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International Biodeterioration & Biodegradation 80 (2013) 1e9

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Batch studies with Exiguobacterium aurantiacum degradingstructurally diverse organic compounds and its potential fortreatment of biomass gasification wastewater

Hansa Jeswani, Suparna Mukherji*

Centre for Environmental Science and Engineering (CESE), Indian Institute of Technology, Bombay, Powai, Mumbai 400076, India

a r t i c l e i n f o

Article history:Received 14 May 2012Received in revised form7 February 2013Accepted 8 February 2013Available online 15 March 2013

Keywords:Exiguobacterium aurantiacumPolynuclear aromatic hydrocarbonsN-heterocyclicsPhenolExopolymeric substancesMonod kinetics

* Corresponding author. Tel.: þ91 022 2576 7854; fE-mail address: mitras@iitb.ac.in (S. Mukherji).

0964-8305/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibiod.2013.02.002

a b s t r a c t

Biomass gasification wastewater primarily consists of phenolics, nitrogen-heterocyclics and polynucleararomatic hydrocarbons (PAHs). Biodegradation of these compounds present individually as sole substrateis studied in batch cultures in presence of Exiguobacterium aurantiacum. It demonstrated good potentialfor degrading phenol, pyridine, quinoline, benzene and naphthalene present at initial concentration (Cin)of 500 mg l�1. E. aurantiacum could also utilize 3- and 4-ring PAHs, phenanthrene, fluoranthene andpyrene as sole substrate present at 100 mg l�1 (Cin). While significant increase in absorbance wasobserved on 3 and 4-ring PAHs, the increase in number concentration of viable cells and extent ofdegradation was relatively low. E. aurantiacum could effectively degrade a synthetic biomass gasifierwastewater comprised of these compounds with a total COD of 1326 mg l�1 and biokinetic studiesrevealed applicability of Monod’s kinetics for culture growth on gasifier wastewater. The half velocityconstant (Ks) and maximum specific growth rate (mmax) were 651 mg l�1 and 1.86 d�1, respectively. Allcomponents in the wastewater were degraded simultaneously and compounds with comparable con-centration depicted comparable degradation rates. E. aurantiacum could degrade the organics in biomassgasification wastewater even when the ammoniacal-nitrogen concentration was increased up to1000 mg l�1. Bioaugmentation with E. aurantiacum can significantly enhance biological treatment ofbiomass gasification wastewater.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Biomass gasification wastewater is produced by wet scrubbingmethods employed to clean producer gas from gasifiers. The char-acteristics of tar and wastewater generated are dependent on thetype of biomass, gasifier configuration, temperature of the gasifierand oxygen content in the gasifiers (Devi et al., 2003). The targenerated during biomass gasification is known to consist of phe-nols, cresols, heterocylics and aromatics ranging from single to 5ring polynuclear aromatic hydrocarbons (PAHs), i.e., benzene toperylene (Kinoshita et al., 1994). The constituents in wastewateroriginate from the tar through solubilization during the wetscrubbing operation and presence of phenolics, nitrogen-heterocyclics and PAHs (Tian et al., 2006) has been reported. Manyof these constituents are known to be carcinogenic and mutagenic(Samanta et al., 2002). The wastewater generated from biomass

ax: þ91 022 2576 4650.

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gasification has chemical oxygen demand (COD) ranging from920 mg l�1 to 160,000 mg l�1. Generally for smaller installationswith no recycling of scrubbing water the COD lies between920 mg l�1 to 4000 mg l�1 (Lata et al., 2006) while larger in-stallations where scrubbing water is recycled can havemuch higherCOD (Jansen et al., 2002). Moreover, ammoniacal nitrogen contentin biomass gasifier wastewater is often high and is reported to varybetween 242 and 16,000 mg l�1 (Maxham and Wakamiya, 1980;Lata et al., 2006; Tian et al., 2006). Treatment of wastewater be-comes essential due to the presence of various toxic and xenobioticcompounds such as phenolics, heterocyclics and high molecularweight PAHs (HMW PAHs). Physicochemical treatment usingcoagulation, flocculation and adsorption on activated carbon is re-ported to yield good removal efficiency (Mehta and Chavan, 2009).Biological treatment in suspended and attached growth aerobic andanaerobic processes such as activated sludge process and upflowanaerobic sludge blanket (UASB) process have also been attempted.COD removal efficiency varying from 50 to 85% is reported over theHRT range 1e4.5 days (Maxham and Wakamiya, 1980; Lata et al.,2006; Tian et al., 2006; Graber et al., 2009).

H. Jeswani, S. Mukherji / International Biodeterioration & Biodegradation 80 (2013) 1e92

Exiguobaterium aurantiacum is an alkaliphilic extremophile(Collins et al., 1983). Extremophiles can be used in various appli-cations due to the presence of rare enzymatic activities whichenable them to gain resistance to xenobiotics; to produce expoly-meric substances; and to produce specific metabolites for detoxi-fying metal compounds (Kalin et al., 2005). Environmentalbiotechnologists are particularly interested in extremophilic mi-croorganisms as they can degrade toxic components present in theenvironment under extreme conditions (Kalin et al., 2005; Brakstadand Bonaunet, 2006; Takeuchi and Sugio, 2006). E. aurantiacum hasbeen reported to degrade aliphatic hydrocarbons and phenol(Mohanty and Mukherji, 2008a). It has a tendency to form longchains and is pleomorphic (Collins et al., 1983). Specific strains ofE. aurantiacum have been used to bring down the pH of highlyalkaline industrial wastewater from pH of 12 and above to theneutral range (Kulshreshtha et al., 2010); to biodegrade pesticidessuch as simazine, atrazine, methyl parathion (Lopez et al., 2005);and is also reported to transform arsenate to arsenite (Andersonand Cook, 2004).

Biodegradation of nitrogen heterocylics and two and three ringsPAHs have been reported for a microbial mixed consortia andPseudomonas sp. (Foght and Westlake, 1988; Liu et al., 1992). Bur-kholderia sp. and Mycobacterium vanbaaleni (Chain et al., 2006;Nam et al., 2006) have been reported to produce multiple dioxy-genases, degrading heterocylics. However, the degradation of het-erocylics and PAHs by E. aurantiacum has not been reported earlier.In the present study, the potential of E. aurantiacum to degrade awide variety of compounds including heterocyclics, phenolics andPAHs was first explored as single component in batch cultures.Based on its potential for degrading these xenobiotic compoundsacross various group types, it was applied for the degradation of asimulated biomass gasifier wastewater containing a combination ofthese compounds. A study was conducted for determining thebiokinetics for degradation of this wastewater by E. aurantiacum.These studies were conducted to reveal the potential for using thisculture for bioaugmentation during biological treatment of gasifierwastewater.

2. Materials and methods

2.1. Chemicals

Naphthalene, phenanthrene, fluoranthene, pyrene and nutrientbroth were procured from Sigma Aldrich (Mumbai, India). Quino-line, pyridine, benzene and phenol were obtained from Merck Ltd.(Mumbai, India) and were of analytical grade. The nutrients inbacterial mineral media were either purchased from S. D. FineChem. Ltd. (Mumbai, India) or Merck Ltd. (Mumbai, India) andwere of analytical grade. The source of osmium tetra-oxide andglutraldehyde used for scanning and transmission electron mi-croscopy was Himedia, Mumbai, India. HPLC grade methanol anddichloromethane (DCM) was obtained from Merck (Mumbai,India).

2.2. Bacterial cultures

The strain of E. aurantiacum (AS1) used was earlier reported tobe capable of utilizing aliphatic hydrocarbons and diesel oil as solesource of carbon and energy (Mohanty andMukherji, 2008a). It wasisolated from oil contaminated soil obtained from a tanker refuel-ing station in an airport using diesel as sole substrate (Mumbai,India). The culture identified based on 16S rDNA analysis (Banga-lore Genei, Bangalore, India) and has been submitted to IMTECHChandigarh (MTCC 5638).

2.3. Culture maintenance

E. aurantiacum was maintained on mineral medium (Mukherjiet al., 2004) supplemented with 150 mg l�1pyrene provided assole substrate. Pyrene dissolved in DCM was first added to theconical flask (500 ml) and DCM was allowed to evaporate over 8 hat room temperature before addition of 100 ml mineral medium.Subsequently, 5% (v/v) inoculum of E. aurantiacum culture grownup to end of log phase and adjusted to unit absorbance was addedto each flask. The flasks were maintained in a rotary shaker (TrishulEquipments, Thane) at 30 �C and 120 rpm.

2.4. Bacterial growth studies and extent of degradation

E. aurantiacum was grown individually on components of syn-thetic wastewater, i.e., phenol, quinoline, pyridine, benzene, naph-thalene, phenanthrene, fluoranthene and pyrene provided as solesubstrate. The initial concentration used was 500mg l�1 for phenol,heterocyclic compounds, benzene and naphthalene. At 500 mg l�1

total concentration, much of the naphthalene remained insoluble,while the other compounds were completely soluble. For phenan-threne, fluoranthene and pyrene, the initial concentration used was100mg l�1.Much of these substrates remained insoluble since thesePAHs have extremely low aqueous solubility. The extent of degra-dation at end of log phase was quantified using HPLC analysis.

For growth and degradation studies with naphthalene, phen-anthrene, fluoranthene and pyrene the substratewas added to eachflask after dissolving in DCM, and the solvent was subsequentlyallowed to evaporate completely at room temperature beforeadding themineral medium (100ml). The experimental flasks wereinoculated with 5% E. aurantiacum having absorbance of unity at600 nm. No inoculum was added to the control flasks. The flaskswere incubated in a rotary shaker at 30 �C and 120 rpm. At regularinterval of time, 3 mL aliquot was sampled from each flask forgrowth measurements and an identical volume of sterile mineralmedium was replenished into the flask so as to maintain constantvolume in the culture broth. For each sample, the absorbance at600 nm was measured using a visible spectrophotometer (Spec-tronic 20, Genesys, USA) and the number concentration of viablecells was also determined. Multiple batch flasks were set up simi-larly for the degradation studies. Residual PAH in the flasks wereanalyzed by sacrificing the flasks at the desired time interval andextracting with DCM (2:1) as suggested by Mohanty and Mukherji(2012). The mixture was centrifuged for 20 min at 12000 rpm in acooling centrifuge at 4 �C (C24, REMI, Mumbai), the organic phasewas separated and passed through sodium sulfate. Subsequently,DCMwas removed by evaporation at 60 �C and the same volume ofmethanol was added.

For growth and degradation studies with phenol, heterocyclicsand benzene the substrate was directly added to mineral mediumbefore inoculating and incubating the cultures as discussed above.To determine the extent of degradation, residual concentrationwasestimated in the beginning and at the end of the log growth phaseafter removing the cells by centrifugation and filtration through0.2 mmMillipore filter. Later 50%methanol was added to the filteredsamples, and HPLC analysis was performed.

The %loss in the experimental flasks and in the control flaskswith respect to the initial concentration (Cin) was determined. Allstudies were conducted in duplicate and the error bars representstandard error.

2.5. Growth kinetics on synthetic wastewater

For the batch biokinetic study, a nutrient supplemented syn-thetic wastewater was prepared by adding phenol (250 mg l�1),

H. Jeswani, S. Mukherji / International Biodeterioration & Biodegradation 80 (2013) 1e9 3

pyridine (280 mg l�1), quinoline (280 mg l�1), benzene(200 mg l�1), naphthalene (60 mg l�1), phenanthrene (0.5 mg l�1),fluoranthene (0.2 mg l�1) and pyrene (0.12 mg l�1) to the mineralmedium. The synthetic wastewater was formulated using compo-nents that are reported for typical wood based gasifiers (Tian et al.,2006; component characteristics are provided in supplementarymaterial, Table S1). The total COD was about 1326 mg l�1.E. aurantiacum was sub-cultured twice on phenol (500 mg l�1) asthe carbon source before starting up the experiment. The culturewas centrifuged, pelleted and washed twice in 2% phosphate bufferand adjusted to unit absorbance at 600 nm. Duplicate flasks withvarying dilutions of the synthetic wastewater were inoculated withit. The COD (S, mg l�1), absorbance at 600 nm and number con-centration of viable cells (N, CFU/ml) were noted at varying timeintervals up to 84 h. The number concentration of viable cells wereobtained by plating on nutrient agar plates.

A correlation was developed between number concentration ofviable cells (N, CFU ml�1) and volatile suspended solids (VSS, Xmg l�1, determined using standard methods, APHA, 1998) and be-tween absorbance and VSS (X, mg l�1). At each dilution of thesynthetic wastewater, linear regression was performed forln(XXo�1) vs time data over the log growth phase to obtain thespecific growth rate (m) (h�1). Here N and X are number concen-tration of viable cells (CFU ml�1) and VSS (mg l�1) at a particulartime point and No and Xo are the corresponding values at timet ¼ 0. Linear regression was performed using m�1 versus S�1 datagenerated experimentally and values of the half velocity constant(Ks mg l�1) and mmax (h�1) based on Monod’s model were deter-mined using the slope and intercept values. A m versus ‘S’ plot wasused to test the goodness of fit between the Monod’s model andexperimental m values. Yield coefficient was determined as theslope of a plot of DX versus DS, where increase in biomass (DX) anddecrease in COD (DS) was determined over the log growth phase foreach dilution of the synthetic wastewater. The biokinetic parame-ters were expressed in terms of COD rather than in terms of ulti-mately degradable organics.

The biokinetic study was repeated to determine the removal ofindividual components over time in the synthetic wastewater usingHPLC analysis. For eachwastewater dilution, the degradation rate ofeach component was determined by linear regression over theactive phase.

2.6. Tolerance to ammoniacal nitrogen

Tolerance of E. aurantiacum for ammoniacal nitrogen wasstudied since biomass gasifier wastewater is reported to containhigh amounts of ammoniacal nitrogen. Duplicate flasks containingsynthetic wastewater, mineral nutrients and varying amount ofammonium chloride 564 mg l�1, 705 mg l�1, 846 mg l�1 and1410 mg l�1 were inoculated with E. aurantiacum culture. COD,Kjeldhal nitrogen (ammoniacal nitrogen and organic nitrogen),nitrate and nitrite nitrogen (APHA, 1998) were measured after in-cubation in a rotary shaker for 7 days.

2.7. Analytical methods

HPLC analysis (Jasco, Japan) was performed using a C18 RestekPinnacle II PAH columnwith pore size 110�A, particle size 4 mm, anddimension 300 mm (L) � 4.6 mm (ID). The volume of sampleinjected was 20 ml. Phenol, pyridine, quinoline and benzene weredetected using a diode array detector (DAD) set at 254 nmwhereasnaphthalene, phenanthrene, fluoranthene and pyrene weredetected using a fluorescence detector (FLD). An excitation wave-length of 254 nm and emission wavelength of 350 nmwas used fordetection of naphthalene and excitation and emission wavelength

of 240 nm and 420 nm, respectively, was used for detection ofphenanthrene, fluoranthene and pyrene. For degradation studieswith individual components of gasifier wastewater, the flow ratewas maintained as 1 ml/min and an isocratic methanol-watergradient elution was employed, i.e., 100:0 for insoluble organicsand 80:20 for soluble organics. Gain in FLD was set as 1. Theretention time for naphthalene, phenanthrene, fluoranthene andpyrene were 5.8, 6.6, 7.2 and 8.1 min, respectively. The retentiontime for phenol, pyridine, quinoline and benzene were 1.8, 2.5, 2.4,2.8 min, respectively. For the study with synthetic wastewaterrequiring simultaneous separation and quantification of all thecomponents, the flow rate was reduced to 0.8 ml/min and themethanol-water gradient elution was altered as follows: 80:20 at0 min, 90:10 at 7 min and 100% methanol at 9 min. Initially theexcitation and emission wavelength in FLD were set for naphtha-lene while after 10 min they were altered for detection of the otherPAHs. Similarly the gain was set at 8 initially and was increased to16 after the first 10 min. The retention time of phenol, pyridine,quinoline, benzene, naphthalene, phenanthrene, fluoranthene andpyrene were 3.8, 4.2, 4.8, 5.2, 7.8, 12.8, 14.6, 15.1 min, respectively.The total run timewas 18min. Concentration of various compoundsin the samples was quantified based on external standards ofknown concentration. Good linearity was observed in the standardcurve for each of the components (R2 � 0.92; R20.05 � 0.77 wherethe degrees of freedom (n) � 3, R20.05 is the critical value of R2 for95% confidence level). The detection limits for phenol pyridine,quinoline, benzene, naphthalene, phenanthrene, fluoranthene andpyrenewere 0.75mg l�1, 1.6 mg l�1, 1.6 mg l�1, 0.6 mg l�1,1.2 mg l�1,0.003 mg l�1, 0.006 mg l�1 and 0.0036 mg l�1 respectively.

For quantification of VSS gravimetric method was used whereasfor COD the closed reflux method was performed using a CODdigester (HACH, Germany). Ammoniacal nitrogen and organic ni-trogen was determined by Kjeldahl distillation. Nitrate nitrogenand nitrite nitrogen were determined using the brucine sulphatemethod and diazotization method, respectively. These parameterswere analyzed as described in Standard Methods (APHA, 1998) andstandard error was determined based on duplicate analysis.

3. Results

3.1. Growth and degradation study

Growth studies of E. aurantiacum on individual componentspresent in synthetic wastewater provided as sole substrate werecarried out using 500 mg l�1 phenol, quinoline, benzene, naph-thalene, pyridine and benzene. Growth was measured as absor-bance at 600 nm (Supplementary material, Fig. S1) and by platecounting. The results are illustrated in Fig. 1a. The inoculum for allthe growth studies was grown on pyrene (100mg l�1) up to the endof log phase. E. aurantiacum did not exhibit any lag phase in pres-ence of phenol (500 mg l�1) and naphthalene (500 mg l�1) assubstrate. It exhibited the highest growth rate on phenol andnaphthalene and the end of log phase occurred at around 24 h. Forgrowth on naphthalene, accumulation of a yellow colored inter-mediate was observed during the initial 24 h. HPLC analysis usingfluorescence detector indicated an additional peak in the aqueousphase which eluted at 4.8 minwhile naphthalene eluted at 5.8 min.The growth on pyridine and quinoline were almost comparable andend of log phase occurred after 80 h. A sudden drop of CFU countwas observed for growth on pyridine at the end of 108 h. Forbenzene, there was a lag phase of 24 h and the end of log phaseoccurred at 64 h. There was a drop in CFU after 84 h, however theabsorbance remained constant. At the end of log phase, Ln(NNo�1)was least for growth on benzene. A good correlation (R2 � 0.94,R20.05 ¼ 0.9 where n ¼ 2) was observed between Ln (NNo�1) and

Table 1Extent of degradation of individual compounds by E. aurantiacum.

Sr. No. Compound Initialconcentration(Cin, mg l�1)

Timeduration

Finalconcentration(Cfe, mg l�1)

Bioticloss (%)

Abioticloss (%)

1 Phenol 500 72 h 23.1 (�10) 91.9 3.53 Pyridine 500 96 h 89.4 (0.0) 76.9 4.34 Quinoline 500 84 h 100.3 (�0.9) 79.9 12.55 Benzene 500 96 h 100.2 (�1) 80 9.26 Naphthalene 500 60 h 38.2 (�0.4) 72.5 18.57 Phenanthrene 100 14 days 61.9 (�3) 25.7 12.48 Fluoranthene 100 14 days 58.5 (�0.2) 23.1 18.49 Pyrene 100 14 days 65.9 (�1.5) 16.8 17.3

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Fig. 1. Batch growth of E. aurantiacum on (a) 500 mg l�1 of phenol, naphthalene,quinoline, pyridine and benzene and (b) 100 mg l�1 of phenanthrene, fluoranthene andpyrene provided as sole substrate.

H. Jeswani, S. Mukherji / International Biodeterioration & Biodegradation 80 (2013) 1e94

absorbance in the log growth phase for the various compounds(supplementary material, Fig. S2a). A correlation could not bedeveloped for phenol and naphthalene due to insufficient mea-surements in the log growth phase. For some compounds, relativelyhigher absorbance at 600 nm was observed possibly due to inter-ference caused by formation of intermediates. Growth studies withfluoranthene, phenanthrene and pyrene were conducted using100 mg l�1of each substrate as depicted in Fig. 1b. Growth onphenanthrene and pyrene was much lower than on fluoranthene.The Ln(NNo�1) versus absorbance relationship derived for thesePAHs in the log growth phase also showed good correlation(R2 � 0.93, R20.05 � 0.9 where n � 2) (Fig. S2b). No visible colordevelopment was observed for growth on these PAHs. For growthon the 3e4 ring PAHs the increase in Ln(NNo�1) was significantlylower than for the other compounds although the increase inabsorbance was comparable to those of the other compounds. Therelatively high absorbance may be due to loss of viability of cellsgrown on the PAHs.

The extent of degradation over the log growth phase was deter-mined for each individual compound provided as sole substrateand the results are shown in Table 1. The overall loss observed forphenol, quinoline, pyridine, benzene and naphthalene was in arange of 79e96% whereas for 3e4 ring PAHs overall loss was in therange of 34e42%. In many cases the aqueous samples diluted inmethanol showed additional peaks indicating intermediate metab-olite accumulation. An additional peak was observed during batchdegradation of phenol, pyridine and quinoline. With DCM extractedsample an additional peak was observed for naphthalene while noadditional peakswere observed for phenanthrene,fluoranthene andpyrene. Table 1 and Fig.1a,b indicate that E. aurantiacum is capable of

utilizing all these compounds across various group types as solesubstrate.

Transmission electron microscopic (TEM) images indicatedpresence of extracellular polymeric substances surrounding thecells and demonstrated their tendency to form chains. Negativestaining with India ink indicated capsule formation around the cellgrown on phenol and pyrene.

3.2. Batch biokinetic study for E. aurantiacum on synthetic biomassgasifier wastewater

The relationship between VSS (X, mg l�1) and number concen-tration of viable cells (N, CFU ml�1) for E. aurantiacum grown onsynthetic wastewater containing phenol, N-heterocyclics and 1e4ring aromatic hydrocarbons was obtained as shown in Fig. 2a.Biomass growth and COD reduction was monitored for 5 dilutionsof synthetic wastewater (1326, 1152, 864, 504, 288 mg l�1 of COD).The COD reduction profile and growth profile were obtained forvarious dilutions of synthetic wastewater (supplementary Fig. S3).COD reduction in the log growth phase was accompanied by in-crease in VSS. The percentage COD reduction in 68 h for1326 mg l�1, 1152 mg l�1, 864 mg l�1, 504 mg l�1, and 288 mg l�1

initial COD values (S) were 92.4%, 90.2%, 86.6%, 90% and 70%,respectively. Growth measured using number concentration ofviable cells (N) was converted to VSS (X, mg l�1) based on a cor-relation and plots of Ln(XXo�1) versus time corresponding tovarious dilutions were used to determine the specific growth rate(m) (h�1) using data corresponding to the log growth phase. Linearregression was performed using m�1 vs S�1 data (Fig. 2b) and thehalf velocity constant (Ks) and maximum specific growth rate(mmax) based onMonod’s Model was determined from the slope andintercept. Good linearity (R2 ¼ 0.998, R20.05¼ 0.77 where n¼ 3) wasobserved in the m�1 vs S�1 plot. Based on the slope and interceptvalues, mmax was determined as 0.077 h�1 or 1.86 d�1 and Ks wasdetermined as 651mg l�1. These values were used to determine thegoodness of fit to Monod’s model as observed for growth ofE. aurantiacum on synthetic wastewater by plotting model gener-ated m and the experimentally derived m versus COD (S) as depictedin Fig. 2c. The standard error in mmax was observed as 0.15 d�1 andthat for Ks was observed as 15 mg l�1. The yield coefficient wasobtained as 0.416 (SE ¼ 0.043) mg-VSS mg-COD�1 using a plot ofbiomass generated in the log growth phase (X-Xo) to COD reductionover the same period (S-So) across the various dilutions. The VSSwas determined based on a VSS versus Ln(NNo�1) correlation thatwas developed. Although absorbance at 600 nm was measuredthese values were not used for determining biomass. When log-growth phase data across the various dilutions of syntheticwastewater was pooled and Ln(NNo�1) versus absorbance wasplotted, good linearity was not observed over the entire range.Thus, for degradation of gasifier wastewater with a diverse range ofcompounds across various group types, absorbance cannot serve as

y = 2E-06x + 32.586R² = 0.9516

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Fig. 2. Batch biokinetic study for E. aurantiacum on synthetic wastewater.

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a direct measure of microbial growth. The single componentstudies also revealed that the slope of Ln(NNo�1) versus absorbanceover the log growth phase changed as the type of substrate wasaltered (Fig. S2aeb).

The component decay profiles were obtained for variouswastewater dilutions as shown in Fig. 3. All components weresimultaneously degraded by this culture. The substrate utilizationrate (-dSidt�1) corresponding to various substrate concentrationwas determined over the active phase for each component. Theactive phase was between 0 and 3 days for all components otherthan naphthalene and phenanthrene where it was between 0 and 2days. The substrate utilization rate for each component was foundto increase linearly with increase in component concentration.Thus, the dependence of utilization rate of specific components tocomponent concentration could not be fitted to Monod’s model.The utilization rate of various components present at comparableconcentration in the gasifier wastewater was similar as depicted inFig. 4aeb. A good correlation (R2 ¼ 0.89, R20.05 ¼ 0.197 wheren ¼ 18) was observed between substrate utilization rate and sub-strate concentration for naphthalene, benzene, quinoline, pyridineand phenol over the concentration range 15e280 mg l�1. Thus, theutilization rate of these components in gasifier wastewaternormalized to concentration is a constant and is w0.2 � 0.017(mean � SE) d�1. A good correlation (R2 ¼ 0.985, R20.05 ¼ 0.33where n ¼ 10) was also observed across the three and four ringHMW PAHs, phenanthrene, fluoranthene and pyrene over theconcentration range (0.03e0.5 mg l�1). The degradation rate ofHMW PAHs normalized to concentration was 0.477 � 0.019 d�1.Phenol removal over seven days was in the range of 89e92% in thevarious dilutions. Removal of pyridine, quinoline, and benzenewere in the range of 83e100%, 87e100% and 82e100%, respectively,over the seven day duration of the study. Removal of the PAHs,naphthalene, phenanthrene, fluoranthene and pyrene were in therange of 84e96%, 95e99%, 83e96% and 84e93%, respectively, over

seven days in the various dilutions. Thus, E. aurantiacum couldeffectively degrade all the components in gasifier wastewater.

3.3. Tolerance to ammoniacal nitrogen

Ammoniacal nitrogen levels are often high in gasifier waste-waters. Hence the effect of increasing ammoniacal nitrogen con-centration on COD removal by E. aurantiacum was studied. Thecarbon source was same as in synthetic wastewater consisting ofphenol, heterocyclics and PAHs in all the flasks. COD, ammoniacalnitrogen along with organic nitrogen, nitrate and nitrite nitrogenwere measured at the end of 7 days. Fig. 4a depicts %COD removalversus the ammoniacal nitrogen concentration. Linear regressionrevealed a distinct drop in %COD removal as the ammoniacal ni-trogen concentration increased; however, the drop was only 1% forevery 100 mg l�1 increase in NH4eN. An ANOVAwas carried out tostudy the effect of change of ammoniacal nitrogen on COD removalefficiency using Statistica 8.1. The p-value obtained was 0.46thereby suggesting that there is no significant effect of ammoniacalnitrogen on COD removal efficiency. Hence, it can be concluded thatE. aurantiacum can tolerate high concentrations of ammoniacalnitrogen and its COD removal capacity is only marginally affectedby high nitrogen levels. Generation of NO�

3 was less than 25 mg l�1

for the maximum NH4eN concentration and NO�2 accumulation

was not observed for any of the samples. A good correlation(R2 ¼ 0.984, R20.05 ¼ 0.77 where n ¼ 3) was observed between finalNH4eN and organic nitrogen concentration and initial NH4eNconcentration across the samples (Fig. 5).

4. Discussion

E. aurantiacum could use all compounds as sole source of carbonand energy as indicated by significant increase in absorbance andnumber concentration of viable cells compared to control. The

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100%_WW 75%_WW 50%_WW 25%_WW

(g)

0.00

0.04

0.08

0.12

0.16

0 2 4 6 8

Pyr

ene

(mg

l-1)

Time (d)

100%_WW 75%_WW 50%_WW 25%_WW

(h)

Fig. 3. Degradation of specific components in various dilutions of synthetic wastewater (a) phenol (b) pyridine (c) quinoline (d) benzene (e) naphthalene (f) phenanthrene (g)fluoranthene and (h) pyrene.

H. Jeswani, S. Mukherji / International Biodeterioration & Biodegradation 80 (2013) 1e96

growth rate is seen to vary depending on the growth substrate.Growth rate on 3 and 4 ring PAHs was low as compared to growthrate on monoaromatic, 2-ring PAH, heterocyclics and phenolicswhich are characterized by higher aqueous solubility. Trend invariation of absorbance versus time appeared linear rather thanexponential over the entire range for fluoranthene (Fig. S1). Suchlinear growth on PAH crystals have been reported due to masstransfer limitations (Volkering et al., 1992). The relatively highabsorbance together with only a small increase in number con-centration of viable cells observed at end of log phase after culturegrowth on 3 and 4 ring PAHs may have been due to loss of cellviability due to toxic effect of these compounds and their degra-dation products on the cells. The results illustrated in Fig. 1 andTable 1 highlights that although this culture can utilize 3 and 4-ringPAHs as sole substrate, the degradation rate of these PAHs andculture growth on these PAHs are much lower than other culturesreported in the literature (Mukherji and Ghosh, 2012). In contrast,this culture shows high growth rate on phenol and naphthaleneand intermediate growth rate on pyridine, quinolone and benzenealong with good degradation of these compounds within 60e96 h.While this culture may not be recommended for removal of

wastewater containing only 3e4 ring PAHs, it would be advanta-geous to utilize this culture for treating gasifier wastewater wherephenolics and N-heterocyclics coexist with PAHs. This is demon-strated through the culture growth and degradation studies usingsynthetic gasifier wastewater. Utilization of this culture in anattached growth bioreactor along with other cultures was found toresult in effective treatment of the synthetic wastewater at HRT of24 h (Jeswani and Mukherji, 2012).

E. aurantiacum, originally isolated using diesel and reported todegrade aliphatic hydrocarbons, was also found to degrade a widerange of aromatic compounds. No study has previously reportedthe simultaneous degradation of such a wide variety of compoundsby a pure culture. This culture was reported to form a capsule whengrown on diesel and exhibited a tendency for chain formation(Mohanty and Mukherji, 2008b). Similar observations were madefor cultures grown on phenol and pyrene as substrate. The culturepossibly produces enzymes with broad substrate specificity. Spe-cific enzymes are secreted by microorganisms to degrade thesecompounds under aerobic conditions. The first step in degradationof PAHs is the dihydroxylation step catalyzed by the dioxygenaseenzyme. For, E. aurantiacum to degrade 2 through 4 ring PAHs, the

0

20

40

60

80

100

0 50 100 150 200 250 300

-d

Si d

t -

1(m

g l

-1

d

-1)

Concentration, Si(mg l

-1)

Phenol Pyridine Quinoline Benzene Naphthalene

(a)

0

0.05

0.1

0.15

0.2

0.25

0.3

0 0.1 0.2 0.3 0.4 0.5 0.6

-d

Si d

t -

1(m

g l-

1 d

-1)

Concentration, Si(mg l

-1)

Phenanthrene Fluoranthene Pyrene

(b)

Fig. 4. Variation in substrate utilization rate with substrate concentration for (a)phenol, pyridine, quinoline, benzene and naphthalene and (b) phenanthrene, fluo-ranthene and pyrene in synthetic biomass gasifier wastewater and its dilutions.

H. Jeswani, S. Mukherji / International Biodeterioration & Biodegradation 80 (2013) 1e9 7

dioxygenase secreted must have broad substrate specificity, elsethis organism possibly has multiple PAH degrading genes produc-ing dioxygenases with different substrate specificities dependingon the substrate as reported for Burkholderia sp. G3 (Tittabutr et al.,2011). Typically cultures that can degrade HMWPAHs are adverselyaffected by low molecular weight PAHs, such as, naphthalene, thiswas not observed for E. aurantiacum. Some bacterial species whichcan degrade phenolics and PAHs include Bacillus sp., Pseudomonassp., Acinetobacter sp., Achromobacter sp. and Rhodococcus sp.However, some of these microorganisms suffer from substrate in-hibition at higher concentration of phenol. For microorganisms,such as, Rhodococcus, even 50 mg l�1 was reported to cause growthinhibition (Prieto et al., 2002; Polymenakou and Stephanou, 2005)whereas an Acinetobacter sp. could effectively utilize phenol up to aconcentration of 1600 mg l�1 (Wang et al., 2007). E. aurantiacumcould grow on 2000 mg l�1 phenol (Jeswani and Mukherji, 2009).

y = -0.010x + 98.91R² = 0.785

0

20

40

60

80

100

250 500 750 1000 1250 1500

CO

D R

emov

al (

%)

Initial NH4N (mg l-1)

(a)

Fig. 5. Effect of initial NH4eN concentration on percentage

Batch biokinetics for the synthetic wastewater was performedusing COD measurement. The residual COD gives a better measureof overall degradation as opposed to the residual concentration ofeach individual compound, since some compounds may only bepartially transformed during biodegradation. In this wastewaterCOD was entirely contributed by the hazardous organic com-pounds, i.e., phenol, benzene, quinoline, pyridine, naphthalene,phenanthrene, fluoranthene and pyrene. COD decrease withbiodegradation was found to be accompanied by simultaneousutilization of all the compounds. Although E. aurantiacum couldutilize all the components as sole substrate when present individ-ually, for the synthetic wastewater study all the components maynot have been utilized as growth supporting substrate. For degra-dation of wastewater, biochemical oxygen demand (BOD) or COD iscommonly used for depicting the kinetics although all componentsin the wastewater may not support culture growth. The mmax valuewas determined as 1.86 d�1. The Ks value of 651 mg l�1 indicatesthat for overall COD much lower than this value, degradation isessentially linear, whereas for overall COD much higher than thisvalue degradation is unaffected by the COD values. Thus,E. aurantiacum demonstrates good potential for degradation of thesynthetic gasifier wastewater and it utilizes compounds in thiswastewater as substrate. In a biokinetic study on coal gasificationwastewater using activated sludge process Luthy and Tallon (1980)reported a decay coefficient as 0.01 d�1 and yield coefficient as 0.1for initial COD of 2500 mg l�1. Haldane kinetics which takes intoaccount the inhibitory effect of toxic substrates has been commonlyused for representing biokinetics for growth on phenol. For variouspure and mixed microbial consortia, the range of mmax values arereported to vary from 0.026 h�1 to 0.48 h�1, Ks values range from2.2 to 76.45 mg l�1 and the inhibition constant (Ki) values rangefrom 54 to 868 mg l�1 for phenol concentration range 50e2500 mg l�1 (Bajaj et al., 2009). However, in a study on phenoldegradation by Pseudomonas putida (Bakhshi et al., 2011) theMonod’s model was reported to provide a good fit and the values ofKs and mmax were reported as 220.5 mg l�1 and 0.0069 h�1,respectively, for phenol concentration up to 1000 mg l�1.

The Ks values obtained in this study are higher than thosetypically reported in the literature. In general when Ks values arehigh for comparable mmax values, it implies that the wastewater isinherently difficult to degrade and utilize as growth substrate. Noprevious studies have reported kinetic coefficients for biomassgasifier wastewater which is a mixture of various difficult todegrade organic constituents. Although some kinetic data is avail-able for phenol degradation, often the cultures studied do notconform to Monod’s model and Haldane’s model incorporating aninhibition term is used instead. When different models are used,the Ks term is not directly comparable across models. Moreover theKs values are affected by choice of units in which substrate con-centration “S” is expressed. Although, “S” is expressed as COD inthis work, it is often expressed as 5-day BOD, ultimate BOD

y = 0.919x - 304.8R² = 0.984

0

200

400

600

800

1000

250 500 750 1000 1250 1500

Fin

al N

H4N

+ o

rg.

nitr

ogen

(m

g l-1

)

Initial NH4N (mg l-1)

(b)

removal of COD and Kjeldhal nitrogen in the effluent.

H. Jeswani, S. Mukherji / International Biodeterioration & Biodegradation 80 (2013) 1e98

(degradable organics) or as concentration of a specific component.The kinetic parameters obtained are also affected by the organicstrength of the wastewater. The kinetic coefficients, mmax and Ksobserved for biokinetic studies with a wood based and husk basedbiomass gasifier wastewater bioaugmented with E. aurantiacumwere found to be 3.9 and 3.76 d�1, and 4344 and 2250 mg l�1,respectively (unpublished results). Although these coefficientswere different for the various wastewaters, the m versus COD plotsfor husk based wastewater almost coincided with that of the syn-thetic wastewater. Thus the differences in values of the biokineticcoefficients are strongly dependent on the initial COD. The initialCOD was 1326, 7680, and 4850, for synthetic wastewater, woodbased gasifier wastewater and husk based gasifier wastewater,respectively.

In this synthetic wastewater, 80% of the COD was contributed byphenol and heterocylics, 17% was contributed by benzene andnaphthalene and only 3% was contributed by 3e4 ring PAHs.Studies with pure compounds as sole substrate not only showedgood degradation of the predominant compounds such as phenol,quinoline, naphthalene, pyridine and benzene but also somedegradation of the less predominant components. Hence, a goodreduction of COD in the synthetic wastewater may also be expectedif significant accumulation of intermediates and adverse substrateinteraction effects does not occur. In the study using syntheticgasifier wastewater significant degradation of all the componentsin thewastewater was observed. Thus, there is no adverse substrateinteraction effect. Favorable substrate interaction effects, such asgrowth on easily degradable substrates (phenolics, naphthaleneand heterocyclics) possibly facilitated the degradation of the 3 and4 ring PAHs. Similar findings have been reported in the literaturefor binary andmulti-substrate studies with PAHs (Guha et al., 1999).

At concentration exceeding 800 mg l�1, ammoniacal-nitrogencan have a toxic effect on bacteria and various other organisms(Cheung et al., 1995). A mixed microbial consortia was able toremove NH4eN with a concentration ranging from 350 to850 mg l�1 (Liu et al., 1996) in an anaerobic, anoxic and oxic systemwith a 32 h HRT without having an impact on COD removal with98% efficiency. In this study removal of ammoniacal nitrogen wasnot high however E. aurantiacum was able to tolerate ammoniacalnitrogen as high as 1000 mg l�1 without affecting the COD removalin aerobic system.

5. Conclusion

Potential of E. aurantiacum to degrade biomass gasificationwastewater consisting of phenol, N-heterocyclics and PAHs wasanalyzed. A wide range of compounds including phenol, n-het-erocyclics, benzene and 2e4 ring PAHs could be utilized as solesubstrate by E. aurantiacum. Phenol, N-heterocyclics and benzenedepicted high overall loss in batch cultures in the range of 79e95%within 96 h. Amongst PAHs, maximum overall loss was observedfor naphthalene (92.4% in 60 h) while that for pyrene was the least(34.8% in 14 days). Good degradation accompanied with culturegrowth was observed for a synthetic biomass gasifier wastewaterwhere the COD was solely contributed by phenol, N-heterocyclicsand PAHs. Batch biokinetic studies revealed the applicability ofMonod’s model for growth of E. aurantiacum on this wastewater. Allthe components were degraded simultaneously and the degrada-tion rate of each compound was dependent on its concentration inthe wastewater. Increase in ammoniacal nitrogen concentrationhad no significant effect on COD removal. This study confirms thatE. aurantiacum has a good potential to satisfactorily degrade a widevariety of compounds commonly present in gasifier wastewaterand hence bio-augmentation with this culture can enhance effi-ciency of biological treatment processes.

Conflict of interest

There is no actual or potential conflict of interest.

Acknowledgments

This work was partly funded by a sponsored project fromDepartment of Biotechnology, New Delhi, Grant No. BT/PR14807/BCE/08/839/2010. The authors acknowledge Ms Shail Safi, projectstaff at IIT Bombay for help with experimental work and Sophisti-cated Analytical Instrumentation Facility-Centre for Research inNanotechnology and Science (SAIF-CRNTS) at IIT Bombay forproviding the Transmission Electron Microscopy facilities.

Appendix A. Supplementary data

Supplementary data related to this article can be found athttp://dx.doi.org/10.1016/j.ibiod.2013.02.002.

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