The Indigo Blue Dye Decolorization Potential of Immobilized Scenedesmus quadricauda

The Indigo Blue Dye Decolorization Potential of ImmobilizedScenedesmus quadricauda

Mathias Ahii Chia & Ojone Anne Odoh & Zakari Ladan

Received: 13 December 2013 /Accepted: 26 February 2014 /Published online: 14 March 2014# Springer International Publishing Switzerland 2014

Abstract In this study, Scenedesmus quadricaudaABU12 was immobilized with sodium alginate to de-termine its potential for decolorizing indigo blue dyeunder different incubation conditions. The microalgawas incubated at different pH (6.5–9.5), biomass con-centrations (0.1–1.0 g l−1), dye concentrations (12–75 mg l−1) and temperatures (25–40°C). The concentra-tion of biomass used significantly determined the rate ofdye decolorization, as the lowest biomass concentration(0.10 g) was able to completely decolorize the dye byday 3, while the highest biomass concentration(1.00 g l−1) attained 100 % decolorization on day 4.Neutral pHs supported the highest dye decolorizationrates compared alkaline pHs. The rate of dye decolori-zation had a linear relationship with the concentration ofthe dye in solution as increasing dye concentration in themedium significantly reduced the rate of decolorization

(p<0.05). At 25°C, the rate of dye decolorization wasconsistently higher from day 2 to the end of the exper-iment. Infra-red analyses of the algal biomass and thedye solution was done in Kbr by pressing between flataperture plates of sodium chloride and scanning from4,000 to 625 cm−1. This revealed the presence of func-tional groups associated with the biomass and dye thatprovided possible explanations for the decolorization ofthe dye under the different incubation conditions. Theseresults showed that immobilized S. quadricauda is ca-pable of decolorizing indigo blue dye at low biomasswhen immobilized with sodium alginate. However, thiswas dependent on the incubation temperature and dyeconcentration.

Keywords Color removal . Textile effluents .

Microalgae . Sodium alginate . Encapsulation

1 Introduction

Indigo dye, which is a Vat dye, is very popular andlargely employed on cellulosic fibers like cotton. Andlike other dyes used in dyeing processes, indigo dyescreate dye rich effluents that cause environmental prob-lems (Kiliç et al. 2011; Chia and Musa 2014). Most ofthe naturally occurring indigo derivatives are insolublein water and considered recalcitrant but may becomesoluble in the presence of reducing agents (Balan andMonteiro 2001).

Decolorization and degradation of textile dyes andtheir associated wastewater can be done by

Water Air Soil Pollut (2014) 225:1920DOI 10.1007/s11270-014-1920-2

M. A. Chia (*)Laboratório de Cianobactérias, Escola Superior deAgricultura Luiz de Queiroz, University of São Paulo,Piracicaba, SP CEP 13418-900, Brazile-mail: [email protected]

M. A. Chiae-mail: [email protected]

O. A. OdohDepartment of Biological Sciences, Ahmadu BelloUniversity,Zaria 810001, Nigeria

Z. LadanNational Research Institute for Chemical Technology,Basawa, Zaria 810001, Nigeria

physicochemical methods like ozonization, floccula-tion–filtration and alkalinization with calciumhydrosulfite. The overall cost of these processes coupledwith cost of regeneration, secondary pollutants, limitedversatility, and interference by other wastewater constit-uents as well as other residual sludge generation limittheir usage (Mohan et al. 2007). Biological treatmentsprovide a cost effective alternative for removing dyesfrom wastewater (Davies et al. 2005; Chu et al. 2009).Various microorganisms ranging from fungi, bacteriaand algae have been the subjects of many investigationsas potential candidates for the decolorization and deg-radation of various dyes (Donmez 2002; Aksu 2003;Chen et al. 2003). A number of studies have suggestedthat microalgae are ideal biosorbents for wastewatertreatment systems (Malik 2004; Gronlund et al. 2004;Ozer et al. 2006). However, these biosorbents haveseveral limitations, such as low resistance to chemicalsand heat.

To overcome the problem of high temperatures andresistance to chemicals, immobilization of microorgan-isms has been proposed and investigated as a viablemeans of decolorization and degradation of dyes (Fanget al. 2004; Couto et al. 2004: Pazarlioglu et al. 2005;Chen et al. 2005; Chu et al. 2009). These studies haveshown that immobilization provides an effective alter-native for high decolorization and degradation rates,which suggests that the technique requires extensiveinvestigations for its improvement and application forspecific dye types. The immobilization of dye removingmicroorganisms provides important advantages, such asexposure to higher dye concentrations without loss ofcell viability, a better environment for dye removalactivity, and protection of microorganisms againstchanges in temperature, pH, and other toxic compoundsof wastewater. Surprisingly, despite the extensive use ofindigo blue dye, nothing is known about the about theapplicability of immobilized microalgae for the decol-orization of this recalcitrant dye. Scenedesmusquadricauda immobilized into Ca-alginate was investi-gated in this study. We examined the dye decolori-zation rate at different pH levels, dye concentra-tions, biomass concentrations and temperatures,achieving the optimum conditions for immobilizedS. quadricauda cells. We hypothesized that chang-ing incubation conditions will affect the quantityas well as the rate of decolorization of indigo bluedye. Therefore, this study was aimed at investigat-ing the potential of sodium alginate immobilized

S. quadricauda for the decolorization of indigoblue dye.

2 Material and Methods

2.1 Algal Culture Conditions

S. quadricauda ABU12 was used as the test organismfor the decolorization of indigo blue dye. The microalgawas isolated from an unprotected and polluted freshwa-ter pond that receives effluents from different sourcesespecially during rainfalls via surface (Chia et al. 2011a;Chia and Musa 2014) in Zaria, Nigeria. Isolation andpurification of the green microalga was carried outaccording to Anderson and Kawachi (2005). Themicroalga was cultured in OECD—Organisation forEconomic Co-operation and Development (1984)).The medium was sterilized by autoclaving at 121°Cfor 30 min. The pH of the culture medium was adjustedto 7 prior to autoclaving. Cultures were maintained at 23±2°C under continuous lighting at an intensity of120 μmol m−2 s−1 with white fluorescent lamps.Cultures were shaken daily to prevent clumping. Thealgal cell culture density was monitored spectrophoto-metrically at 500 nm and microscopically using cellcounts on a daily basis (Chia and Musa 2014).

2.2 Dye

Indigo blue dye (Fig. 1) in pure form was obtained fromEmmybiz Dye and Chemicals International Limited,Sabon Gari, Zaria, Nigeria. The identity of the dye wasconfirmed using Infra-red and UV–VIS methods (see“Analytical methods”).

2.3 Immobilization

The alga was grown using the batch culture systemcontaining OECD medium and harvested in the expo-nential growth phase. It was concentrated by centrifu-gation, and resuspended in 2 % (w/v) sodium alginate

Fig. 1 Chemical structure of the Indigo blue dye used in theexperiments

1920, Page 2 of 9 Water Air Soil Pollut (2014) 225:1920

solution (obtained from Macrocystis pyrifera; SigmaA-7128). Afterwards, 1 g of centrifuged cells was ho-mogeneously mixed into 3 ml sodium alginate solutionto make beads. The mixture was then placed into 20 %(w/v) calcium chloride solution under magnetic stirring.The beads were stirred in the solution for at least 2 h tocomplete gel formation. Ca-alginate gel immobilized S.quadricauda particles were then collected by filtration,washed three times with distilled water, and stored in theculture medium without the dye and then used in theexperiment.

2.4 Dye Decolorization Experiments

To determine the effect of initial pH on dye decoloriza-tion, the OECD medium was prepared at pH 6.5, 7.5,8.5, and 9.5 containing 25 mg l−l of indigo blue dye and0.1 g l−1 immobilized alga. This phase of the experimentwas monitored over 4 days to observe the effect of pHon dye decolorization.

The influence of biomass concentration on the decol-orization of the dye was investigated at pH 7.5 becausethis was the pHwith the highest decolorization of indigoblue dye. Different biomass concentrations rangingfrom 0.1 to 1.0 g l−1 were exposed to it to indigo bluedye at a concentration of 25 mg l−1.

Experiments were carried out to evaluate the effect ofinitial dye concentration on dye decolorization. In thiscase, OECD medium was prepared at the optimum pH(7.5) value with 12, 25, 50 and 75 mg l−1 of indigo bluedye having a biomass concentration of 0.1 g l−1.

The effect of temperature on dye decolorization wasinvestigated in another series of experiments. 0.1 g ofimmobilized algae was grown in 25 mg l−1 initial dyeconcentrations at the optimum pH. The cultures werethen incubated at 25°C, 30°C 35°C and 40°C.

The control in each experiment had Erlenmeyerflasks containing the dye with cell-free beads preparedto enable the observation of any reaction of media andCa-alginate with dye. All experiments were carried outin triplicate. The experiments were monitored on a dailybasis.

2.5 Analytical Methods

The identification of the raw dye was done using spec-troscopic methods by measuring its absorbance at dif-ferent wavelengths (Fig. 2) using a SpectrumLab 7525UV–VIS spectrophotometer (B. Bran Scientific and

Instrument Company, England). In addition, Fouriertransform infrared (FTIR) analysis was used to deter-mine the different functional groups of the dye as ameans of confirming its identification (Table 1). About1 mgwas weighed in a small agate mortar with a drop ofnujol and Kbr. The mull was then pressed between theflat plates of sodium chloride. Furthermore, the dye insolution was analyzed using the liquid membrane meth-od by dripping several drops of it onto a Kbr apertureplate and pressing onto another aperture plate. Care wastaken to avoid the formation of air bubbles within theplates when dye solution samples were analyzed. Beforerunning a scan on each sample type, a background scanwas carried out. All scanning was done from 4,000 to625 cm–1 as previously described by Chia et al. (2011b,2012) using a Shimadzu FT-IR Model 8400 s spectro-photometer (Shimadzu, Japan). FTIR analyses of algalsamples were determined to judge the potential of thecells to decolorize indigo dye effluent (Table 1).

2.6 Data Treatment

The dye decolorization was determined by the percent(%) ratio absorbance reduction as given in the followingequation (Eq. 1) according to Kiliç et al. (2011):

D ¼ 100 Ci−Ctð ÞCi

; ð1Þ

where D is the decolorization of dye (in %), Ci is theinitial concentration of the dye and Ct is the dye con-centration along the time.

Percentage decolorization by blank beads was deter-mined as described below.

Fig. 2 The UV–VIS absorption spectrum used in identifying theindigo blue dye used in the experiments

Water Air Soil Pollut (2014) 225:1920 Page 3 of 9, 1920

Dye decolorization capacity is the concentration ofthe pollutant in the biomass and can be calculated basedon the mass balance principle from Eq. 2:

qm ¼ C0 − C f

� �=Xm ð2Þ

Note that qm (the maximum specific dye decoloriza-tion) represents the maximum amount of dye removedper unit dry weight of microbial cells (milligrams pergram), Xm maximum dried cell mass (grams per liter),and C0 and Cf the initial and final concentrations (mil-ligrams per liter), respectively.

The influence the different conditions on the decol-orization of indigo blue dye was tested for statisticalsignificance using two-way ANOVA and the means ofsignificantly different main effects were compared atp<0.05.

3 Results and Discussion

The application of microbial consortia presents consid-erable advantages over the use of pure cultures degra-dation of synthetic dyes. Although, most studies haveused bacteria and fungi for the biodegradation of organicpollutants, recent studies have indicated that in additionto providing oxygen for aerobic bacterial biodegraders,microalgae can also biodegrade organic pollutants di-rectly either as free cells or as immobilized composite(Fang et al. 2004; Lima et al. 2004; Kılıç et al. 2007).This is supported by the results this study that greenmicroalgae provide a reliable alternative for the decol-orization and degradation of organic pollutants like in-digo blue dye.

The UV–VIS spectrum of the dye showed that it hadan absorption maximum around 600 nm (Fig. 2). Theidentity of the dye was confirmed using FTIR analyses,which showed the chemical structure using the function-al group peaks as signatures/fingerprints (Table 1). Theresults confirmed that the NH, C–H, C=O, C=C, andPh functional groups representing amino, alkyl, carbon-yl, alkenyl and phenyl groups were present in the dyesamples used. The analyses of the S. quadricaudashowed that it had the following functional groups,NH, C=O, C=C and C–H present on its biomass(Table 1). The presence of the different functionalgroups associated with the algal biomass will allowvarious chemical reactions like electrophilic aliphaticand aromatic substitution to take place between the dyesand S. quadricauda. The amino group found in thestructure of indigo blue dye and the cell surface of themicroalga imply that they can be involved in reactionslike reductive amination, alkylation and conversion intoamides. These reactions and other types are capable offacilitating the uptake of the dye by the microalga and/ordegradation of the dye, thereby resulting in its decolor-ization. However, the extent of the decolorization isdependent on a number of factors such as biomassconcentration, initial pH, initial dye concentration andincubation temperature.

The effect of biomass concentrations on dye decol-orization was examined at four different biomass con-centrations to find suitable biomass concentrations forindigo dye decolorization. Maximum percentage decol-orization of the dye was 69.84 % and 65.75 % at 0.10 gand 1 g after 24 h (day 1), respectively. By day 4, 0.1 and1 g l−1 treatments had resulted in complete decoloriza-tion (100 %) and the maximum dye decolorization

Table 1 FTIR analysis for theindigo blue dye and Scenedesmusquadricauda biomass used in thedecolorization experiments

NH amino, C–H alkyl group, C=O carbonyl group, C=C alkenylgroup, Ph phenyl group

Chemical characteristics Samples

Wave number (cm−1) Functional group Indigo blue dye Scenedesmusquadricauda

3,500–3,100 R-NH 3,458.48 3,425.69

3,000–2,800 C–H 2,929.97

2,800–2,000 C=O

C=C

2,366.74 2,324.30

2,000–1,665 C–H – 2,092.83

1,680–1,600 C=C 1,633.76 1,647.26

1,200–900 C–H 1,195.91 –

900–600 Ph 626.89 –


potential per unit biomass of the dye (Fig. 3; Table 2).The changes in decolorization rate per time and between

the biomass concentrations were significantly different(p<0.05, Table 3). However, when compared to theother biomass concentrations, and taking into consider-ation the rate of dye decolorization over the experimen-tal period, the 0.1 g l−1 biomass concentration presentedthe best results as it gave consistently higher decolori-zation rates from day 2 and attained 100 % decoloriza-tion at day 3. With the same amount of dye in solution,the ability to completely decolorize with much lessbiomass is a plus. A possible explanation for this be-havior may be because at a low biomass concentration,the surface area per algal cell per unit area increases

Fig. 3 The effect of biomassconcentration on thedecolorization (%) potential ofimmobilized Scenedesmusquadricauda at 25 mg l−1 initialdye concentrations after 4 days ofincubation

Table 2 Comparison of maximum specific dye decolorization(qm) by immobilized Scenedesmus quadricauda under differentconditions

C0 (mg l−1) qm (mg g−1)

Biomass concentration (g l−1)

0.1 26.67 32.80

0.25 27.07 23.20

0.5 25.20 18.00

1 30.06 31.60

Initial dye concentration (mg l−1)

12 14.00 13.90

25 22.07 19.93

50 80.80 44.80

75 84.47 46.53

Initial pH

6.5 32.66 17.74

7.5 20.92 14.00

8.5 22.33 4.93

9.5 22.50 1.20

Temperature (°C)

25 21.47 26.00

30 21.07 10.2

35 22.73 4.47

40 30.53 6.80

C0 represents the average initial dye concentration and qm is themaximum dye decolorization capacity per unit algal biomass

Table 3 Two-way ANOVA summary table showing the effect ofbiomass concentration, dye concentration, initial pH and temper-ature after 4 days on the decolorization of indigo blue dye

Parameters F value P value

Biomass concentration

Time (days) 30.57 0.0001

Biomass concentration 19.95 0.0001

Initial dye concentration

Time (days) 5.16 0.004

Dye concentrations 77.01 0.001

Initial pH

Time (days) 1.85 0.154

Initial pH 6.90 0.001

Temperature

Time (days) 8.48 0.001

Temperature 11.43 0.001


thereby increasing the decolorization potential of themicroalga. The increased surface area is further enhancedby the entrapment of the cells in sodium alginate.Immobilization has the added advantages over free cellssuch as increased metabolic activities and metaboliteproduction, which will have further enhanced the decol-orization potential of S. quadricauda even at low bio-mass. Kiliç et al. (2011) and Daneshvar et al. (2007) statethat the rate of dye decolorization is directly proportionalto the amount of biomass present in the medium, hencethe need to encourage conditions that promote higherbiomass production in dye removal or decolorization

processes. However, from our results it can be shownthat the case is not always so because wewere still able toobtain good decolorization at low biomass when themicroalga was immobilized. Our results still agree withtheir findings to some extent because aside the lowestconcentration, indigo decolorization increased with in-creasing biomass production with the highest biomasshaving the highest decolorization rate at the end of theexperiment.

The results for the effect of medium pH on dye decol-orization by immobilized S. quadricauda are shown inFig. 4. The highest dye decolorization percentage was

Fig. 4 The effect of initial pH onindigo blue dye decolorization(%) by Scenedesmusquadricauda at 25 mg l−1 initialdye concentration after 4 days ofincubation

Fig. 5 The decolorization ofindigo blue dye by Scenedesmusquadricauda as a function ofdifferent initial dyeconcentrations


attained at 6.5 and 7.5 compared to the other pH levels(p<0.05, Table 3). However, the optimum pH for indigodye decolorization was 7.5. Published results shows thatthe optimum pH for dye decolorization by differentmicroorganisms varies depending on the dye typeand the specific organism involved. For example,Ertugrul et al. (2008) showed that 7.5 to 8.5 werethe optimum pH attained in treatment of ReactiveBlack B and Remazol Blue dyes by an immobilizedPhormidium sp., while Tian et al. (2013) showedthat the optimum pH obtained were 4.0 and 5.0 forthe decolorization of indigo dye using rot fungi.However, for most algae, there is an isoelectricpoint that is pH 3.0, and at pH below this point(and mostly within the acidic pH range), the H+

ions compete effectively with dye cations, causinga decrease in color decolorization (Daneshvar et al.2007). This implies that as H+ concentrations arereduced, i.e., from the neutral to alkaline pH, thehigher the dye decolorization and biodegradationpotential of algae (Kiliç et al. 2011). At higher pHthan the isoelectric point, the biomass surfacebecomes negatively charged, which facilitatesthe positively charged dye cations through elec-trostatic force of attraction thereby increasingtheir decolorization. Despite immobilization of theS. quadricauda strain used in this study, it wasnot able to increase decolorization of the dye atalkaline (8.5 to 9.5) pH. We had expected thatimmobilization will enhance the tolerance of themicroalga to alkaline condition that would have

increased the possibility of applying it for thedecolorization of this dye at much higher pH.However, the case may be different for other dyes.

Our results show that as the dye concentration in themedium increased the rate of decolorization byS. quadricauda decreased (Fig. 5). Within the first24 h, the lowest dye concentration was completelydecolorized whereas by day 4 (the end of the experi-ment) only about 60 % of 50 and 75 mg l−1 initial dyeconcentrations were decolorized. These results showthat initial dye concentration plays a significant role inthe decolorization potential of immobilized microalgae.Despite the reduced decolorization percentage at thehigher dye concentrations, with more than 50 %decolorization percent, the results are promisingfor the application of sodium alginate immobilizedS. quadricauda. Similar to these findings, Ertugrulet al. (2008) demonstrated that at different tested dyeconcentrations (9.1 to 82.1 mg l−1), dye decolorizationpotential decreased with increased dye concentrationduring the incubation period of Phormidium sp. In ad-dition, Kiliç et al. (2011) showed the same behavior byGonium spp. grown in several synthetic reactive dyes.Therefore, confirming the well established fact that dyedecolorization is dependent on the initial concentrationof the dye solution even when immobilized microalgalbiomass is used.

The results of the effect of different temperatures ondye decolorization by immobilized S. quadricauda areshown in Fig. 6. The percentage dye decolorization washighest at 40°C within the first 24 h, but with the

Fig. 6 The influence oftemperature on the indigo bluedye decolorization (%) potentialby Scenedesmus quadricauda at25 mg l−1 initial dyeconcentration during theincubation period


progression of the experiment, i.e., from day 2, percent-age decolorization was highest at 25°C, which wasclosely followed by the 30°C treatments. This meansthat after 24 h, the shielding effect sodium alginateimmobilization provides to the microalga is not able tomaintain the decolorization rate as exposure temperatureincreases beyond 30°C. These results agree very wellwith those of Tian et al. (2013), who showed that 28°Cwas the optimum temperature for decolorization of in-digo dye. Ertugrul et al. (2008)), for example, showedthat at 45°C, the cyanobacterium Phormidium sp. yieldover 50 % decolorization of textile dyes, which is dif-ferent to what was obtained in this study at high tem-peratures having <50 % decolorization of indigo bluedye. This may be related to the fact that thePhormidium strain studied was thermophilic innature, hence its ability to withstand higher tem-perature treatments. It is important to note that thetemperatures with the highest decolorization ratesin our study simulate indigo and azo dye effluentat ambient temperatures (Manu and Chaudhari2003; Chia and Musa 2014), which demonstratestheir applicability under real life conditions.

In conclusion, our results agree with the hypothesisof this study that changing dye exposure conditions willaffect the ability of immobilized S. quadricauda todecolorize indigo blue dye. The highest decolorizationoccurred at pH 6.5 and 7.5 pH, and biomass 0.1 and1.0 g l−1. On day 3, 0.1 g l−1 completely decolorized thedye, while 1.0 g l−1 completely decolorized the dye onday 4. High temperatures, pHs and dye concentrationsresulted in reduced decolorization by immobilizedS. quadricauda ABU12.

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The Indigo Blue Dye Decolorization Potential of Immobilized Scenedesmus quadricauda