Bioresource Technology 101 (2010) 4371–4378

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Bioresource Technology

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Pilot-scale removal of chromium from industrial wastewater usingthe ChromeBac™ system

Wan Azlina Ahmad, Zainul Akmar Zakaria *, Ali Reza Khasim, Muhamad Anuar Alias,Shaik Muhammad Hasbullah Shaik IsmailDepartment of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 October 2009Received in revised form 17 January 2010Accepted 21 January 2010Available online 25 February 2010

Keywords:DetoxificationElectroplatingAcinetobacterCr(VI)Pineapple wastewater

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.01.106

* Corresponding author. Tel.: +60 7 5534546; fax: +E-mail address: zainul@kimia.fs.utm.my (Z.A. Zaka

The enzymatic reduction of Cr(VI) to Cr(III) by Cr(VI) resistant bacteria followed by chemical precip-itation constitutes the ChromeBac™ system. Acinetobacter haemolyticus was immobilized onto carriermaterial inside a 0.2 m3 bioreactor. Neutralized electroplating wastewater with Cr(VI) concentration of17–81 mg L�1 was fed into the bioreactor (0.11–0.33 m3 h�1). Complete Cr(VI) reduction to Cr(III) wasobtained immediately after the start of bioreactor operation. Together with the flocculation, coagula-tion and filtration, outflow concentration of less than 0.02 mg Cr(VI) L�1 and 1 mg total Cr L�1 werealways obtained. Performance of the bioreactor was not affected by fluctuations in pH (6.2–8.4), Cr(VI)(17–81 mg L�1), nutrient (liquid pineapple waste, 1–20% v/v) and temperature (30–38 �C). Standbyperiods of up to 10 days can be tolerated without loss in activity. A robust yet effective biotechnologyto remove chromium from wastewater is thus demonstrated.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The incomplete removal of chromium from industrial waste-waters is of concern due to its potential hazards. The Cr(VI) spe-cies is of particular importance as pollutant due to its persistent,stability and highly soluble nature. Chemical reduction followedby precipitation is the most common Cr(VI) treatment techniqueused in the industry (Cushnie, 1985). Such processes have its dis-advantages whereby large volumes of sludge are generated, therelease of obnoxious gases such as H2S and also high operatingcost. Cr(VI) is highly toxic to most organisms whereas Cr(III) isrelatively innocuous. In cellular systems, Cr(VI) is reduced toCr(III) by physiological reducing agents such as NAD(P)H andFADH2 whereby free radicals in the forms of reactive oxygen spe-cies are generated (Hojo et al., 2000). Microorganisms such asbacteria has long been reported to have the ability to reduceCr(VI) as one of its resistance mechanisms. Species such as Acine-tobacter sp., Pseudomonas sp., Arthrobacter rhombi, Bacillus coagu-lans, E. coli ATCC 33456 and Desulfomicrobium norvegicum weretested in various operational arrangements either in the sus-pended or attached systems (Elangovan and Philip, 2009). Theuse of fixed-film bioreactors for Cr(VI) reduction was first re-ported by Chirwa and Wang (1997) where a continuous-flow lab-oratory-scale biofilm reactor was demonstrated to reduce Cr(VI)

ll rights reserved.

60 7 5566162.ria).

without the need to constantly supply fresh Cr(VI) reducing cells.Ekenberg et al. (2005) reported the use of biofilm formed on plas-tic carriers to treat Cr(VI) containing leachate originating fromferrochromium slags where anaerobic column system was oper-ated in upward flow. Krishna and Philip (2005) reported theCr(VI) reduction via anaerobic condition carried out by biofilmformed on Fujino media consisting of Cr(VI)-resistant microbialconsortium during the treatment of leachate from Cr(VI)-contam-inated soil.

We have previously shown that laboratory-scale bioreactorsare capable of reducing Cr(VI) from industrial electroplatingwastewater whereas the resulting Cr(III) can be removed viacaustic precipitation technique (Zakaria et al., 2007). However,promising results obtained from the laboratory-scale does notnecessarily equates to success in large-scale operating conditions.This is because important operational parameters are normallycontrolled in the laboratory and huge fluctuations in the natureof the wastewater avoided. Hence, it is necessary to assess therobustness of the system at a larger scale where it will be sub-jected to fluctuations in wastewater parameters, as typicallyencountered in a factory compound. This study describe the inoc-ulation and operation of a Cr(VI)-reducing pilot-plant based onour laboratory results reported earlier (Zakaria et al., 2007). Sincethe period of the report, we have accommodated the system withan improved chromium removal techniques and the system beingtermed as the ChromeBac™ system (Ref: The Malaysian PatentOffice – 08022573).

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2. Methods

2.1. Design and operation of the pilot plant

The schematic diagram for the 0.2 m3 pilot plant of the Chrome-Bac™ system to remove chromium from industrial electroplatingwastewater is shown in Fig. 1.

In the 0.15 m3 mixing tank, raw Cr(VI) wastewater wasmixed with liquid pineapple waste (LPW) to a final LPW con-centration of between 1–20% (v/v). The pH of the mixturewas adjusted to 7.0 ± 0.5 using controller-regulated dosing of12.5% (v/v) NaOH prior to transferring into the 0.3 m3 holdingtank. Upon reaching the capacity of the holding tank, the neu-tralized and LPW amended Cr(VI) wastewater entered the0.2 m3 bioreactor gravitationally in a down flow mode at0.11–0.33 m3 h�1. Effluent collected from the bioreactor thenran through a flocculation (alum) and coagulation (anionicpolymer) setup where the Cr(III), total Cr, other heavy metals,color, odor and organic contents were removed from thewastewater. Sludge formed was dried at the sludge dryingbed prior to safe disposal to a local waste management com-pany, Kualiti Alam Sdn. Bhd, Bukit Nenas, Negeri Sembilan.The treated effluent was passed through powdered activatedcarbon units (Vtotal of 0.3 m3) prior to discharge to the nearbywater system. The Cr(VI) and total Cr values were always mon-itored at the bioreactor inlet, bioreactor outlet, treated effluentand final discharge effluent. Redox potential, pH and tempera-ture were monitored using electrode measurements at the bio-reactor inlet and outlet sections. Cr(VI) concentration wasdetermined using the DPC method (APHA 4500, Greenberget al. 2005) while total Cr was determined using the AAS spec-trophotometer (Perkin Elmer A Analyst 400). The organic con-tents were determined using the COD and BOD kits (HACH,USA) and measured at 620 nm using UV–vis spectrophotometer(HACH DR4000, DR5000). The treated effluent was protectedfrom the presence of Cr(VI) and total Cr by a bypass whichwas automatically triggered at positive ORP readings of thebioreactor outlet (+mV).

Fig. 1. Schematic representation of the experimental setup for the Cr(VI) reduction systemixing tank, D – holding tank, E – bioreactor, F – receiving tank, G – flocculation and cactivated carbon column.

2.2. Preparation of bacterial inoculum

Primary bacterium used to inoculate the bioreactor, Acinetobac-ter haemolyticus (A. haemolyticus), was isolated from the Cr(VI)-containing wastewater from a batek (textile-related) manufactur-ing premise in Kota Bharu, Kelantan, Malaysia (Zakaria et al.2007). The nucleotide sequence was deposited in GenBank andwas assigned with Accession No. of EF369508. Single colony waspicked from fresh nutrient agar plate and inoculated into 250 mLnutrient broth (NB) and grown for 24 h, 200 rpm at 30 �C. This seedculture was then inoculated into a fresh NB medium (2 L) andgrown for a further 24 h in the same condition. Then the 2 L cul-tures of A. haemolyticus were transferred into two 16 L fermenta-tion vessels containing 10 L of NB added with antifoaming agent.The fermentation was carried out for 16 h at 30 �C with an initialstirring rate of 100 rpm that increased gradually to 250 rpm in or-der to obtain oxygen saturation level of 20%. Neutral pH of the fer-mentation liquid was controlled using 1 M NaOH and 1 M H2SO4.Following this, the 20 L cultures were transferred into two 150 Lfermentation vessels containing 100 L of the NB medium. It wasfermented using similar operating conditions as the 20 L fermenta-tion. The resulting 200 L cultures were then transferred into a ster-ile 250 L polycarbonate container for immediate inoculation intothe bioreactor.

2.3. Inoculation of the bioreactor

The bioreactor was packed with rubber wood sawdust (RWS) ascarrier material which was first sieved through a 25 mm2 pores.The RWS has specific surface areas of 3.0025, 5.8345 and1.9806 m2 g�1 determined using the BET, Langmuir and SinglePoint (at P/Po = 0.2002) methods plus an average pore diameterof 694.03 nm. Upon packing inside the bioreactor, the RWS wasfirst rinsed using sufficient amount of tap water until a clear efflu-ent was obtained. This is to remove large particulate substancesfrom the RWS that may clog the bioreactor. Then, the bioreactorwas inoculated with 200 L bacterial cultures with continuouscirculation for 3 days at 0.33 m3 h�1 to allow initial bacterial

m using a 0.2 m3 bioreactor; A – nutrient tank, B – raw Cr(VI) wastewater tank, C –oagulation section, H – sludge drying bed, I – sludge collecting tank, J – powdered

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attachment. Then, 200 L of 20% (v/v) LPW in water (pH 7.0 ± 0.2)was pumped into the bioreactor for 3 days using similar flow rateas the bacterial inoculation, to aid in the early-stage formation ofthe biofilm. The spent medium was collected and disinfected priorto disposal.

2.4. Flocculation, coagulation and filtration of the bioreactor effluent

The bioreactor effluent/outlet collected in the receiving tankwas transferred into five 200 L drums, used as the precipitationtank. Cr(III) along with other metallic ions and organic compoundswas precipitated via pH adjustment, coagulation and flocculation.NaOH, 12.5% (v/v) was used to adjust the pH of the effluent to7.0 ± 0.2, prior to the addition of alum (flocculation) and anionicpolymer (coagulation). Various combinations of 0.5 kg L�1 alum(0.4–1.5 L) and 2.67 g L�1 of polymer (0.4–2.0 L) were used to meetthe effluent discharge limit as stipulated in the EnvironmentalQuality Act (1974) – Environmental Quality (Sewage and IndustrialEffluents) Regulations 1978 (Environmental Quality Act and Regu-lations Handbook, 1996). The amount of Cr and organic-rich pre-cipitates generated was estimated at 10–15% (v/v) of the effluenttreated. The treated effluent was then passed through the PACunits to remove remaining color and odor prior to discharge tothe nearby drainage system. Sludge obtained was then air-driedto a 35% dry weight prior to disposal at Kualiti Alam Sdn. Bhd.The sludge produced from the ChromeBac™ was compact andwas determined to have a density of 1.02 ± 0.03 kg m�3.

2.5. Analysis on biofilm developed inside the bioreactor

The Field Emission Scanning Electron Microscope (FESEM) wasused to determine the development of biofilm on RWS in the bio-reactor. Sample preparation is as follows; the biofilm-containingRWS was sampled from the middle section of the bioreactor after1 week, 1 and 2 months. The samples were immersed in 2.5% (v/v) glutaraldehyde for 1–2 h. It was then washed using deionisedwater before immersed in 2% (v/v) osmium tetraoxide (OsO4) in0.1 M Phosphate Buffer Saline (PBS) for about 1 h. It was againwashed with deionised water. The RWS was then dehydrated usingincreasing concentrations of absolute ethanol before overnightdrying in a desiccator. The dried RWS was then mounted on a sam-ple holder before viewing under the electron microscope (Hitachi4500, FESEM).

Besides this, the isolation of bacterial species from the biofilmwas also carried out using the culture enrichment technique. TheRWS sample was obtained from five different locations inside thebioreactor. It was then transferred (1 g) into 10 mL of sterilizeddeionised water. The mixture was sonicated (1 min, 50 W, 100 A),vortexed (1 min) and inoculated (2.5 mL) into 25 mL of fresh NBmedium and grown for 48 h, 200 rpm at 30 �C. The bacterial iso-lates were then transferred onto NA plates and incubated for24 h at 30 �C. Microbiological count was carried out and the singlecolonies obtained were also evaluated for its Cr(VI) reducing abilityusing 50 mg Cr(VI) L�1. The Cr(VI) reduction was assessed as fol-lows; the single colonies obtained were grown in a series of250 mL Erlenmeyer flasks containing 25 mL of NB for 12 h,200 rpm at 30 �C. Then, 50 mg Cr(VI) L�1 from the Cr(VI) stocksolution was added into the flasks and incubated further for 24 h,200 rpm at 30 �C. At the end of the assessment, the Cr(VI) wasdetermined using the DPC technique. Stock Cr(VI) solution wasprepared by dissolving 2.829 g K2Cr2O7 (294.18 g mol�1) in 1 L ofdeionised water. The pH of Cr(VI) solution was adjusted to 7.0using 0.1 M NaOH or 0.1 M HCl before filter-sterilized using a0.45 lm Whatman filter paper.

2.6. Determination of Cr(VI), data recording and analysis

The Cr(VI) concentration was determined colorimetrically at540 nm using the diphenylcarbazide (DPC) method with a detec-tion limit of 5 lg L�1. The method is as follows; in a 10 mL volu-metric flask, 1 mL of sample was mixed with 9 mL of 0.2 MH2SO4. Then 0.2 mL of freshly prepared 0.25% (w/v) DPC in acetonewas added to the volumetric flask. The mixture was then vortexed(Maxi Mix-II Thermolyne) for about 15–30 s and let to stand be-tween 10–15 min for full color development. The red–violet to pur-ple color formed was then measured at OD540 using distilled wateras reference. The instrument used was calibrated using 0.4–2.0 mg L�1 Cr(VI) prepared from Cr(VI) stock solution(1000 mg L�1). The data for pH, temperature and the ORP valueswere monitored by the DAQFactory process control software pack-age and stored on disk. Process data was then exported to Micro-soft Excel ‘07’ and processed by standard procedures. Thesoftware also offers rapid trouble-shooting options and quick refer-ence for operational parameters/conditions.

3. Results

3.1. Operation of the pilot-plant

A. haemolyticus was circulated for 3 days inside the bioreactor toallow initial bacterial attachment. This was followed by 3 days cir-culation of 20% (v/v) of LPW at pH 7.0 to enhance initial stage ofbiofilm formation. Fig. 2 shows the Cr(VI) concentrations for the in-let and outlet of the bioreactor after 2 months of bioreactor opera-tion i.e. bioreactor operating at full capacity.

The bioreactor was operated with a flowrate of 0.11–0.33 m3 h�1 depending on the concentrations of Cr(VI). The extentof Cr(VI) reduction by the bioreactor was monitored from the oscil-lations in the ORP values, which can roughly be correlated withCr(VI) concentrations. The bioreactor maintained its completeCr(VI) reducing capability even after 2 months of operation as de-picted in Fig. 2. This was indicated by the drop in the ORP valuesfrom positive (indicating the presence of more than 0.05 mg L�1

Cr(VI)) to negative values. All ORP measurements of the Cr(VI) val-ues were verified using the DPC technique. There were also in-stances where the ORP values were negative prior to entering thebioreactor (Fig. 3A and B).

This can be attributed to the abiotic reduction from the organicconstituents present in the LPW especially when more than 10% (v/v) of LPW was used in the wastewater mixture. Throughout thebioreactor operation, the inlet pH fluctuated irregularly between7.5 ± 1.0 while the outlet pH between 6.7 ± 0.5. The fluctuation inthe inlet pH was due to two factors namely the volume of thewastewater and the high concentration of NaOH i.e. 12.5% (v/v)used to neutralize the wastewater. Low fluctuation in Cr(VI) con-centrations (17–81 mg L�1) assisted in the exceptional Cr(VI)reduction by the bioreactor (Fig. 4).

Complete Cr(VI) reduction was achieved at the first Cr(VI) in-flow of 17 mg L�1 at 0.33 m3 h�1 without the need to recirculatethe Cr(VI) wastewater into the bioreactor. This shows fast start-up for the bioreactor i.e. complete Cr(VI) reduction was achievedimmediately after inoculation without the need to adapt the biore-actor’s content to the Cr(VI) wastewater. However, three cycleswas needed to completely reduce 72 mg Cr(VI) L�1 in the secondbatch of wastewater even though the flowrate was decreased to0.16 m3 h�1. Decreasing the incoming flowrate is normally appliedto enhance the Cr(VI) reduction process by increasing the contacttime between cells in the bioreactor and the wastewater contents(Cr(VI), DO, nutrients). With time, the number of cycles needed forCr(VI) reduction was reduced which eventually leads to a single

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Fig. 3. Cr(VI) reduction profiles of the bioreactor in the ChromeBac™ system; profiles shown are actual data recorded during the treatment of a particular batch of Cr(VI)wastewater after (A) – 1 week, early stage of operation (B) – 1 month, bioreactor operating near its full capacity; Cr(VI) concentrations is represented by the ORP (mV) values;� – ORP inlet, 4 – ORP outlet, h – pH inlet, X – pH outlet.

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cycle of treatment only (batch 5). Wastewater batches of 6 and 7demonstrates the full capacity of the bioreactor where althoughmore than 70 mg Cr(VI) L�1 was present in the incoming wastewa-ter, no recirculation was needed to completely convert Cr(VI) tothe undetectable level i.e. complete conversion to the Cr(III) forms.One thing worth mentioning is the ability of the bioreactor contentto withstand a long standby period without losing its Cr(VI) reduc-tion capacity. This is clearly demonstrated from the treatment ofwastewater batches of 9 and 10 where a 14 days gap was allowedbefore the 10th batch was pumped into the bioreactor. The 14 daysoperation gap was due to non-operation of the Cr(VI) wastewatersampling site. This condition is really advantageous from the oper-ation point of view as this would reduce the need to re-supply thebioreactor with fresh cells or having to increase nutrient supple-mentation to promote bacterial growth, after a long standby peri-od. Electron microscopy (FESEM) examinations on the formation ofbiofilm on RWS packed inside the bioreactor showed gradual colo-nization of the RWS with biofilm containing microorganisms invarious shapes and sizes. With time, biofilm formed may be

subjected to colonization by ubiquitous Cr(VI) resistant bacteriaand other microorganisms. At this time, it can be stated that theinvading microorganisms were beginning to have a role in the bio-film as evident from its good survival ability. This could explain thehigh Cr(VI) reduction capacity of the bioreactor. AAS analysis of theinlet and outlet fractions of the bioreactor after 1 week of opera-tion indicated the presence of high concentration of Cr in the outletfraction (Fig. 5).

Slight difference between total Cr (35 mg L�1) and Cr(VI)(42 mg L�1) in the inlet fraction can be explained from the highersensitivity of the DPC method to detect Cr(VI) compared to theAAS technique (for total Cr) which is prone to interference due tothe various detection wavelengths available. Initially, the decreasein the total Cr present in the outlet fraction (12.5 mg L�1) to the in-let fraction (35 mg L�1) indicates that the Cr species was adsorbedby the RWS packed inside the bioreactor. However, on other occa-sions, a higher total Cr value were detected in the outlet fractions(it is worthy to note that total Cr in the outlet fraction should con-sist of Cr(III) species only as no Cr(VI) species was detected). This

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shows that Cr binding by the RWS in the bioreactor has achievedits breakthrough point i.e. where most of the available Cr bindingsites on the RWS have been saturated.

3.2. Flocculation, coagulation and filtration of the bioreactor effluent

The bioreactor outlet fraction was treated using the coagula-tion/flocculation techniques to remove pollutants such as Cr, otherheavy metals and organic matters present in the wastewater priorto final treatment using the polishing unit. The coagulation stepwas carried out at a pH of 7.0 ± 0.2 using alum (0.5 kg L�1) with aratio of 0.8–2 L alum per 200 L of the outlet fraction. Upon the for-mation of fine particles in the wastewater, 0.4–1 L of anionic poly-mer (2.67 g L�1) was added to initiate the flocculation process.Flocs formed were let to settle for about 8 h before transferred into

the sludge settling tank. The treated wastewater was then passedthrough the powdered activated carbon (PAC) units to remove col-or, odor and remaining Cr and heavy metals present. Throughoutthe bioreactor run; no Cr(VI) was detected in the final dischargedwater, complete removal of total Cr after the coagulation/floccula-tion steps, more than 95% removal of organic matters (in the formsof COD and BOD) and satisfactory removal of color and odor afterpassing through the PAC units. Table 1 shows example for pollu-tants removal profile using the coagulation/flocculationtechniques:

The lower total Cr value (25.53 mg L�1) in the bioreactor outletfraction shows that substantial portions of Cr in the wastewaterwere retained by the column contents i.e. biofilm and RWS. Thehigh color reading (178) was due to the strong yellow–green ofthe LPW in the wastewater mixture. A slight increase in color

Table 1Profile for the removal of pollutants using the coagulation/flocculation at initial Cr(VI) in wastewater of 62.38 mg L�1.

Bioreactor outlet After coagulation/flocculation After PAC Final discharge Percent removal (%)

Cr(VI), mg L�1 0 0 0 0 100aTotal Cr, mg L�1 25.53 0.76 0.48 0.21 99.21Turbidity (FAU) 36 18 43 27 25Color (ADMI) 178 66 144 67 62.36pH – – – 7.22 –

a Total Cr is the sum of Cr(VI) and Cr(III) present in the solution; if no Cr(VI) was detected in a particular wastewater fraction, total Cr determined should constitute of Cr(III)species only.

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and turbidity after passing through the PAC unit can be attributedto the washing out of microparticles from the PAC itself. Examplefor the overall characteristics of the Cr(VI)-wastewater beforeand after treatment using the ChromeBac™ system is shown in Ta-ble 2.

4. Discussion

A number of viable process arrangements has been reported forthe application of microbial technology to reduce Cr(VI) to Cr(III)including aerobic suspended growth system, aerobic attachedgrowth system and anoxic attached growth system (Elangovanand Philip, 2009). From an industrial point of view, the continu-ous-flow and fixed-film bioreactors offers the most reliable modeof application due to its easiness of handling and simple operation.Hence, most of the studies are now directed towards the evalua-tion on the feasibility of the process at the pilot-scale level.Amongst reported studies are as shown in Table 3.

It is preferable to have a good ratio between concentration ofNaOH and the volume of wastewater to ensure well mixing ofthe wastewater mixture to the desired pH value. However duringthe bioreactor operation in this study, the ratio between the con-centration of NaOH and wastewater volume was huge where eventhe smallest dosage of NaOH would cause a tremendous change inthe pH values, which ultimately results in high fluctuation of theinlet pH. Wastewater recycling was needed at the early stage ofcolumn operation which reflects the immaturity of the biofilmcommunity in the bioreactor in terms of cell number and cell

Table 2Characteristics of the Cr(VI)-wastewater before and after treatment using theChromeBac™ system; all units in mg L�1; discharge limit according to MalaysianStandard B (industrial wastewater outside the catchments area).

Parameter Before treatment After treatment Discharge limit

Temperature (�C) 30 30 40pH 5.45 6.23 5.5–9.0BOD 3173 30 50COD 8692 70 100SS 71 36.5 100Hg <0.001 <0.001 0.05Cd <0.001 <0.001 0.02Cr(VI) 29.4 <0.05 0.05As <0.05 <0.05 0.1CN 0.06 <0.05 0.1Pb <0.05 <0.05 0.5Total Cr 49.8 0.70 1Cu 0.07 0.08 1Mn 0.32 0.15 1Ni <0.01 <0.01 1Sn 0.3 <0.1 1Zn 0.11 0.02 1B <0.2 <0.2 4Fe 0.61 0.2 5Phenol <0.02 <0.02 1Chlorine >1.0 <1 2Sulphide <0.2 <0.2 0.5O and G 2.4 3.8 10

survival. The low number of cells present was not expected to carryout complete Cr(VI) reduction due to the low Cr(VI) reduction rateof 2.26 lg Cr(VI) L�1 min�1 mg�1 cell dry wt. of A. haemolyticus asreported previously (Zakaria et al., 2007). Choice of LPW as nutri-ent for the microbial community inside the biofilm was madedue to its abundance and cheap pricing. Even though, other typesof nutrients source such as brown sugar also showed excellent roleto support bacterial growth in a biofilm system (Ahmad et al.2009), the dark coloration and high COD contents in the final efflu-ent off-set its potential application. The important role of the bio-film community inside the bioreactor to carry out the Cr(VI)reduction process can be substantiated from the following obser-vation; as many as 12 morphologically different bacterial colonieswere isolated using the culture enrichment technique from fivedifferent locations of the bioreactor. These colonies were then indi-vidually tested to reduce 50 mg Cr(VI) L�1 via the shake-flask tech-nique. Unfortunately, none of the isolated bacterial coloniesshowed encouraging Cr(VI) reduction ability with the highestbeing 8.2% only. The number of surviving cells in the solution alsodecreases to 102 CFU mL�1 from an inoculation cell concentrationof 108 CFU mL�1. Even though, four of the strains showed directmorphological resemblance to that of A. haemolyticus, furthergenotypic identification on the isolated bacterial strains was notcarried out as it was not within the scope of this study. However,initial works on the genotypic identifications of the bacterialstrains isolated form the biofilm and the influence of different bac-terial species/numbers presence in the biofilm on the overall Cr(VI)reduction performance are currently on-going.

At some points of the bioreactor operation, higher total Cr con-centration was recorded in the outlet fraction as opposed to theinlet fraction. This shows that Cr binding by the RWS in the bio-reactor has achieved its breakthrough point i.e. where most of theavailable Cr binding sites on the RWS have been saturated. Sincethe bioreactor operates in a continuous and dynamic condition,the RWS-bound Cr(III) may be displaced by counter ions suchas Cu(II) and Sn(II), with higher affinity towards binding sitesthan Cr(III), which was determined to be present in the wastewa-ter (data not shown). This can be viewed as advantageous to thesystem as this should ensure Cr will not be accumulated exces-sively by the RWS-biofilm system, hence not interfering withthe overall performance of the bioreactor. The extent of Cr accu-mulation by the biofilm is negligible due to the inability of theCr(III) ions to penetrate the bacterial cell membranes. Besidesthis, the washing-out of the biofilm fragments from the RWS sur-face in the bioreactor due to adverse operating conditions (fluctu-ating pH, Cr(VI), DO concentration, temperature, LPW) may alsocontribute towards the higher total Cr content in the outlet frac-tion. This was evident from the muddy appearance of the outletfractions observed throughout the bioreactor run. Therefore, it isestimated that the bioreactor system can be operated for a longperiod (much longer than the 3 months period as reported in thisstudy) provided performance limiting factors such as Cr(VI) con-centrations, adequate supply of dissolved oxygen and nutrientare controlled.

Table 3Cr(VI) reduction using the continuous-flow and fixed-film bioreactors at pilot-scale level.

Bioreactor Microbes used Time needed (h) Initial Cr(VI) (mg L�1) Cr(VI) reduction (%) Reference

Anaerobic Desulfomicrobium norvegicum 7 20 �100 Brunet et al. (2006)Anaerobic trickling filter Mixed cultures 11 10–20 �100 Ekenberg et al. (2005)Aerobic packed-bed Mixed cultures 3.3 5.5 100 Tziotzios et al. (2008)Aerobic trickling filter Acinetobacter sp. 19 30 100 Dermou et al. (2005)Aerobic trickling filter Mixed cultures 0.3–6 5–100 100 Dermou and Vayenas (2007)Aerobic packed-bed Acinetobacter haemolyticus 3–9 17–81 100 This study

W.A. Ahmad et al. / Bioresource Technology 101 (2010) 4371–4378 4377

In the solution, Cr(III) ions will form a complex with water mol-ecule in the form of hexaaquochromium (III) ion-[Cr(H2O)6]3+

which is fairly acidic in nature with a typical pH of 2–3 (Remoudakiet al., 2003). This ion will react further with water molecule whereone hydrogen ion is lost from one of the water ligand molecules(Eq. (1)):

½CrðH2OÞ6�3þ þH2O ! ½CrðH2OÞ5ðOHÞ�2þ þH3Oþ ð1Þ

The complex acts as an acid by donating a hydrogen ion towater molecules, acting as base, in the solution. The hexaaquochro-mium (III) ion is a ‘‘difficult to describe” violet–blue–grey com-pound. It is always nearly described as green being Cr(III),implying the hexaaquochromium (III) ion, which actually is anover-simplification. This is because the Cr(III) ions also have thecapability to form hexahydroxochromate (III) ion-[Cr(OH)6]3�,which is also green, in basic condition. As a result, Cr(III) ion spe-cies are pH dependent, where it will form different complexes atdifferent pH values. This knowledge is important during the Cr re-moval i.e. coagulation/flocculation process as this shall determinethe use of the most suitable precipitating agent to ensure a stableCr(III) compound is formed. In this study, the different forms of theCr(III) ions present in the bioreactor outlet fraction (violet–blue–grey or green) were not visually detected due to the low concentra-tions of Cr(VI) used i.e. 17–81 mg L�1. Besides, Cr(III) was deter-mined by taking the difference between total Cr and Cr(VI),measured using AAS and the DPC technique respectively. Bothmethods used were not capable of differentiating the differentforms of Cr(III) present in the solution.

Another important aspect to be considered during the Cr re-moval process is the presence of soluble organic matters (SOM)in the bioreactor outlet fraction. These SOMs may originate fromthe following sources namely (1) co-existing with Cr in the waste-water, (2) metabolically produced by the microbes during theCr(VI) reduction process and (3) excess of nutrients added intothe bioreactor. Analysis on the Cr(VI) wastewater showed no traceof organic matters hence, the first possible source can be dis-counted. In solution, the SOM exist in the anionic forms and willbe attracted to the cationic precipitating agent. In this study, pos-sible source for SOM may come not only from glucose, sucrose orfructose but also the unknown organic matter co-existing in theLPW. The presence of organic matters would complicate the pro-cess of coagulation because of its complex structure such as highnegative charge and wide range of molecular weight. Part of the or-ganic matter is dissolved and is unlikely to provide nucleation sitefor the formation of particles. Two important mechanisms nor-mally associated during the organic matter removal process arecharge neutralization and adsorption (Yan et al., 2008). It is alsoestablished that the formation of complexes between organic mat-ters and Cr(III) alters the solubility behaviour of Cr(III), resulting inthe inhibition of Cr precipitation. This was evident from traces of Crpresent in the after coagulation/flocculation, after PAC and finaldischarge fractions. This can be explained as follows; Cr(III) formcomplexes with virtually any species capable of donating electronpairs. The complex formed may be anionic, cationic or neutral andnormally in the hexacoordinate or octahedral coordination. Due to

the octahedral d3 electron configuration, the Cr(III) complexes be-comes inert. Ligand substitution and rearrangement reactions aretoo slow (half time in the order of h). Therefore, if the Cr(III) con-centration is the same or more than the available organic matters,no changes in the overall Cr(III) solubility is expected. However, ifexcess organic matters are present in the solution, the formation ofCr(III) complexes is observed, and Cr precipitation is hindered.

No Cr(VI) was detected throughout the various pollutants re-moval steps indicating a number of conditions namely (1) completeconversion of Cr(VI) to Cr(III) by the bioreactor, (2) the stability ofthe Cr(III) compounds formed and (3) neither the presence of or-ganic and inorganic materials in the LPW-wastewater mixture northe use of alum and polymer contributed towards a highly reducingcondition that may re-oxidize the Cr(III) back to Cr(VI).

5. Conclusion

The technical feasibility of the locally developed Cr(VI) removalsystem i.e. the ChromeBac™ system was demonstrated at the 200 Lpilot-plant scale. The high percentage conversion of Cr(VI) to Cr(III)suggests the feasibility of using a bacterial system as an alternativetreatment for Cr(VI) contamination in the aqueous system. Theability of Acinetobacter haemolyticus, as a primary Cr(VI)-reducingmicroorganisms in the bioreactor, to utilize liquid pineapplewastewater as a nutrient is an excellent example of the substitu-tion of a cheap and readily available industrial waste in place ofexpensive growth medium and could be a significant factor inthe commercial use of a process such this.

Acknowledgements

The authors acknowledge the contributions by Mr. Yahya(FKKKSA) for the clarifier and Mr. Jefri Samin (FKM) for the excel-lent FESEM works. A special note of thanks to Marlini and Nurfadi-lah (for lab analysis) and Mr. Saufi and Mr. J.K. Lee for setting up ofthe pilot-plant. This work is supported by the Ministry of Science,Technology and Innovation (MOSTI), Malaysia under the Techno-fund grant (TF0106B001).

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