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Article history:Received 17 January 2014Received in revised form29 March 2014Accepted 9 April 2014Available online 23 May 2014

most of the industrial wastewaters contain oil-in-water emulsionswhich can lead to severe problems in the different treatmentstages. The presence of O&G in water treatment units will causefouling in process equipment, complication in water discharge re-quirements and problems in biological treatment stages (Ahmadet al., 2006).

available includeel-plate coalesces,media ltration,ery few of theseeet the increasinge expensive initialdevelop effective,nologies that can

serve as an alternative to existing treatment systems. One exampleof current application of alternative treatment system is theponding system which is also the most common treatment systemused by Malaysian palm oil mills to treat POME. One of the mostattractive features for the ponding system is the low capital costwhich can be attributed to the limited requirement for mechanicalmixing, operational control and monitoring (Yacob et al., 2009).This system consists of a series of specically built ponds includingthe anaerobic and facultative ponds incorporated with physico-* Corresponding author.

Contents lists available at ScienceDirect

International Biodeterior

.e

International Biodeterioration & Biodegradation 95 (2014) 33e40E-mail address: [email protected] (Z.A. Zakaria).1. Introduction

The discharge of oil and grease (O&G) containing wastewater tothe environment increases every year due to rapid urbanizationand industrial development. Major industrial sources of oilywastewater include petroleum reneries, metal manufacturing andmachining, food processors, electronic and electrical and palm oilmill efuent (POME). Unlike free or oating oil spilled in the sea,

Amongst existing treatment technologieschemical coagulation, gravity separation, parallgas oatation, cyclone separation, granularmicroltration and ultraltration. However, vtechnologies provide satisfactory solution to mstringent water quality regulations as well as thand operating cost. This makes it imperative toeconomical and sustainable O&G treatment techKeywords:OilGreaseBacteriaBiosurfactantDegradationhttp://dx.doi.org/10.1016/j.ibiod.2014.04.0090964-8305/ 2014 Elsevier Ltd. All rights reserved.a b s t r a c t

The potential of oil and grease (O&G)-degrading ability of three local bacterial isolates was evaluatedusing wastewaters obtained from food processing, electrical and electronic and oil palm (POME) in-dustries. These bacteria were chosen based on its high bacterial adherence to hydrocarbon (BATH),culture turbidity and maximum biosurfactant production (BSF) capabilities. From the 16S rRNA analysis,the food-processing isolate was identied and deposited in GenBank as Serratia marcescens EU555434,electrical & electronic (Aeromonas hydrophila KF049214) and POME (Bacillus cereus KJ605415). Prior toevaluation for its O&G degradation ability (effect of contact time, different concentrations of wastewater,pH and initial organic loading rate), S. marcescens was adapted in used cooking oil while B. cereus inPOME. S. marcescens, with the highest BSF and BATH values, showed maximum oil and grease degra-dation ability (91%) at pH 7.0 after 12 days of incubation and initial organic loading rate of 1.46 101 kg O&G l1 day1. For B. cereus, 100% (v/v) of POME (3012 mg l1 oil and grease) was degraded after 7days of incubation at 200 rpm, 30 C and pH 6 while A. hydrophila was able to degrade 100% (v/v) of4.88 mg l1 of O&G from the electronic wastewater, supplemented with tryptone and lactose after only2 h of incubation at 200 rpm, 30 C at pH 7.0. The role of tryptone and lactose in complete biodegradationof O&G by A. hydrophila is signicant as neither the addition of tryptone or lactose only resulted inenhanced O&G degradation, compared to E&E wastewater only. This nding showed the potential ofusing local aerobic bacterial isolates as an alternative solution to remove the presence of O&G in variousindustrial wastewaters.

2014 Elsevier Ltd. All rights reserved.bDepartment of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, MalaysiaDegradation of oil and grease from highusing locally isolated aerobic biosurfact

Iezzat Emeer Affandi a, Nur Haizarisha Suratman b,Wan Azlina Ahmad b, Zainul Akmar Zakaria a,*a Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 UTM Johor

journal homepage: wwwtrength industrial efuentst-producing bacteria

akilah Abdullah b,

ru, Johor, Malaysia

ation & Biodegradation

lsevier .com/locate/ ibiod

ioratchemical and biological treatments, respectively (Tong and Jaafar,2004; Vijayaraghavan et al., 2007).

Recent study showed that the removal of Chemical OxygenDemand (COD) as well as O&G by aerobic oxidation was higher inanaerobically digested POME as compared to diluted raw POME atHydraulic Retention Time (HRT) of 60 h (Vijayaraghavan et al.,2007). Single culture such as Acinetobacter sp. (KUL8), Bacillus sp.(KUL39) and Pseudomonas sp. (KLB1) showed higher O&G removalcapacity compared to mixed culture (Bhumibhamon et al., 2002).Lipase producing bacteria such as Bacillus subtilis, Bacillus lichen-iformis, Bacillus amyloliquefaciens, Serratia marcescens, Pseudomonasaeruginosa and Staphylococcus aureus has also been reported as apotent agent for lipid degradation from O&G-containing waste-waters such as those emanating from palm oil mill, dairy, slaughterhouse and soap industries (Prasad and Manjunath, 2011). The aimof this study was to investigate the feasibility of using indigenousbacteria to remove O&G in specic industrial wastewater namelyPOME, Electrical & Electronic, Food Processing Industries waste-water in bench-scale laboratory treatment system.

2. Materials and methods

2.1. Industrial wastewater

The O&G wastewater was obtained from the WastewaterTreatment Plant section of three different industries located in theJohor Bahru district, Johor, Malaysia. Each industry has been iden-tied to incorporate O&G in some stages of their operation namelypalm oil mills (mixing pond, POME), electrical and electronic in-dustry, E&E (discharge point) and the food industry (dischargepoint). The wastewater samples were collected in clean and ster-ilized glass containers and the pH of the samples were adjusted topH< 2.0 with either 1 MHCl or 1 MNaOH (to prevent abiotic redoxreaction process that might change the chemical speciation in in-dustrial efuents) and transported to the laboratory using refrig-erated ice chests in dark condition (APHA, 2005). Each wastewatersample was then immediately stored at 4 C prior to use. Theaverage O&G contents in the wastewater was determined to bebetween 1886 and 14,510 mg l1 for POME, 1.45e10 mg l1 (E&E)and 180e79,000 mg l1 (food industry).

2.2. Isolation and screening of O&G degrading bacteria

The O&G degrading bacteria were isolated from POME, E&E andfood industry wastewaters using the following procedures: 2.5 mlof each wastewaters were transferred into a series of 250 ml ofErlenmeyer asks containing 22.5 ml of Nutrient Broth, NB (8 g l1,Merck) and incubated at 30 C, 200 rpm for 24 h (Certomat, B.Braun). Then, one loopful of each bacterial culture broth wasinoculated onto Nutrient Agar, NA plates (20 g l1, Merck) followedby overnight incubation at 30 C (Memmert, USA). The bacterialcolony was then sub-cultured onto fresh NA plates using similarprocedures until a pure culture was obtained.

For POME, the isolated bacteria were further screened for O&Gdegradation properties (lipolytic activity) using the Tween 80peptone agar (Plou et al., 1998). The selected bacterial isolates wereevaluated for its cell surface hydrophobicity properties using thebacterial adherence to hydrocarbon (BATH) test (Rosenberg et al.,1980). The BATH tests were carried out using early exponentialphase cells which were rst harvested by centrifugation at9500 rpm for 5 min (Allegra 25-R Centrifuge, Beckman Coulter)and resuspended in 0.01 M potassium phosphate buffer (pH 7.0) toachieve OD600 value of 0.5. Then, 0.5 ml of either palm oil orparafn oil was added into 5 ml of the cell suspension and vortexed

I.E. Affandi et al. / International Biodeter34for 30 s (Thermolyne Mixer II) and let to stand for 15 min. Theabsorbance of the aqueous phase was recorded at 600 nm and thepercentage of microbial adhesion to substrate was calculated asfollows:

1OD600of aqueousphaseafter theadditionof substrateOD600of initialphasebefore theadditionof substrate

100%

(1)

All 25 bacterial isolates from the E&E wastewater were proledfor growth, cell concentration (CFUml1) and culture turbidity(OD600) using the following methods: one loopful of respective24 h-old bacterial colony was inoculated into a series of 250 mlErlenmeyer asks containing 25 ml of NB. This was followed by24 h incubation at 200 rpm and 30 C where bacterial isolates withthe shortest time to reach early stationary phase and OD600 valuesof greater than 1.0, were earmarked for subsequent experiments.From this evaluation, four bacterial isolates denoted as isolate A8,B1, B6 and B7 were selected for subsequent studies. Bacterial strainwhich showed good growth in minimal basal salt medium withhighest cell count was chosen as potential bacteria to degrade O&Gsince it has capability to grow well in low minimal medium. Thecomposition for the minimal basal salt medium is as follows:(NH4)2SO4 (3.0 g l1, Univar), MgSO4$7H2O (0.5 g l1, Merck),K2HPO4 (0.5 g l1, BDH), KCl (0.1 g l1, Merck) and yeast extract(0.1 g l1, Oxoid). One isolate each from respectivewastewaters thatshows highest turbidity and fastest growthwas identied using the16S rRNA analysis which was carried out by First BASE LaboratoriesSdn. Bhd., Malaysia and Ktrade Enterprise, Malaysia.

For the food industry wastewater, all 10 isolated strains showedgood growth with OD600 of more than 1.0. One of the isolates, i.e.isolate A was evaluated for its lipid-degrading ability using Tween80 peptone agar (peptonee 10 g l1, NaCle 5 g l1, CaCl2e 0.1 g l1,Tween 80 e 5 ml and agar e 18 g l1) where formation of opaquezone around microbial colonies indicates lipolytic activity. Isolate Awas adapted in 1, 3 and 5% (v/v) of cooking oil at 30 C and 200 rpm(Certomat, B. Braun) for 24 hwhile isolates X7 and X10was adaptedin 100% (v/v) of POME in 2 days. Isolates X10, B1 and A weremaintained in LuriaeBertani glycerol medium and stored at 4 C forbiodegradation study.

2.3. Biosurfactant production and activity

The biosurfactant production ability of the bacterial isolate wasevaluated using isolate A by inoculating 5 ml of a 24 h-old culturebroth of isolate A in 500 ml Erlenmeyer ask containing 50 mlmixture of minimal medium (3 g l1 of (NH4)2SO4, 0.5 g l1 KH2PO4,0.1 g l1 KCl, 0.5 g l1 MgSO4$7H2O) and 5% v/v of cooking oil. Themixture was then incubated for 12 h, 30 C and 200 rpm and har-vested at 10,000 rpm, 20 min and 4 C. The resulting supernatantwas extracted with chloroform and methanol (2:1 v/v). The extractwas concentrated using rotary evaporator and determined forbiosurfactant activity using the following procedure (Matsumiyaet al., 2007); 30 ml of the concentrated extract was pipetted ontothe center of a series of petri dishes containing amixture of 50ml ofdistilled water (DW) and 100 ml of cooking oil. One unit of bio-surfactant activity (U) is dened as the diameter of the clearingzone formed after 1 min from the addition of surfactant. The oildisplacement test is an indirect measurement of surface activity ofa biosurfactant sample tested against oil where a larger diameterrepresents a higher surface activity of the test solution.

2.4. O&G biodegradation study

The O&G biodegradation study using different industrial

ion & Biodegradation 95 (2014) 33e40wastewaters was carried out as follows: isolate A (10% v/v) was

The O&G content was measured using the oil and grease

4500 SEM) and TEM (Tecnai G2, Philips) at Institute for MedicalResearch (IMR), Kuala Lumpur, Malaysia.

3. Results and discussion

3.1. Isolation of microorganisms

For the electric and electronic (E&E) industry wastewater, 25distinct bacterial colonies were successfully isolated on NB and NA.Two isolates (isolate X7 and X10) were determined to have thelipolytic activity after screening with Tween Peptone Agar. Onebacterial isolate, i.e. isolate B1 showed the fastest growth in NBwith OD600 of 1.23 after 2 h of contact time as well as good growth(1.66 108 CFUml1 after 24 h) in salt minimal medium (MM). Theisolate is a gram- negative, forming slightly creamy, glistening andraised colonies when grown for 24 h at 30 C on NA.

From the 10 initially isolated bacteria from the food industryefuent (FIE), isolate A showed the highest ability to grow in a

ioration & Biodegradation 95 (2014) 33e40 35determination method as described in APHA2005-5520B (APHA,2005), with n-hexane used as the oil-extraction solvent. The O&Gcontent in the suspension was determined for each sample beforeand after experiment. All tests were carried out at an ambienttemperature of 25 C. All results were analyzed for standard devi-ation (s), which measures the condence interval and variation asdescribed by Eq. (2):

sx Xn

i1

xi x2n 1

vuut (2)

2.5. Electron microscopy analysis

The morphology of isolate A grown in a mixture of 0.5 mL ofcooking oil in 5 mL phosphate buffer was analyzed using theScanning Electron Microscope (SEM) and Transmission ElectronMicroscope (TEM). Sample preparation for SEMwas prepared usingmodied procedure from that described by Zakaria et al. (2007) asfollows: the bacterial cell pellets were obtained by centrifugation at9500 rpm, 4 C for 30 min. The cell pellets were xed in 2.5% (v/v)glutaraldehyde (in phosphate buffer, pH 7.4) for 1e2 h and washedtwice with deionized water. It was then post-xed using 2% (v/v)osmium tetroxide (Fluka, Switzerland) for 1 h and again washedwith deionized water. The cell pellets were subsequently dehy-drated using increasing concentration of ethanol (10%, 30%, 50%,70%, and 100% v/v; 5 min each) and left to dry overnight at 70e80 C in a desiccator.

For the TEM analysis, the ethanol-dehydrated cells wereembedded in 50% and 100% (v/v) of epoxy resin in acetone (Q rc)for 15 min each. Then, the cell pellet was inltrated a second timewith fresh 100% (v/v) epoxy resin and cured at 60 C overnight.Specimens of 90 nm thickness were sectioned from the embeddedblocks using a Leica UltraCut UCT ultramictrotome andmounted on200-mesh copper TEM grids. The specimens were stained withuranyl acetate and post-stained with lead-citrate for 5 min each(Southam et al., 2001; Chaerun et al., 2004). All samples weretransferred into 90 ml of food industry wastewater, isolate B1 (10%v/v) in 90 ml of E&E wastewater and isolate X10 (20% v/v) in 80 mlof POME in a series of 1 l Erlenmeyer asks followed by shaking at200 rpm in an orbital shaker (Certomat M, B Braun, Germany) at30 C for 24 h. POME was sterilized for 15 min at 121 C (200 kPa)prior to use. The bacterial culture broth was sampled at designatedtime intervals (4 h e isolate B1, 7 days e isolate X10 and 12 days eisolate A) and determined for residual O&G contents. Similarexperimental setup was carried out in the following studies: (1)effect of various pH of wastewaters (pH 3e11, adjusted using either1.0 M NaOH or 1.0 M HCl) where samples were analyzed every 2 hfor 4 h (for the E&E wastewater), every 24 h for 7 days (for POME)and every 24 h for 12 days, for the food industry wastewater; (2)effect of nutrient supplementation on O&G degradation whereisolate B1 (10% v/v) was cultivated in E&E wastewater (80 ml) thatwas added with 1 g l1 of either glucose, succinic acid, lactose,tryptone, urea, L-alanine, (NH4)2SO4 or NH4NO3, at pH 7 andmonitored for 4 h and (3) effect of initial organic loading rate (OLR)where isolate A was grown for 12 days in the presence of either1.08 101 kg O&G l1 day1 or 1.46 101 kg O&G l1 day ofcooking oil while 20% (v/v) of isolate X10 was mixed with 20%, 40%,60%, 80% and 100% (v/v) of POME and monitored for 7 days. At theend of the studies, cell concentration (CFUml1) and O&G con-centration were determined. The experiments were carried out intriplicates.

I.E. Affandi et al. / International Biodetermaintained in a desiccator prior to viewing under SEM (Hitachi S-nutrient-limiting environment (Minimal Medium) with OD600 ofmore than 1.0 after 24 h of incubation compared to other isolates.Apart from this feature, isolate A was also the only isolate showinglipolytic activity (distinct formation of opaque zone when grown inTween 80 peptone agar) as well as showing good growth whengrown in 5% (v/v) of cooking oil with cell concentration of108 CFU ml1. Tween, which is the fatty acid esters of polyoxy-ethylene sorbiton, is the preferred substrate for the detection oflipolytic microorganisms in agar media (Sierra, 1957) with Tween80 (the oleic acid monoester of polyoxyethylene sorbitan) being themost widely used compound. It is based on the precipitation (ascalcium salt) of the fatty acid resulting from the hydrolysis ofTween (Plou et al., 1998). The ability of microorganism to hydrolyzeoleic acid is a direct indication for its potential use to degrade O&G-containing wastewater such as POME which is rich in palmitic acidand oleic acid contents (Wu et al., 2009). Result from the 16S rRNAgene sequence analysis is shown in Table 1, hence isolate B1 wastermed as Aeromonas hydrophila (UTM2), isolate A as S. marcescens(C 19320) and isolate X10 as Bacillus cereus (UTM6). The completesequence was deposited in GenBank under Accession number ofKF049214, EU555434 and KJ605415, respectively.

3.2. Characterization of microorganisms

3.2.1. Bacterial adherence to hydrocarbonThe bacterial adherence to hydrocarbon (BATH) was carried out

to determine the degree of cells hydrophobicity toward

Table 1Identication of isolate B1, X10 and A by 16S rRNA gene sequence analysis.

Isolate Species as close relatives Accession no. Percentsimilarity

B1 Aeromonas hydrophila sub sp. dhakensi NR_042155.1 99%Aeromonas hydrophila sub sp. hydrophila NR_074841.1 99%Aeromonas hydrophila NR_043638.1 99%Aeromonas jandaei NR_037013.2 99%

X10 Bacillus cereus strain B234 KF494192.1 99%Bacillus cereus strain 2Y-2 FJ493043.1 99%Bacillus cereus strain YLB-P5 KF376341.1 99%Bacillus cereus strain B236 KF494193.1 99%Bacillus sp. LT3 FJ932655.1 99%Bacillus cereus strain XZM002 FJ932655.1 99%

A Serratia marcescens strain AB244453 99%Serratia marcescens strain AB244433 99%Serratia marcescens strain AB244291 99%Serratia nematophila strain EU036987 99%Serratia marcescens EF415649 99%

Serratia marcescens strain AB270613 99%

iorathydrocarbon. The BATH index is important to evaluate the afnityof a particular bacterial strain to carry out any hydrocarbondegradation process. In this study, B. cereus (UTM6) showed thehighest BATH capacity with a value of 84.51% followed byA. hydrophila (UTM2) (22.86%). The higher hydrocarbon afnity forB. cereus (UTM6) can be attributed to its original location of isola-tion, i.e. POME which contains high concentration of O&Gcompared to A. hydrophila (UTM2) which was isolated from the E&Ewastewater with lower O&G concentration. Being originallyadapted to live in high O&G concentration environment, B. cereus(UTM6) is anticipated to have cell surface properties that wouldallowmigration of hydrocarbon into the inner bacterial cells regionwhere degradation process would take place (Chang and Su, 2002).The use of oil-adapted cells was successful in increasing BATH valuefor bacterial cells as demonstrated by S. marcescens (C 19320)where oil-adapted cells of S. marcescens (C 19320) showed a BATHvalue of 81% compared to the non- adapted cells (44% only). Theformation of distinct white layer at the interface between oil andaqueous layers clearly indicate the afnity of S. marcescens (C19320) toward oil-phase, hence its high BATH value. By introducingan environmental isolate such as S. marcescens (C 19320) into anoil-rich solution, it is anticipated that the fatty acid component onthe cell membrane would be increased. This condition proceedsdue to the ability of the bacterial cells to change its membraneuidity by isomerization of the fatty acids contents from cis totrans-unsaturated fatty acids. The secretion of biosurfactant of thecell surface directly assisted the adhesion of isolates onto the oildroplets.

3.2.2. Production of biosurfactantDue to its highest BATH value, S. marcescens (C 19320) was

further evaluated for its biosurfactant activity using cooking oil assubstrate. Formation of smaller oil droplets at the early stationaryphase of growth indicates initial point for the secretion of bio-surfactant (data not shown). Control set (not inoculated with bac-teria) showed oil droplets with a much bigger diameter andnumbering much less. The crude biosurfactant was also evaluatedusing the oil displacement test where the results clearly indicate amuch superior biosurfactant activity for the crude biosurfactant ofS. marcescens C 19320 with a value of 380 42.4 Uml1 comparedto the commercial surfactant, Tween 80 (79 0.93 Uml1). Controlset (consisted of a mixture of cooking oil-water) did not show anybiosurfactant activity. Higher biosurfactant activity would result in alarger and faster rate of clearing zone formation. Biosurfactantexcreted by the bacteria assisted in the emulsication and encap-sulation of the oil in smaller droplets by reducing the surface tensionof the oil (Kosaric, 2001). This actually, increases the rate of organicsubstrate dissolution, hence its utilization by the isolate(Papanikolaou and Aggelis, 2011). High biosurfactant activity isdesirable in oil and grease biodegradation process as high bio-surfactant activity would result in higher surface area of hydro-phobic water-insoluble substrates followed by increasing thebioavailability of hydrophobic compounds, hence increasing theoverall oil and grease degradationwaste (Ron and Rosenberg, 2002).

SEM analysis of S. marcescens (C 19320) (after a 24 h cultivationin the presence of oil droplets) showed marked changes in the cellmorphology, formation of macropores and cell disintegrationresulting in the formation of exopolymer matrix that would lead tointerconnection of cells into intricate network of coherent mass(Fig. 1). The exopolymer matrices shown in Fig. 1b (arrow) can beattributed to the excretion of biosurfactant by the cells as a meanfor cells anchorage to oil droplets as well as to allow degradationprocess to take place. From the TEM micrographs, translucentglobules of oil droplets (100e200 nm) were clearly discernible at

I.E. Affandi et al. / International Biodeter36the inner region S. marcescens (C 19320) grown in the presence ofoil droplets (Fig. 1a). None of these globules are present in bacterialcells grown without the presence of oil droplet (Fig. 2b).

3.3. Biodegradation of oil and grease

3.3.1. Effect of contact time and concentrationA. hydrophila (UTM2) showed the ability to carry out complete

degradation of O&G (initial concentration of 5.186mg l1) from E&Ewastewater after 4 h of incubation at 30 C. High cell concentrationsof more than 108 CFU ml1 maintained throughout the biodegra-dation study indicate good adaptability of the cells to grow in an oilenvironment (Fig. 3). An interesting point to note is that eventhough A. hydrophila (UTM2) was isolated from a low O&G con-centration environment (1.5e10 mg l1 in E&E wastewater), itshows high adaptation ability to survive and utilize O&G inwastewater containing O&G indicating its potential to be appliedfor biodegradation process.

For the food industry wastewater, S. marcescens (C 19320)showed maximum O&G removal capacity of 89% after 12 days ofincubation at pH 7.0 and 5% (v/v) of cooking oil (Fig. 4). The oilremoval increased gradually with increasing cell concentrations.After 2 days of incubation, 39% of oil was degraded, followed by 72%(4 days), 80% (6 days), 84% (8 days), 85% (10 days) and 89% (12days). High cell concentration of around 108 CFU ml1 was alsomaintained throughout the study. The complexity of compoundspresent in cooking oil induces the excretion of either lipase orbiosurfactant to facilitate the uptake process of the complexnutrient into the cell (Dumore and Mukhopadhyay, 2012).

For POME, B. cereus (UTM6) showed the ability to carry outhigher O&G degradation with increasing POME concentration.More than 90% of O&G were removed from all concentrations ofPOME used (20e100% v/v) with complete removal achieved for20%, 80% and 100% (v/v) of POME after 6 days of contact time at200 rpm and 30 C. A point to note is the ability of B. cereus (UTM6)to degrade O&G even at the highest concentration used, i.e.100% (v/v) POME (correspond to 14,510 mg l1) compared to both 20% (v/v)and 80%(v/v) of POME. As shown in Fig. 5, the cell concentrationsalso remained high throughout the experiment with a value of3.25 108 CFU ml1 (at 100% O&G removal) and1.88 109 CFU ml1 for 40% (v/v) of POME (lowest O&G removal at90.53%). Even though this study has clearly demonstrated the roleof B. cereus (UTM6) in carrying out the O&G degradation process byusing pre-sterilized POME, more studies need to be carried outusing ltered (to remove suspended solids) but non-sterilizedPOME to evaluate potential role of indigenous microbes in O&Gdegradation. This would give better indications on the feasibility ofusing B. cereus (UTM6) for eld application. This is because in non-sterile POME, there is always the possibility of indigenous micro-organisms in POME to proliferate and participate in the O&Gdegradation process as well as consuming the nutrients present(Salihu and Alam, 2012). This situation may or may not work to theadvantage of B. cereus (UTM6) in any attempts to evaluate its O&Gdegrading ability at a bigger scale (open treatment systems/eldapplication) where the effect of other environmental factors such asuctuation in pH, temperature, possible introduction of other pol-lutants to the aqueous environment would be much higher.

Similar condition was observed for the O&G degradation fromthe food industry wastewater with highest O&G removal of 90%was obtained at the highest organic loading rate used, i.e.146 mgO&G l1 day1. This is followed by 85% and 82% removal forOLR of 1.20 101 kg O&G l1 day1 and1.08 101 kg O&G l1 day1, respectively (Fig. 6). Moreover, theinitial OLR can also be related to HRT, thus a good balance betweenthese two parameters has to be obtained for good degradation

ion & Biodegradation 95 (2014) 33e40operation (Khemkhao et al., 2011).

Fig. 1. SEM micrographs of (a) Serratia marcescens (C 19320) without oil (control) and (b) S. marcescens (C 19320) in the presence of oil.

I.E. Affandi et al. / International Biodeterioration & Biodegradation 95 (2014) 33e40 373.3.2. Effect of pHpH plays a very important role in the O&G biodegradation

process. This was clearly demonstrated in this study where com-plete O&G degradation from POME was determined to occur atinitial pH of 6.0, 6.5 and 7.0 whereas insignicant O&G degradation(less than 10%) was recorded at initial pH values of less than 5.5 i.e.pH 5.5 and 5.0, indicated by the absent of surviving cells (Fig. 7).This situation can be directly correlated with the amount of cellconcentration present in the POME where for complete O&Gdegradation, CFU values of more than 108 CFU ml1 were alwayspresent whereas for the insignicant O&G degradation (less than10%), the cell concentrations were less than 105 CFU ml1 Ahmad

Fig. 2. TEM micrographs of S. marcescens (C 19320) growing (a) in the presence of oil anddroplets.

Fig. 3. Biotic and abiotic degradation of O&G in electric and electronic wastewater byAeromonas hydrophila (UTM2).et al. (2009) suggested that minimum cell concentration of105 CFU ml1 is required for efcient bacterial-based bioremedia-tion process. The presence of high concentrations of oil in POMEresulted in very low water activity as well as low pH that directlyaffect bacterial survival (Nwuche and Ogbonna, 2011).

Similar condition was also recorded for the O&G biodegradationfrom the E&E wastewater by A. hydrophila (UTM2) with 100%degradation achieved at initial pH 7.0 (Fig. 8). Increasing ordecreasing the initial pH of the E&E wastewater resulted in uc-tuation of the O&G degradation values between 90% (pH 11) to 75%(pH 3). Nevertheless, this high O&G degradation values can be

(b) without oil droplets; arrow indicates the presence of translucent globules of oil

Fig. 4. Percentage removal of O&G and bacterial survival during biodegradation of foodindustry efuent at different time intervals.

ioration & Biodegradation 95 (2014) 33e40I.E. Affandi et al. / International Biodeter38attributed to the alteration of the O&G structures due to the low/high pH values that would allow abiotic degradation process tooccur (Leahy and Colwell, 1990). Verstraete et al. (1976) reportedthe increase in the degradation rate of gasoline in soil when the pH

Fig. 5. Effect of different POME concentrations on O&G removal and CFUml1 byBacillus cereus (UTM6): (a) 20%, (b) 40%, (c) 60%, (d) 80% and (e) 100%.

Fig. 6. Effect of initial OLR on the removal of oil and grease for S. marcescens (C 19320)during biodegradation of food industry efuent.

Fig. 7. Effect of pH on O&G degradation and cell concentration of B. cereus (UTM6): (a)pH 6, (b) pH 6.5, and (c) pH 7.

resulted in gradual reduction in O&G removal capacity as follows:pH 7.0e77%, pH 6.0e72% and pH 5.0e71% (Fig. 9). Together with thehigh concentration of lipid in the food industry wastewater and thefact that optimum pH for lipase production and activity is 8.0, thisshould directly explain pH 8.0 being the optimum pH for O&Gdegradation by S. marcescens (C 19320). A point to note is the op-timum pH of 8.0 that may prove advantageous for the survival ofbacterial cells during real application where a low pH (due to theaccumulation of high VFA concentration) has been reported toinhibit bacterial growth (Stafford, 1982) resulting in drastic reduc-tion of O&G removal level and possible reactor failure. Thus, theconcentration of volatile fatty acids (VFA) should also be used asone of the parameters to be monitored during bioreactor operation(Buyukkamaci and Filibeli, 2004).

3.3.3. Effect of nutrient supplementationThe percentage of O&G degradation by A. hydrophila (UTM2) in

the presence of carbon and nitrogen supplementation is shown inFig. 10. The addition of either 1 g l1 of lactose, tryptone, (NH4)2SO4

Fig. 8. Effect of pH for oil and grease degradation in electrical and electronicwastewater.

I.E. Affandi et al. / International Biodeterioration & Biodegradation 95 (2014) 33e40 39was increased from 4.5 to 7.4. However, the degradation valuedropped signicantly when the pH increased to pH 8.5.

pH 8.0 was determined as the optimum pH for O&G degradationof food industry wastewater by S. marcescens (C 19320) withhighest O&G removal of 91%. However, decrease in pH values

Fig. 9. Effect of pH on the removal of oil and grease for S. marcescens (C 19320) duringbiodegradation of synthetic food industry efuent.

Fig. 10. Percentage of O&G degradation by Aeromonas hydrophila (Uor succinic acid into E&E wastewater resulted in complete removalof O&G with high concentration of surviving cell between 107 and109 CFU ml1. Other carbon and nitrogen sources supplementationresulted in high O&G degradation, i.e. 91% (NH4NO3), 96% (L-alanine), 94% (urea) and 88% (glucose). Control experiments(without the addition of bacterial cells) showed minimal role ofabiotic degradation with values ranging from 1.33 to 12.96%.Various studies reported the effect of carbon supplementation to-ward the production of biosurfactant by bacteria which ultimatelyincreased the biodegradation process (Nitschke et al., 2005; Goudaet al., 2007; Abouseoud et al., 2007). However, Gouda et al. (2007)reported that the addition of glucose as a co-substrate stronglyinhibits the degradation of kerosene by Gordonia sp. DM. Similarsituation was reported by Leahy and Colwell (1990) where thesupplementation of nutrient would limit the microbial degradationof hydrocarbons.

4. Conclusion

This study demonstrates the potential application of a bacterial-based O&G removal system as a cost-effective and environmental-friendly process. The ability of the bacterial isolates to breakdownO&G is advantageous as this would limit the need to supplementthe system with the normally expensive complex or syntheticTM2) in the presence of carbon and nitrogen supplementation.

growth medium. Nevertheless, more studies need to be carried outsuch as evaluation of the continuous/pilot-scale before any at-tempts on introduction of the system to the industry.

Acknowledgments

The authors would like to thank the Ministry of Higher Educa-tion (MOHE), Malaysia for the MyMaster scholarship award toIezzat Emeer Affandi and Nur Haizarisha Suratman. Our sinceregratitude also to Universiti Teknologi Malaysia for the ResearchUniversity Grant (03H84, 02H84) and Universiti Tun Hussien Onn(UTHM) for the SLAI scheme to Shakilah Abdullah.

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Degradation of oil and grease from high-strength industrial effluents using locally isolated aerobic biosurfactant-producin ...1 Introduction2 Materials and methods2.1 Industrial wastewater2.2 Isolation and screening of O&G degrading bacteria2.3 Biosurfactant production and activity2.4 O&G biodegradation study2.5 Electron microscopy analysis

3 Results and discussion3.1 Isolation of microorganisms3.2 Characterization of microorganisms3.2.1 Bacterial adherence to hydrocarbon3.2.2 Production of biosurfactant

3.3 Biodegradation of oil and grease3.3.1 Effect of contact time and concentration3.3.2 Effect of pH3.3.3 Effect of nutrient supplementation

4 ConclusionAcknowledgmentsReferences