 1999). However, these low-cost adsorbents generally have low adsorption capacities. Using microorganisms as biosorbents for removing dyes offers a potential alternative (Kirby et al., 1995; Nigam et al., 1996). Among these microorganisms fungal biomass can be produced cheaply and obtained as waste from various industrial fermentation processes (Kapoor and Viraraghavan, 1995).
Biosorption of dyes consists of an adsorptive binding of dyes to the inac- tive dead biomass, using purely physicochemical pathways of uptake (Tsezos and Volesky, 1981). The implied mechanisms in biosorption differ qualitatively and quantitatively according to the type of biomass, its origin and its processing.
The use of dead cells in biosorption is more advantageous for water treatment in that dead organisms are not affected by toxic wastes, don’t require a continuous supply of nutriments, and can be regenerated and reused for many cycles (Gadd, 1990).
The purpose of this study was to investigate the ability of pretreated biomass of Rhizopus stolonifer as biosorbent to remove bromophenol blue dye (3,3,5,5- Tetrabromophenol-sulfonephtalein, sodium salt) from aqueous solutions. This dye, which is a triphenylmethane derivative, and its structurally related compounds, e.g.
fluoresceins and xanthenes are widely used as industrial dyes for foods, drugs, cosmetics, textiles, printing inks, and laboratory indicators. Some of these com- pounds have been reported to be genotoxic (Salen, 2000). Effective pretreatment of Rhizopus stolonifer biomass for BPB removal was investigated and the effects of various parameters on the biosorption of dye (pH, temperature, initial dye con- centration, contact time) were conducted in a batch stirred reactor. Langmuir and Freundlich models were applied to experimental data to describe the biosorption equilibrium between the BPB dye and the fungal biomass. The experimental data was also analyzed using the pseudo first-order Largergren and the pseudo second- order kinetic models. The reversibility and the reuse of the fungal biomass in several biosorption-desorption cycles were examined.
2. Materials and Methods
2.1. MICROORGANISM AND GROWTH CONDITIONS
Rhizopus stolonifer was isolated from dye-contami- nated wastewater. It was iden- tified based on the visual observation of isolates grown on PDA plates, micro- morphological studies in slide culture (Riddell, 1950) at room temperature, and the taxonomic keys described in Hoog and Guarro (1995).
Stock cultures were routinely maintained on LB agar supplemented with 1%
of glucose. Precultures of Rhizopus stolonifer were prepared by inoculating plugs
(diameter 0.5 cm) from the growing zone of fungus on agar plate to 50 ml of LB liquid medium supplemented with 1% of glucose. Then, cells were cultivated statically at 25◦C for 24 hours. Afterwards, the precultures were homogenized aseptically using a potter homogenizer (200 rpm), and the homogenats were used as an inoculum for liquid cultures.
Liquid cultures consisted of 150 ml of LB supplemented with 1% of glucose in 250 ml Erlenmeyer flasks. These were inoculated at 1% with homogenized preculture and incubated aerobically at 25◦C on a rotary shaker at 150 rpm for 2 days.
2.2. PREPARATION OF MICROORGANISM BIOMASS
After a growth period, the biomass was harvested by filtration and washed thor- oughly with distilled water. Some of the fungal biomass was dried overnight at 50◦C and used as a native biosorbent to remove dye from the dye solution. Other fungal biomass was pretreated using 3 different methods as follows:
– Autoclaved for 20 min at 121◦C and 18 spi, and then dried overnight at 50◦C.
– Contacted with 0.1 M NaHCO3solution for 1 h at room temperature.
– Contacted with 0.1 M NaOH solution for 1 h at room temperature.
The biomass after each chemical pretreatment was washed with generous amounts of distilled water until the pH of the wash solution was close to that of distilled water, and all biomasses were subsequently autoclaved for 20 min at 121◦C and 18 spi, and then dried overnight at 50◦C. The dry biomass was ground to a powder using a mortar, and powder particles including between 300 and 500μm, which was defined by the sieve size, were used as biosorbent in this study.
2.3. DYE SOLUTION PREPARATION AND DYE ANALYSIS
The dye used in this study was bromophenol blue (FW=691.9,λmax=433 and 590 nm) an anionic triphenylmethane dye, supplied by Sigma-Aldrich Co. (St. Louis, USA). Its chemical structure is shown in Figure 1.
Dye solutions were prepared by dissolving accurately weighted dye in dis- tilled water. In order to compare dye removal on the same basis, the pH of all the samples was adjusted to 2 before measurement with 1 mM HCl solution.
The concentrations of BPB dye in the biosorption medium were measured spec- trophotometrically at 433 nm. Dilution of the dye solution was conducted where required.
Figure 1. Chemical structure of bromophenol blue.
2.4. BATCH BIOSORPTION STUDIES
Dye uptake studies were conducted in 250 ml Erlenmeyer flasks containing 100 ml of dye solutions with 1 g biomass/L. The flasks were agitated on a shaker at 150 rpm and left to equilibrate for 20 h at 25◦C. The solution pH before and during the sorption tests was adjusted with 1 mM NaOH and 1 mM HCl solutions. Aliquots of dye solutions were collected at predetermined time intervals to determine the residual dye concentration in the solution. Before analysis the samples were cen- trifuged at 5000 rpm for 5 min and the supernatant liquid was analyzed for any remaining dye. All the experiments were carried out in duplicates. Dye adsorbed by the biomass was calculated according to a material balance.
2.5. DESORPTION STUDY
In the desorption experiments, 0.1 g of dye-loaded biomass was mixed with 100 ml of NaOH solution (0.1 M) in 250 ml Erlenmeyer flask under agitation of 150 rpm.
The released dye concentration was evaluated as described above.
3. Results and Discussion
The biosorption of bromophenol blue (BPB) by Rhizopus stolonifer biomass was conducted for 20 h in order to determine the effect of time on biosorption. As shown in Figure 2, more than 90% of dye was removed by these dried cells in the first 4 hours of contact. Equilibrium was established in 15 h. This suggests that for theses fungus biomasses the uptake of dye occurs predominantly by surface binding and those available sites on the biosorbent are a limiting factor for the biosorption.
The results in Figure 2 clearly indicate that the biosorption behavior and biosorp- tion capacities of Rhizopus stolonifer biomass were different depending of the pre- treatment method. Native biomass of Rhizopus stolonifer showed a high biosorption capacity of 280 mg/g at 400 mg/l of initial BPB concentration. Compared to native cells, all pretreatments used in this study increased biosorption capacities. Auto- claving increased the sorption capacity of the fungal biomass (317 mg/g at 400 mg/l
Figure 2. Uptake kinetic of bromophenol blue by native and different pretreated biomasses of Rhi- zopus stolonifer.
initial BPB concentration) by disrupting the fungal structure (Fu and Viraraghavan, 2002). This disruption may cause an increase of surface area and increase in poros- ity of the particles, and thus latent sites, consequently increasing the dye adsorption (Gallagher et al., 1997). NaHCO3pretreatment also increased the biosorption ca- pacity of the fungal biomass (352 mg/g at 400 mg/l initial BPB concentration).
This could be because bicarbonate ion can provide protons in water (Fu and Vi- raraghavan, 2002). The protons could neutralize negative charges on the surface of the biomass and change the part of the negatively charged surface to positively charged. In this study, NaOH pretreatment increased biosorption capacity for BPB to the highest extent (385 mg/g at 400 mg/l initial BPB concentration). This could be because NaOH treatment can remove proteins and glucans from the cell wall thereby increasing the percentage of chitin/chitosan in the wall cell fraction, since chitin/chitosan was suggested as the predominant biosorbent of the dyes (Gallagher et al., 1997; Aksu, 2005). Thus, the fungal biomass pretreated with NaOH was used in further studies.
The pH value of the solution was an important controlling parameter in the biosorption process (Waranusantigul et al., 2003). In fact, solution pH influences both the cell surface dye binding sites and the dye chemistry in water. The variation of equilibrium uptake with pH, at 800 mg/l of an initial BPB concentration at 25◦ provided in Figure 3. The maximum uptake was at pH 2 and then declined with further increase in pH. The enhancement of uptake of BPB dye at acidic pH may be explained in terms of electrostatic interactions between the biomass and the dye.
With a diminishing pH, increasing numbers of weak base groups in the biomass become protonated and acquire a net positive charge (O’Mahony et al., 2002). These charged sites become available for binding to anionic groups, such as the BPB dye used in this study. Hydrogen ions also act as a bridging ligand between the fungi cell wall and the dye molecule (Banks and Parkinson, 1992; Fu and Viraraghavan, 2001).
The effect of temperature on the equilibrium capacity of NaOH pretreated fun- gal biomass for BPB was investigated in the temperature range of 25–55◦C. As shown in Table I, the biosorption of BPB dye increased slightly with an increasing
TABLE I
Effect of temperature on the equilibrium dye sorption capac- ity of Rhizopus stolonifer
Temperature (◦C) qeq(mg/g)
25 710
35 718
45 716
55 717
Figure 3. Effect of pH on the equilibrium dye sorption capacity of Rhizopus stolonifer.
temperature up to 35◦C (from 710 to 718 mg/g), and then remained stable with further increases in temperature. These results suggest that BPB dye biosorption on tested biomass was essentially chemical sorption. Similar results have been re- ported by Hu (1996) concerning the effect of temperature on the removal of six reactive dyes by tree Gram-negative bacteria (P. luteola, E. coli and Aeromonas sp.) suggesting the feasibility of directly using dead biomass in the dyeing wastewater to absorb dyes without decreasing the temperature of wastewater (Hu, 1996).
The effect of initial dye concentration on the dye sorption capacity of tested biomass was investigated between 100 and 1200 mg/l at different pH. As shown in Figure 4, the equilibrium sorption capacity of the biomass increased by increasing
Figure 4. Bromophenol blue adsorption isotherms for Rhizopus stolonifer biomass at different pH.
the initial dye concentration up to 1200 mg/l while the removal percentage of dye showed the opposite trend. At pH 4 the biomass exhibited the lowest uptake capacity (340 mg/g). The biomass reached quasi-saturation at a relatively low concentration of 600 mg/l. In contrast at pH 2, the biomass was capable of removing more than 88% of coloring material up to 800 mg/l initial dye concentration and did not appear to reach saturation over the concentration range involved. By increasing the initial dye concentration from 200 to 1000 mg/l the equilibrium sorption capacity increased from 191 to 864 mg/g. At pH 3, the uptake also increased by increasing the initial dye concentration, and tended to saturation at high dye concentration (1000 mg/l). When the initial dye concentration increased from 200 to 1000 mg/l the loading uptake of the biomass increased from 194 mg/g to 750 mg/g while the adsorption yield decreased from 97% to 75%.
In order to describe the biosorption equilibrium between the BPB dye and the fungal biomass, the experimental data were fitted to Langmuir (1918) and Fre- undlich (1907) models.
The Langmuir model is described by the following equation:
qeq =qmaxbCeq/(1+bCeq) (1)
Where qeqis the adsorbed dye quantity per gram of biomass at equilibrium (mg/g).
Ceq is the concentration of the dye solution at equilibrium (mg/l). qmaxis the max- imum amount of dye per unit weight of biomass to form a complete monolayer on the surface bound at Ceq (mg/g). b is a constant related to affinity of the binding sites (l/mg).
The Freundlich model equation is of the form:
qeq =k.C1/neq (2)
Where k and n are the Freundlich constants characteristic of the system.
The maximum uptake capacity qmax and the equilibrium constants b in the Langmuir model as well as k and n in the Freundlich model at different pH were calculated, and are presented in Table II. In view of the values of linear regression
TABLE II
Regressed Langmuir and Freundlich sorption model param- eters at different pH
Langmuir parameters Freundlich parameters
qmax(mg/g) b (l/mg) r2 k n r2
PH 21111 0.026 0.99 80.26 2.16 0.88
PH 3769 0.086 0.98 138.89 3.04 0.80
PH 4333 0.054 0.98 59.97 3.42 0.94
coefficients in the table, the Langmuir model exhibited a better fit to the biosorption data than the Freundlich model. The maximum adsorption capacity at pH 2 was 3.5- fold higher than at pH 4 which dropped from 1111 to 333 mg/g when pH increased from 2 to 4.
In order to analyze the biosorption kinetics of BPB dye, the pseudo first-order (Largergren, 1898) and the pseudo second-order (McKay and Ho, 1999) were applied to experimental data.
The first-order rate expression of Largergren is expressed as follows:
log(qeq−q)=logqeq−k1,adt/2.303 (3)
Where q is the amount of adsorbed dye on the biosorbent at time t, and k1,ad is the rate constant of Largergren first-order biosorption.
The pseudo second-order kinetic rate equation is expressed as:
t/q =1/
k2,adqeq2
+t/qeq (4)
Where k2,ad is the rate constant of second-order biosorption.
The first-order rate constant k1,ad and qeq were determined from the slopes and the intercepts of plots of log(qeq−q) versus t obtained at different initial con- centrations of dye (200, 400 and 600 mg/l). The obtained results are represented with their correlation coefficients in Table III. It can be seen from these results that the pseudo first-order Largergren model, as reflected by the low correlation coefficients obtained and the disparity between the theoretical and experimental equilibrium dye sorption capacities, was not suitable to describe the biosorption kinetics of BPB on the studied biomass. Using equation 4, t/q was plotted against t at 200, 400, and 600 mg/l initial dye concentration and second-order adsorption rate k2,ad and equilibrium uptake values qeq were determined from the slop and intercept of the plots. The values of the parameters k2,ad and qeq and correlation coefficients are also represented in Table III. The correlation coefficients for the second-order kinetic model were close the unity for all the concentration studied and the theoretical qeqvalues also agreed well with the experimental qeqvalues. These results suggest that the process of dye adsorption followed the second-order rate kinetics.
In order to study the reversibility of BPB sorption, the dye loaded biomass was eluted by various eluants; NaHCO3, HCl, NaOH, C2H5OH. It was established that the NaOH (0.1 M) solution was effective in BPB desorption. The experimental results for the elution of BPB loaded biomass with various initial dye loading by 0.1 N NaOH are represented in Figure 5. It is evident that the elution percentage values are close to unity indicating that the sorbed dye can be completely and easily eluted from the biomass.
To show the reusability of the biosorbent, the same regenerated biomass was reused for up to 5 sorption-desorption cycles. As shown in Figure 6 the uptake
TABLEIII Comparisonofthefirstandsecondorderrateconstantsandcalculatedandexperimentalqeqvaluesobtainedatdifferent initialbromophenolblueconcentrations First-ordermodelparametersSecond-ordermodelparameters C0(mg/l)qeq,exp(mg/g)k1,ad(1/min)qeq,cal(mg/g)r2k2,ad×102(mg/g/min)qeq,cal(mg/g)r2 2001910.007900.950.0241920.99 4003850.0071250.810.0193840.99 6005680.0061340.740.0175880.99
Figure 5. Comparison of the initial bromophenol blue loading and the dye elution with 0.1 M NaOH.
Figure 6. Desorption rates of dye obtained for successive biosorption-desorption cycles.
capacity did note change during the repeated sorption-desorption cycles. These results exhibited that the fungal biomass could be repeatedly used in dye biosorption without any detectable losses in their initial sorption capacity.
This study reveals that NaOH pretreated biomass of Rhizopus stolonifer is an effective biosorbent for BPB dye removal. The mechanisms and kinetics of BPB biosorption on fungal biomass depend on the experimental conditions particu- larly the medium pH and dye concentration. The loaded biosorbent can be conve- niently regenerated with NaOH solution (0.1 N). The high efficiency of biosorption and elution, low biomass damage, and stability of over prolonged operation time make this process an economical effective alternative technique for dye pollution monitoring.
Acknowledgments
This work was supported by the grant of 2003 Post Doctoral Program, Chonbuk National University and the Oriental Medecine R&D Project, Ministry of Health
& Welfare, Republic of Korea (0405-OM00-0815-0001).
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