phenol degradation by chlorella

A Comprehensive Study on Chlorella pyrenoidosafor Phenol Degradation and its PotentialApplicability as Biodiesel Feedstock and Animal Feed

Bhaskar Das1 & Tapas K. Mandal2 & Sanjukta Patra3

Received: 23 July 2014 /Accepted: 27 April 2015# Springer Science+Business Media New York 2015

Abstract The present work evaluates the phenol degradative performance of microalgaeChlorella pyrenoidosa. High-performance liquid chromatography (HPLC) analysisshowed that C. pyrenoidosa degrades phenol completely up to 200 mg/l. It could alsometabolize phenol in refinery wastewater. Biokinetic parameters obtained are the fol-lowing: growth kinetics, max (media)>max (refinery wastewater), Ks(media)KI(refinery wastewater); degradation kinetics, qmax(media)>qmax (refinery wastewater), Ks(media)KI(refinery wastewater). The microalgae could cometabolize the alkane componentspresent in refinery wastewater. Fourier transform infrared (FTIR) fingerprinting ofbiomass indicates intercellular phenol uptake and breakdown into its intermediates.Phenol was metabolized as an organic carbon source leading to higher specific growthrate of biomass. Phenol degradation pathway was elucidated using HPLC, liquid chro-matographymass spectrometry (LC-MS) and ultravioletvisible (UVvisible) spectro-photometry. It involved both ortho- and meta-pathway with prominence of ortho-pathway. SEM analysis shows that cell membrane gets wrinkled on phenol exposure.Phenol degradation was growth and photodependent. Infrared analysis shows increased

Appl Biochem BiotechnolDOI 10.1007/s12010-015-1652-9

Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1652-9)contains supplementary material, which is available to authorized users.

* Sanjukta [email protected]

Bhaskar [email protected]

Tapas K. [email protected]

1 Centre for the Environment, Indian Institute of Technology Guwahati, Guwahati 781039, India2 Department of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati 781039,

India3 Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, India

intracellular accumulation of neutral lipids opening possibility for utilization of spentbiomass as biodiesel feedstock. The biomass after lipid extraction could be used asprotein supplement in animal feed owing to enhanced protein content. The phenolremediation ability coupled with potential applicability of the spent biomass as biofuelfeedstock and animal feed makes it a potential candidate for an environmentally sustain-able process.

Keywords Chlorella pyrenoidosa . Phenol degradation . Kinetic parameters . cis . cis-muconicacid .Ortho-pathway . FTIR fingerprinting . Lipid accumulation . Protein accumulation

Introduction

Phenol widely used in industrial processes of petroleum refineries, resin plants, cokingoperations, etc. are released in wastewaters. The phenol concentration in the wastewaterof petroleum refinery have been estimated to be 1388 mg/l [14], 180 mg/l in cokewastewater from a steel facility [5], and 70 mg/l in resin industry wastewater [6]. Phenolis water soluble, so it can easily reach water sources downstream from dischargescausing harmful effects to aquatic flora, fauna, and humans. Phenol released into theaquatic ecosystems is biodegraded by the naturally occurring microflora as bacteria,fungi, as well as algae. However, the studies related to phenol biodegradation by algaeare much less than that available concerning bacteria and fungi. Phenol degradation bymicroalgae have been reported by strains of Chlorella sp., Scenedesmus obliqus andSpirulina maxima [7], Ochromonas danica [8], Ankistrodesmus braunii and Scenedesmusquadricauda [9], Chlorella vulgaris [10, 11], Chlorella VT-1 [10], Volvox aureus, Lyngbalagerlerimi, Nostoc linkia, and Oscillatoria rubescens [11]. However, none of the abovestudies have reported algal growth and degradation kinetics in phenol. Knowledge ofgrowth and substrate utilization kinetics is essential to better understand the role playedby microalgae during the natural biodegradation process in phenol-polluted aquaticecosystems. The microbial phenol mineralization capability is completely dependent onactivity of metabolic pathway involving a cascade of phenol metabolizing enzymes. Themetabolic pathways involved in phenol biodegradation have been well studied in bacteria[1214] and fungi [1517]. The only complete pathway of algal phenol mineralizationhas been reported in batch cultures of achlorophyllus algae O. danica [8]. Recently,phenol oxidation to catechol by different green algal species as V. aureus, N. linkia andO. rubescens [11] have been reported. Oxidation of catechol by C. vulgaris and V. aureushave also been reported; however, the product of oxidation was not determined [11]. Thepresent research work provides evidence that green algae possess an enzymatic mecha-nism for phenol removal as in other microbial systems. Thus, the metabolic mechanismof phenol mineralization for green unicellular algae abundantly found in aquatic waterbodies deserves adequate attention. To fulfill the lacuna of literature, we have undertakenthis work with multi-fold objectives: (a) to evaluate the biomass growth and phenoldegradation performance in nutrient media and refinery wastewater by kinetic modeling,(b) to understand the enzymatic mechanism and pathway elucidation, and (c) to evaluatethe biochemical parameters of the algal biomass for further applications. Chlorellapyrenoidosa (NCIM 2738) was chosen as the model organism since it is one of the mostpredominant green microalgae in aquatic ecosystems and water treatment plants.

Appl Biochem Biotechnol

Materials and Methods

Chemicals

MgSO4,CaCl2, K2HPO4, FeSO4, Na2EDTA, H3BO3, MnCl2.4H2O, ZnSO4.7H2O,Na2MoO4.2H2O, CuSO4.5H2O, and phenol were of analytical grade obtained fromMerck, India. Catechol and cis,cis-muconic acid standards were of high-performanceliquid chromatography (HPLC) grade obtained from Sigma-Aldrich, India. Fouriertransform infrared (FTIR) grade KBR was obtained from Spectrochem, India. Glu-taraldehyde solution, 25 % for electron microscopy, was obtained from Himedia,India.

Microorganism and its Culture Condition

Axenic culture of C. pyrenoidosa (NCIM 2738) obtained from NCIM Pune was culturedin Fogs medium (pH 7.5) containing 0.2 g/l MgSO4, 0.2 g/l K2HPO4, 0.1 g/l CaCl2.H20,1 ml/l micronutrient solution, and 5 ml/l Fe-EDTA solution. The micronutrient solutionis composed of 2.86 mg H3BO3, 181.0 mg MnCl2.4H20, 22.0 mg ZnSO4.7H2O, 39 mgNa2MoO4.2H2O, and 8 mg CuSO4.5H2O dissolved in a final volume of 100 ml distilledwater. Cultures were maintained on an orbital shaker operated at 140 rpm with illumi-nation of 3500 lx for a photoperiod of 14 h light/10 h dark in order to simulate thenatural light/dark cycle. For growth of microorganisms in refinery wastewater, sampleswere collected in sterile sample bottles from Indian petroleum refinery and thentransported in ice packs to the laboratory. The oil components in refinery wastewaterwere determined by China National Standard Method [18]. The amount of phenol wasdetermined in 0.2-m filtered water sample by HPLC as described below. The pH ofrefinery wastewater was not adjusted for the degradation experiments. All other growthand culture conditions were similar as mentioned above.

Biomass Growth, Phenol Degradation Analysis, and Kinetic Modeling

The phenol biodegradation capabilities of the microalgae were analyzed in the concen-tration range of 25200 mg/l. The lowest phenol concentration was chosen to be 25 mg/las it is lethal to aquatic organisms like fishes [19]. The upper level of phenol concen-tration was chosen to be 200 mg/l, keeping in mind the phenol concentration found inrefinery wastewater [14]. A control media without phenol was maintained with all otherconditions similar. To account for any abiotic loss of phenol, phenol media without algalcells was incubated under the same culture conditions. To determine photodependency ofphenol metabolism, a similar experiment was carried out in the dark. Samples werecollected at regular intervals of 24 h, and growth analysis was carried out by dry weightanalysis. For measurement of residual phenol concentration, the samples were filteredusing a 0.2-m membrane filter. The filtrate was quantified for phenol concentration byHPLC (Prostar, Varian, USA) equipped with an ultravioletvisible (UVvisible) detectoroperating at 270 nm and a C-18 column. HPLC analysis was performed using mobilephase of acetonitrile/water (70:30) at a flow rate of 0.5 ml/min. To verify differences intotal chlorophyll concentration between phenol-degrading and control biomass,


extraction was performed as per Cuaresma et al. [20]. The total chlorophyll in extractswas determined following the modified Arnons equation [21] as follows:

Chlb 16:72A665 9:16A652 dilution factor mg=l Chla 34:9A652 15:28A665 dilution factor mg=l Chltot Chla Chlb mg=l

All experiments were carried in triplicates, and the mean values and standard error werecalculated using Origin Pro8 and reported in the respective plots

The biomass growth at various initial concentrations of phenol was utilized for calculatingthe specific growth rates, (day1) according to the following equations:

InN2InN1 t1t1

1

where N1 (mg/l) and N2 (mg/l) are biomass growth at time t1 (day) and t2 (day) [22].The growth kinetics of C. pyrenoidosa in phenol was studied. The experimental data were

analyzed with several available growth kinetic models like Haldane [23], Yano [24], Webb[25], Aiba [26], and Edward [27] to select a suitable kinetic model that can represent growthpattern of C. pyrenoidosa. From the experimental data of specific growth rate (, day1) withrespect to various initial concentration of phenol (S0), the model equations were solved usingnonlinear regression method and the values of kinetic parameters of different models weredetermined based on highest regression coefficient and least standard deviation value.

The experimental data on the substrate degradation were utilized for calculating the specificdegradation rate, q (day1) according to the following equations:

q 1x

dSodt

2

where x and S0 are biomass (mg/l) and phenol concentration (mg/l ) at time t (day) [28].The phenol degradation kinetics of C. pyrenoidosa was studied, and the experimental data

were analyzed with several available degradation kinetic models that can represent presentexperimental data. The model equations were solved to determine the degradation kineticparameters using present experimental values of q (day1) for various phenol concentrations(So) [29].

For growth and degradation kinetics study in refinery wastewater log phase C. pyrenoidosacells at the concentration of 220 mg/l was inoculated to refinery wastewater (0.2 m filtered).Biomass growth was determined by dry cell weight analysis. The changes in the nature ofother oil components of the wastewater were characterized after treating the sample with themicroalgae. The specific growth and degradation rates were calculated according to Eqs. 1 and2 as mentioned above. Experimental growth and degradation kinetic data were fitted to thekinetic models, and the biokinetic parameters were estimated.

Analysis of Effect of Phenol on Cell Surface Morphology

The effect of phenol on the cells of C. pyrenoidosa was studied by imaging with scanningelectron microscope (1430vp, Leo, Germany). The surface of the algal biomass treated with200 mg/l phenol for a period of 48 h were observed by SEM and also compared with controlcells. Sample preparation was carried out as per protocol given by Sadiq et al. [30].


Whole Cell Finger Printing of Biomass

The whole cell fingerprint of the biomass was analyzed by FTIR with two objectives: (a) todetermine whether phenol is bioaccumulated in the cell or biodegraded and (b) to analyze thebiomolecular changes in the algal cell during the phenol removal process. Algal cells activelydegrading 125 mg/l phenol were taken for this study. The harvested biomass was washed anddried in vacuum desiccator. One volume of dried algal sample was blended with 100 volumeof dried KBr powder and pressed into tablets before measurement. The spectral acquisitionwas performed using a FTIR spectrometer (IR Affinity, Shimadzu, Japan) by means of 500scans with 4 cm1 of spectral resolution over the wave number range of 4004000 cm1. Thespectrum was submitted to a 15-point smoothing filter for noise reduction. The characteristicpeak areas were obtained using IR Solution FTIR software (Shimadzu, Japan).

Characterization of the Phenol Degradation Pathway

Log phase algal biomass growing in Fogs media supplemented with 125 mg/l phenol washarvested, washed thrice, and grounded in potassium phosphate buffer solution (50 mM) usinga mortarpestle in an ice bath. The extract was centrifuged at 10,000 rpm for 10 min at 4 C,and the supernatant obtained was used for enzyme activity assays. Total protein in thesupernatant was determined as per procedure given by Lowry et al. [31].

Phenol hydroxylase activity was analyzed in a assay mixture containing 50 mM potassiumphosphate buffer solution (pH 7.2), 88 g protein, 1.5 M phenol, and 1.5 MNADPH. Heat-inactivated enzyme extract served as control. The incubation was stopped at equal intervalswith 20 l of 0.6 M perchloric acid. Samples were analyzed for phenol utilization andconcomitant accumulation of the reaction product catechol by HPLC.

The ortho-cleavage of catechol (hydroxylation product of phenol) to cis,cis-muconic acid iscarried out by catechol-1,2-dioxygenase. Catechol-1,2-dioxygenase activity was analyzed in areaction mixture containing 50 mM potassium phosphate buffer pH 7.2, 88 g protein, and1.5 M catechol. Catechol cleavage to cis,cis-muconic acid was analyzed by HPLC. Catechol-2,3-dioxygenase is responsible for meta cleavage of catechol to 2-hydroxymuconic semialde-hyde (2-HMS). Catechol-2,3-dioxygenase activity was determined by increase in absorbanceat 375 nm due to accumulation of the reaction product 2-HMS (E375=14,700 mol L

1 cm1).The breakdown products of cis,cis-muconic acid and 2-HMS were identified by

electrospray ionization liquid chromatographymass spectrometry (LC-MS) (Make: Agilent,Model: Infinity LC system) in negative charge mode. To characterize the metabolites, thecatechol dioxygenase assay was carried out as mentioned above. The LC-MS was operatedusing acetonitrile/water (60:40) mixture as solvent at a flow rate of 0.5 ml/min with detector at270 nm. The m/z signals corresponding to the metabolites were identified using the TandemMass Spectrum database (open sourceCentral Drug Research Institute, India).

Neutral Lipid Analysis

The success of microalgal system to serve as an efficient biodiesel feedstock depends on itshigh neutral lipid productivity [32]. To determine neutral lipid accumulation, algal cells werestained with a microwave-assisted Nile red staining method as per Chen et al. [33]. Forstaining, the cell density was chosen to provide 0.06 (OD750) as optimized for the stainingprotocol by Chen et al. [33]. Fluorescence from stained algal cells were measured on a


multimode microplate reader (Infinity, Tecan, Switzerland) at excitation and emission wave-lengths at 490 and 580 nm, respectively. The excitation and emission standards were deter-mined based on pre-scan characteristics of neutral lipid standard, triloein (Himedia, India). Astandard curve of triloein (R2=0.99) in the concentration range of 5100 g/ml was used forquantification of neutral lipids. The Nile red stained cells were also observed under afluorescence microscope (CX41, Olympus, Japan) as per Greenspan et al. [34] and imagedat 100 magnification.

Results and Discussion

Biomass Growth and Phenol Degradation

The effect of phenol concentration on biomass growth profile and experimental specificgrowth rate of C. pyrenoidosa have been shown in Fig. 1a and b, respectively. The growthcurves show that there is no lag phase in control and low concentration of phenol (25 mg/l)cultures. However, lag phase is observed from 50 mg/l phenol, and the phenol concentrationspreceding it as seen from Fig. 1a. Lag phase is followed by exponential growth phase, which issimultaneously followed by phenol utilization by the biomass. After the exponential growthperiod, phenol is depleted, and the microalgae enters the stationary phase. The biomass growtheven when phenol is depleted may be explained by biotransformation of phenol into itsmetabolic intermediates, which serves as growth substrates until fully utilized [35]. Li et al.[35] worked on phenol degradation by Pseudomonas putida LY1 and observed similar resultsof appearance of lag phase with increased phenol concentration, simultaneous phenol trans-formation in the exponential phase, appearance of stationary phase concomitant with phenoldepletion, and increase in biomass even after complete phenol utilization. From the growthcurve, the specific growth rate of 0.16 day1 of control culture was found to be comparativelylower as compared to specific growth rate achieved in presence of phenol (Fig. 1b). Thishigher specific growth rate is probably because of phenol utilization as an organic carbonsource by C. pyrenoidosa. The specific growth rate was found to increase with increase insubstrate concentration until the highest value of 0.65 day1 was attained at phenol concen-tration of 125 mg/l (Fig. 1b). The highest total chlorophyll content of 27.03 mg/l in 125 mg/lphenol cultures is 26.07 % higher than that of 21.44 mg/l total chlorophyll in control culturesfurther supporting the high biomass growth rate in phenol (Supplementary Fig. 1a and b).However, the growth rate was found to decline with increase in phenol concentration beyond125 mg/l, suggesting growth inhibition effect of phenol. Utilization of phenol as an organiccarbon source by algae has also been reported by Semple and Cain [8] and Lika and Papdakis[36]. They suggested that phenol can be metabolized into organic end products like pyruvateand CO2, which can contribute to biomass growth.

To determine the phenol degradation profile, the residual phenol was estimated by HPLCwith a retention time of 19.4 min. HPLC data shows complete phenol degradation as shown inFig. 2a. The specific degradation rate increases with phenol concentration until a maximumrate of 0.29 day1, which was achieved at 125 mg/l phenol (Fig. 2b.). It is due to the highestspecific growth rate of the microalgae at 125 mg/l phenol. Beyond this phenol concentration(125 mg/l), progressive decrease in specific degradation rate could be well explained byinhibited biomass growth. Mathur and Mazumdar [37] observed similar phenomena duringphenol degradation by P. putida. They reported that both specific growth and degradation rate


increases with increase in phenol concentration until a maximum value of 100 mg/l. They alsoreported that both growth and degradation rate declines due to substrate inhibition of phenolconcentration beyond 100 mg/l.

To negate the effect of any abiotic factors in phenol removal, the loss of phenol from culturemedia without C. pyrenoidosa was determined, and a 1 % abiotic loss of phenol was foundwithin 4 days as compared to complete removal of phenol in C. pyrenoidosa inoculatedcultures. This proves that C. pyrenoidosa is solely responsible for phenol removal from thesample. To determine if the process of phenol degradation in C. pyrenoidosa isphotodependent, microalgal cells were incubated in phenol under dark condition. During thisprocess, the biomass growth and phenol degradation have been found to be negligible ascompared to that in light/dark cycle (Supp. Fig. 2). C. pyrenoidosa could metabolize only 7 %phenol in 3 days under dark condition, which is almost negligible as compared to light/darkcycle (81.56 % phenol removed within 3 days). It resembles the observation of Papazi andKotzabasis [38]. They reported that phenol biodegradation is a photoregulated response in thealgae Scenedesmus obliquus. They showed that phenol biodegradation by S. obliquus was

Fig. 1 a Biomass growth profile of Chlorella pyrenoidosa in various initial phenol concentrations. bVariation inspecific growth rate of Chlorella pyrenoidosa in various initial phenol concentrations

Fig. 2 a Phenol degradation profile of Chlorella pyrenoidosa. b Specific degradation rate of C. pyrenoidosa fordifferent phenol concentrations


reduced to 5 % in dark. Scraag [10] reported that there is no growth as well as phenoldegradation by C. vulgaris under dark condition, which is in accord with the present obser-vations. Attempts were also made to understand the dynamics of biomass growth and phenoldegradation of C. pyrenoidosa in refinery wastewater. The refinery wastewater was quantifiedto contain 23.33 mg/l phenol. The pH of the refinery wastewater was noted as 7.9. Intending tounderstand the natural growth and phenol degradation profile of C. pyrenoidosa, the experi-ment in refinery wastewater was carried out without attempting to meet the nutritionalrequirement of the microalgae and maintaining the actual pH of the refinery wastewater.Figure 3a shows the growth and phenol degradation ability of the microalgae in refinerywastewater. The biomass growth profile indicates an initial lag phase of 2 days unlike that in25 mg/l phenol supplemented Fogs media where no lag phase was observed. After the lagphase, the biomass grows exponentially on the fourth day and then enters the stationary phaseon the 5th day with a final biomass of 339 mg/l. There is no phenol removal by C. pyrenoidosawhen it is in the lag phase of growth (till second day). After the second day, the microalgaeuptakes phenol, which is coincident with exponential growth of the algal biomass.C. pyrenoidosa mineralized 38.32 % of phenol in refinery wastewater by 7 days unlikecomplete mineralization of 25 mg/l phenol concentration by third day in Fogs media. Agarryet al. [39] reported inhibition of complete mineralization of 30 mg/l phenol in refinerywastewater by Pseudomonas aeruginosa and Pseudomonas fluorescens, which correlates wellwith present finding. P. aeruginosa mineralized 94.5 %, while P. fluorescens mineralized69.4 % of initial phenol concentration. However, contrary to the present work, they addedmineral salt medium to refinery wastewater to meet the nutritional requirement of themicroorganism for proper growth. Refinery wastewater may contain other constituents thatmay prove inhibitory to the phenol degradation potential of the microorganisms [39]. Char-acterization of the nature of oil present in refinery wastewater by UV-spectophotometry shownin Fig. 3b indicates that the refinery wastewater before treatment (day 0) consists of bothalkane (absorbance at 215230 nm) and aromatic compounds (absorbance at 250260 nm).Since the peak of maximum absorbance is around 215 nm, the nature of oil in refinerywastewater is clean oil. Dotted line (Fig. 3b) represents the oil component characteristics inwastewater after treatment with C. pyrenoidosa for 8 days, indicating the degradation of bothalkanes and aromatic compounds. This adds to the potential of the algal candidate.Cometabolism of other substrates along with phenol may have slowed the phenol degradationrate.

Growth and Degradation Kinetic Modeling: Biokinetic Parameter Evaluationand Performance Assessment

The behavior of biodegradation rate is a strong function of biomass growth rate. Any growthmedium where the microbial population can double itself faster will potentially result in higherbiodegradation rate [39]. Understanding of a microorganisms degradation and growth kineticswill bring out its potential for phenol biodegradation [40]. Thus, an attempt has been made tofind out the mathematical relationship between (a) growth rate of biomass and substrateconcentration and (b) phenol degradation rate and its initial concentration. The results obtainedby solving the various growth kinetic models have been tabulated in Table 1. From this table, itis clear that Yano model yielded comparatively high R2 value and least SD value confirmingthat Yano model best fitted the experimental data. A comparative plot of experimental andmodel predicted specific growth rates has been shown in Fig. 4a. The value of KS (half


saturation coefficient) indicates the affinity of the microorganism to the substrate. The value ofKI (substrate inhibition constant) signifies the degree of resistance of microorganism to thetoxic effect of the substrate [41].

The substrate consumption rate is the most important parameter for denoting microbialdegradative performance [42]. Initial phenol concentration has strong influence on specificdegradation rate making kinetic analysis of substrate consumption essential [39]. The value ofkinetic parameters obtained by solving the various degradation kinetic models has been shownin Table 2. The specific degradation rate predicted by the various kinetic models at differentinitial concentration of phenol has also been plotted graphically in Fig. 4b. Table 2 indicatesthat Yano model yielded the highest correlation coefficient (R2) and the least standarddeviation (SD) among all other models and thus best fitted the experimental data.

On the basis of the encouraging results in cultured condition, we attempted to verify theapplicability of C. pyrenoidosa for phenol removal from refinery wastewater. The kineticparameters obtained for various growth kinetic models have been shown in Table 3. Table 3shows that Haldane model yielded the highest correlation coefficient (R2) and the leaststandard deviation (SD) and thus best fitted the experimental data. Table 4 describes thekinetic parameters of degradation kinetic models, and the Haldane model shows both highestcorrelation coefficient (R2) and least standard deviation (SD). Thus, the Haldane modelrepresents appropriately the phenol degradation behavior of the microalgae in refinery waste-water used in the present study.

Fig. 3 a Biomass growth and phenol degradation by C. pyrenoidosa in refinery wastewater. b Oil characteristicsof refinery wastewater before (day 0) and after treatment (day 8) with C. pyrenoidosa

Table 1 Estimated value of growth kinetic parameters of C. pyrenoidosa (NCIM 2738) in phenol-containingnutrient media

Model max (day1) Ks (mg/l) KI (mg/l) K (mg/l) R

2 SD

Haldane 5.572 444.1 24.46 0.94 0.049

Yano 4.344 410.5 214.5 32.26 0.97 0.035

Webb 5.394 480.74 15.44 519.7 0.92 0.055

Aiba 7.15 472.5 132.7 0.95 0.045

Edward 28.1 111.5 105.3 0.95 0.044


Comparison of the biokinetic parameters may give the indication of how C. pyrenoidosabehaved under two significantly different culture conditions of nutrient sufficient media andrefinery wastewater. For this reason, a comparison was made between the respective biokineticparameters of best fit kinetic models representing the biomass growth and phenol degradationbehavior. Kinetic modeling of the experimental biomass growth data suggests that max(0.017 day1) and KI (10.46 mg/l) is lower in refinery wastewater (first row in Table 3) ascompared to that in nutrient media (second row in Table 2). The Ks value (600.1 mg/l) inrefinery wastewater (first row in Table 3) is higher than that in nutrient media (second row inTable 2). While degradation kinetic modeling shows lowered qmax (0.012 day

1) and KI(53.24 mg/l) values in refinery wastewater (first row in Table 4) as compared to that in nutrientmedia (second row in Table 2). The Ks value (300.99 mg/l) in refinery wastewater (thirdcolumn of first row in Table 4) is higher compared to that in nutrient media (thirrd column ofsecond row in Table 2). Lower max values in refinery wastewater is probably due to the lackof optimal nutrient factors for growth as well as other growth inhibitory constituents, whichmay be present in refinery wastewater. Maximum degradation rate (qmax) is also lower inrefinery wastewater due to the lower specific growth rate as mentioned above. Therefore,efficient phenol utilization is less in refinery wastewater as compared to that in nutrient mediacontaining optimal biomass growth conditions. Secondly, cometabolism of alkanes in refinerywastewater along with phenol (aromatic) as discussed in Biomass growth and phenoldegradationmay lead to decrease in phenol degradation rate (qmax). Ks being inversely relatedto affinity of microbial system for substrate, a higher Ks value indicates its lower affinity to the

Fig. 4 aGrowth kinetic model fitted to experimental batch growth data of C. pyrenoidosa. bDegradation kineticmodel fitted to experimental batch degradation data of C. pyrenoidosa

Table 2 Estimated value of degradation kinetic parameters during phenol biodegradation by C. pyrenoidosa(NCIM 2738) in phenol-containing nutrient media

Model qmax (day1) Ks (mg/l) KI (mg/l) K (mg/l) R

2 SD

Haldane 0.55 89.99 100.24 0.73 0.05

Yano 0.76 170.60 250.6 86.54 0.81 0.04

Webb 0.30 77.93 100.9 350.06 0.70 0.07

Aiba 0.71 58.13 200.40 0.65 0.08

Edward 2.78 77.94 100.9 0.76 0.06


substrate [43]. Higher Ks value suggests a decreased affinity for phenol of C. pyrenoidosa inrefinery wastewater compared to that in nutrient media. Higher Ks value in refinery wastewaterexplains inhibition of complete phenol mineralization unlike complete mineralization inmedia. C. pyrenoidosa utilizes phenol less efficiently in refinery wastewater due to decreasedaffinity for phenol. KI value is involved in quantification of the effect of toxicity of acompound during the biodegradation process. A higher KI value implies less sensitivity ofthe microbe to substrate inhibition. Lower KI value in refinery wastewater suggests highsensitivity of C. pyrenoidosa to toxic effect of phenol compared to that in nutrient media.This can be understood from the fact that optimal growth conditions in media helps themicroalgae counter the inhibitory effect of phenol in a better way. On the other hand, lack ofoptimal biomass growth factors and possible presence of other additional inhibitory constitu-ents in refinery wastewater compromises the ability of C.pyrenoidosa to resist the inhibitoryeffect of phenol.

Characteristics of Cell Surface Morphology on Phenol Treatment

The SEMmicrograph (Supp. Fig. 3a and b.) shows that phenol exposure affects the membranemorphology of cells. When cells were exposed to 200 mg/l phenol for 48 h, the cell surfacewas found to be wrinkled. Accumulation of phenol in the hydrophobic part of the membraneleads to disturbance in the interactions between the acyl chain of phospholipids. This causesmodification of membrane fluidity and may lead to swelling of the bilayer [44].

FTIR Fingerprinting Analysis

The FTIR spectrum (Supp. Fig. 4a) depicts the whole-cell fingerprint of the biochemicalchanges in response to phenol. Adsorption at 34443419 cm1 is due to stretching of OHgroups of alcohol, phenol, or carboxyl OH and hydrogen vibration of the amide NH [45].

Table 3 Estimated growth kinetic parameters of Chlorella pyrenoidosa (NCIM 2738) in refinery wastewater

Model max (day1) Ks (mg/l) KI (mg/l) K (mg/l) R

2 SD

Haldane 0.017 600.1 10.46 0.96 0.025

Yano 4.344 600.5 150.5 10.26 0.96 0.412

Webb 0.356 580.7 5.44 530.7 0.82 0.113

Aiba 0.019 572.5 70.7 0.84 0.023

Edward 0.03 50.94 70.9 0.34 0.05

Table 4 Estimated degradation kinetic parameters for Chlorella pyrenoidosa (NCIM 2738) during phenoldegradation in refinery wastewater

Model qmax (day1) Ks (mg/l) KI (mg/l) K (mg/l) R

2 SD

Haldane 0.012 300.99 53.24 0.99 0.02

Yano 0.018 220.6 200.6 40.54 0.48 0.04

Webb 0.107 350.93 100.9 400.06 0.11 0.09

Aiba 0.054 208.13 30.4 0.33 0.05

Edward 0.058 77.94 100.9 0.19 0.08


Infrared spectrum shows that the percent transmittance in this region decreases (Supp. Fig. 4a)while peak area increases (Supp. Fig. 4b) with phenol incubation attaining prominence on thethird day. However, there is no such prominent transmittance (Supp. Fig. 4a) or area (Supp.Fig 4b) variation within this wave number range in control cells. The results suggest increasedintracellular phenol accumulation with incubation time. Since the microalgae completelyremoved 125 mg/l phenol from the culture medium by the second day (Fig. 2a), its highintracellular accumulation is quite evident. An increased peak area indicates increase inconcentration of functional groups whose stretching/bending is responsible for the peak. Thus,peak area differences have been successfully used to monitor change in concentration ofdifferent biomolecules as monosaccharides in Enterobacter cloacae [46], amide I and II,cellulosic compounds, nonstructural carbohydrates in different barley varieties [47], as wellas erythromycin quantification in pharmaceutical formulations [48]. Similarly, comparativelyhigher percent transmittance as well as low peak area due to low intracellular phenol uptake onday 1 is explained by lower phenol removal rate till day 1. The first metabolic intermediate ofphenol degradation pathway is catechol. The metabolic intermediates formed by breakdown ofcatechol contains carboxylic acid group (COOH) [49]. The infrared spectra region 17541710 cm1 is associated to carbonyl group vibration in COOH group [44]. Transmittance(Supp. Fig. 4a) in this region is found to decrease, and peak area (Supp. Fig. 4c) increases withphenol incubation, which is not evident in control cells. This shows that the accumulatedintracellular phenol is metabolized into intermediate products downstream of catechol. Wharfeet al. [49] reported similar findings of an increased infrared absorbance due to carbonyl groupvibration of intermediate products of phenol metabolism in a microbial consortium. Theinfrared region 14401380 cm1 is attributed to CH bending of aliphatic groups. Decreasedpercent transmittance (Supp. Fig. 4a) and an increased peak area (Supp. Fig 4d) in this regionsuggest an increased accumulation of aliphatics in phenol-incubated cells. Intracellular accu-mulation of aliphatic intermediates of phenol metabolism may be associated to the decreasedtransmittance and increased peak area. Thus, infrared analysis suggests intracellular uptake ofphenol by C. pyrenoidosa, and then phenol is broken down into intermediate products. Thisbreakdown of intracellular uptaken phenol into its intermediate metabolites confirms theprocess of phenol removal to be biodegradation.

Elucidation of Phenol Degradation Pathway

The phenol metabolic pathway was characterized by identifying the different intermediatesproduced during phenol degradation using HPLC, UVvisible spectrophotometry, and LC-MS. Supplementary Fig. 5a and b suggests appearance of catechol peak (11.9 min) withprogressive decrease in phenol peak (10.69 min), which confirms catechol accumulation inmedia (extra cellular). The present results also accord with the observation reported by El-Sheekh et al. [11] for different algal species as V. aureus, N. linkia, and O. rubescens. It provesthat phenol is degraded through an enzymatic pathway in C. pyrenoidosa. Intracellular phenolhydroxylase activity was determined by HPLC (Fig. 5a). Control incubations carried out by

Fig. 5 a Hydroxylation of phenol to catechol by phenol hydroxylase activity. b Ortho-cleavage of catechol tocis,cis-muconic acid by catechol-1,2-dioxygenase activity. c Meta cleavage of catechol to 2-hydroxymuconicsemialdehyde (2-HMS) by catechol-2,3-dioxygenase activity. d LC-MS analysis of catechol dioxygenase assaymixture at 0 min (before incubation). e LC-MS analysis of catechol dioxygenase assay mixture at 20 min (afterincubation). f Proposed pathway of phenol degradation in Chlorella pyrenoidosa (NCIM 2738)


Phenol

Catechol

Phenol hydroxylase

Catechol-1,2-dioxygenase

cis,cis-muconate

-ketoadipate

Catechol-2,3-dioxygenase

2-hydroxymuconic semialdehyde

Citrate cycle

Cis-2-hydroxypenta-2,4-dienoate

2-hydroxymuconate semialdehyde hydrolase

Acetaldehyde Pyruvate

a b c

d e

f


heat-killed enzyme extract showed no phenol hydroxylation activity (no catechol and nophenol utilization). Similar observations have also been reported in the literature for algae[8], fungi [50, 15] and bacteria [12, 51]. Catechol can be ortho-cleaved (if it follows ortho-pathway) or meta-cleaved (if it follows meta-pathway) by catechol-1,2-dioxygenase andcatechol-2,3-dioxygenase, respectively. Catechol-1,2-dioxygenase activity was characterizedby identifying its ortho-cleavage product namely cis,cis-muconic acid using HPLC as shownin Fig. 5b. The reaction product was identified to be cis,cis-muconic based on identicalretention time of 4.2 min with that of standard cis,cis-muconic acid. Control incubationscarried out by heat-killed enzyme extract showed no catechol-1,2-dioxygenase activity. Cat-echol-2,3-dioxygenase activity was also determined by identifying 2-hydroxymuconic semi-aldehyde as the meta-cleavage product of catechol, using UV-visible spectrophotometry asshown in Fig. 5c. Control incubations by heat-killed enzyme extract showed no accumulationof meta-cleavage product 2-hydroxymuconic semialdehyde, indicating no catechol-2,3-dioxygenase activity. Both catechol-1,2-dioxygenase and catechol-2,3-dioxygenase activitysuggests that phenol metabolism involves both ortho- as well as meta-pathway. The catabolicefficiencies of phenol hydroxylase, catechol-1,2-dioxygenase, and catechol-2,3-dioxygenasewere estimated on basis of specific enzyme activities, which are tabulated in Table 5. Com-paratively higher activity of catechol-1,2-dioxygenase against that of catechol-2,3-dioxygenasesuggests efficiency of ortho- over meta-pathway in C. pyrenoidosa. Similar results aboutsimultaneous activity of meta as well as ortho-pathway were reported in P. fluorescens PU1[12]. They reported higher meta-activity over ortho-activity. Cai et al. [52] also reportedsimilar findings of coexistence of both ortho- and meta-pathway in Fusarium species. Sempleand Cain [8] reported involvement of meta pathway in golden brown chrysophyte algaO. danica, whereas most eukaryotes generally utilize ortho-pathway [53]. Evidence of or-tho-activity in other eukaryotes as Trichosporon cutaneum [15], Penicillium sp. [54], Fusar-ium sp., Aspergillus sp., Penicillium sp. and Graphium sp. [55], and Candida sp. [17]correlates with the present finding.

The breakdown products of cis,cis-muconic acid (ortho-pathway) and 2-hydroxymuconicsemialdehyde (meta-pathway) were identified by LC-MS analysis. In order to identify thetarget metabolites, we are analyzing the catechol dioxygenase assay mixture both at the startand end of the reaction, and the chromatograms are shown in Fig. 5d and e, respectively.Figure 5d shows a highly abundant (abundance=95,828) m/z signal of 131. The m/z signal of131 is consistent with the molecular ion mass of [M+Na2H] adduct of catechol. Two morem/z signals of low abundance at 177 (abundance=2989) and 195 (abundance=3060) are alsonoted. The m/z signal at 177 corresponds the molecular ion mass of [M+K2H] adduct ofcis,cis-muconic acid (ortho-cleavage product of catechol). Similarly, the m/z signal at 195 isconsistent with the molecular ion mass of [M+Cl] adduct of -ketoadipate, a metabolite ofthe ortho-pathway. The occurrence of adduct ions are common occurrence in LC-MS analysis.Biological samples generally have high endogenous concentration of various salts, while other

Table 5 Enzyme activities in C. pyrenoidosa (NCIM 2738) cell lysate grown in phenol-containing media

Enzyme assayed Activity (U) Specific activity (U/mg)

Phenol hydroxylase 833 11.41

Catechol-1,2-dioxygenase 514 7.04

Catechol-2,3-dioxygenase 13 0.18


salts may be added during sample preparation. This justifies the high probability of formationof adduct ions during LC-MS of biological samples [56]. One probable source of potassiumleading to formation of potassium ion adduct in our samples is possibly the utilization ofpotassium phosphate buffer during cell free extract preparation. The chloride ion adductformed is one of the commonly formed metal adduct ion during negative ion electrosprayanalysis [56]. Figure 5e shows that the abundance of the m/z signal 131 decreases (abun-dance=3236) after 20-min incubation of the reaction mixture, which confirms utilization ofcatechol. The increase in abundance of m/z signal 177 (abundance=263,655) shows increasein accumulation of ortho-cleavage product of catechol, i.e., cis,cis-muconic acid. This indi-cates active ortho-pathway for phenol metabolism. Similarly, the increase in abundance of m/zsignal at 195 (abundance=373,154) shows increase in accumulation of ortho-pathway inter-mediate, -ketoadipate. Figure 5e shows an additional abundance at m/z 338 (abundance=35,223) with a molecular ion mass identical to [3MH] adduct of cis-2-hydroxypent-2,4-dienoate, a metabolite from the meta-cleavage pathway. Identification of adduct ion of cis-2-hydroxypent-2,4-dienoate suggests the presence of meta-pathway along with ortho-pathway.The literature also supports present observation as Tsai et al. [17] identified a LC-MS signal atm/z 163 corresponding to molar mass of sodium adduct of cis,cis-muconic acid in the enzymeactivity reaction mixtures, which is due to active ortho-pathway in Candida albicans TL3.

On the basis of the enzyme activity and metabolite analysis study, the pathway proposed forphenol degradation in C. pyrenoidosa has been shown in Fig. 5f. In the pathway, phenolhydroxylase is involved in initial attack on phenol hydroxylating phenol to catechol. Theresulting catechol is ortho-cleaved by catechol-2,3-dioxygenase as well as meta-cleaved bycatechol-1,2-dioxygenase. Catechol-1,2-dioxygenase ortho-cleaves catechol into its reactionproduct cis,cis-muconic acid. Determination of 3-oxoadipate, a metabolite downstream ofcis,cis-muconic acid in the ortho-pathway indicates breakdown of cis,cis-muconic acid.Catechol also undergoes meta-cleavage into 2-HMS by catechol-2,3-dioxygenase activity. 2-Hydroxymuconate semialdehyde hydrolase causes hydrolysis of 2-HMS to cis-2-Hydroxypenta-2,4-dienoate in the meta-pathway. Thus, both ortho- as well as meta-pathwayis involved in phenol degradation in C. pyrenoidosa. However, ortho-pathway is significantlyactive over meta-pathway. The proposed pathway is found to possess similarities with algal[8], fungal [17, 52, 55], as well as bacterial [12, 51] catabolic mechanisms.

Biomolecular Characterization of the Biomass for Potential Applications

This section analyses the usefulness of the phenol-degrading algal biomass as potentialfeedstock for applications as biodiesel and animal feed. Microalgal biomass with high lipidand protein content could serve as biodiesel feedstock [57] and protein supplement in animaldiet, respectively [58]. Liu et al. [59] characterized lipid content in algal strains in a bid toidentify high lipid producing strains. They reported C. pyrenoidosa to be one of the best oilproducers whose total lipid content varies between 18.67 % to as high as 52.08 % of drybiomass. Chlorella sp. have been reported to have high protein content between 51 and 58 %of dry biomass, and so it is one of the species selected for large scale production [60]. Thebiochemical characteristic of the algal biomass was analyzed using FTIR. FTIR analysisshown in Supp. Fig. 4a depicts a snapshot of the changes in biomolecular level in responseto phenol stress. The infrared region 28752850 cm1 corresponds to symmetric stretching ofCH2 and CH3 of lipids [61]. With phenol incubation, there is prominent decrease in transmit-tance (Supp. Fig. 4a) and increase in peak area (Supp. Fig. 6a) in this region indicating higher


cellular lipid accumulation compared to control biomass. Gracia et al.[57] reported increasedlipid production in Phaeodactylum tricornutum UTEX-640 when cultured mixotrophicallywith carbon sources glycerol and fructose. Kong et al. [62] reported stimulation of lipidbiosynthesis in C. vulgaris when mixotrophically cultured in glucose and glycerol. Thesefindings accords with present finding of increased lipid accumulation in the presence of phenolas additional carbon source. However, high neutral lipid accumulation in algal biomass isnecessary for its commercial applicability as biodiesel feedstock [32]. The profile for neutrallipid biosynthesis in C. pyrenoidosa cells (Fig. 6a) shows 50 % increased neutral lipidaccumulation in phenol-degrading biomass as compared to control on fourth day of incuba-tion. Hamed and Klock [63] reported similar results of enhanced neutral lipid accumulation inChlorella sorokiniana during mixotrophic culture on glycerol. Fluorescence microscopy ofphenol-degrading cells (Fig. 6b) showed enhanced yellow gold fluorescence of neutral lipidbodies in cell cytoplasm compared to that from control cells (Fig. 6c). This further supports thefinding of enhanced neutral lipid accumulation in phenol-degrading biomass. Therefore, thealgal biomass (after phenol biodegradation) could serve as potential raw material for biodieselproduction. Although mixotrophic cultivation allows microalgae to accumulate higher propor-tion of lipids within less time, its commercial applicability is hindered by high substrate cost.The cost of carbon source represents 50 % of the cost of the medium used in mixotrophic algalcultivation. This makes the process of production of algal biomass feedstock for biodieselcostly [64]. Thus, cheap alternative carbon sources for mixotrophic cultivation of algae couldhelp commercialize algal biodiesel by reducing the cost of production of algal biomassfeedstock. The present study shows that C. pyrenoidosa accumulates high proportion of lipidswhile metabolizing phenol, which is a waste product of various industrial processes. Thus,mixotrophic cultivation of C. pyrenoidosa using industrial waste phenol as a carbon sourcecould provide exciting possibility to decrease the production cost of algal biomass feedstockfor biodiesel.

Fig. 6 a Neutral lipid accumulation in phenol-degrading and control biomass of C. pyrenoidosa. b Fluorescencemicroscopy image of Nile red stained phenol-degrading C. pyrenoidosa cells. c Fluorescence microscopy imageof Nile red stained control cells of C. pyrenoidosa


The infrared region 1200900 cm1 corresponds to symmetric stretching of COC ofpolysaccharides [61]. The infrared region 16101685 cm1 is associated to C=Ostretching of proteins [61]. Phenol incubation causes increased intracellular proteinsynthesis compared to control as evident from decreased transmittance (Supp. Fig. 4a)as well as increased peak area (Supp. Fig. 6b) in this region (16101685 cm1) inphenol-degrading biomass. Thus, the protein rich algal biomass (after phenol degrada-tion) could be used as an animal feed supplement. Since the amino acid profiles ofmicroalgal protein is comparable to other food proteins, it could serve as a proteinsupplement in animal feed [65]. Although microalgae have been widely used as proteinsource to supplement animal diets, recent research trend in this area is supplementationof animal diets with defatted (lipid extracted) microalgal biomass from biodiesel pro-duction processes [66]. Since phenol-degrading biomass of C. pyrenoidosa is lipid aswell as protein rich (increased accumulation of lipid and protein during the phenoldegradation process), the biomass after lipid extraction (for biodiesel production) couldserve as a protein supplement in animal diets.

Conclusion

This study showed photodependent phenol degradation capability of C. pyrenoidosa withcomplete degradation till 200 mg/l phenol concentration under the optimal nutrientconditions of Fogs medium. The maximum specific rate of degradation was achievedat 125 mg/l phenol due to maximum specific growth rate at this concentration. However,the strain could metabolize 38.32 % of 23.33 mg/l phenol along with removal ofaliphatics from petroleum refinery wastewater. Biokinetic parameters obtained by kineticmodeling shows the differences in growth and phenol degradation dynamics ofC. pyrenoidosa in nutrient media and petroleum refinery wastewater. Low max valuesobtained by kinetic modeling in refinery wastewater is probably related to lack ofoptimal growth factors as well as other inhibitory constituents, which may be presentin refinery wastewater. Cometabolism of alkanes along with decreased max values maybe responsible for decreased phenol degradation rates (low qmax value) as well asdecreased phenol affinity (high Ks value)in refinery wastewater. SEM analysis indicatesthat the cellular membrane morphology gets wrinkled on phenol exposure.C. pyrenoidosa metabolizes phenol simultaneously by both ortho- as well as meta-pathway. The ortho-pathway is significantly predominant over the meta pathway. Thephenol-degrading biomass has 50 % higher neutral lipid accumulation compared tocontrol cells, suggesting exciting possibility to utilize the spent biomass as biodieselfeedstock. The defatted biomass could additionally serve as animal feed owing to itsenhanced protein content. Thus, the mixotrophic growth of C. pyrenoidosa on industrialwaste phenol could prove to be an environmentally sustainable process as it will causeremediation of the toxic waste phenol along with generation of biodiesel feedstock withdecreased production costs solving a major bottleneck in commercialization of algalbiodiesel.

Acknowledgments Bhaskar Das acknowledges Indian Institute of Technology, Guwahati, for providing re-search fellowship to pursue doctoral studies at the Centre for the Environment, Indian Institute of Technology,Guwahati. The present work is not financially supported by any funding agency.


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A...AbstractIntroductionMaterials and MethodsChemicalsMicroorganism and its Culture ConditionBiomass Growth, Phenol Degradation Analysis, and Kinetic ModelingAnalysis of Effect of Phenol on Cell Surface MorphologyWhole Cell Finger Printing of BiomassCharacterization of the Phenol Degradation PathwayNeutral Lipid Analysis

Results and DiscussionBiomass Growth and Phenol DegradationGrowth and Degradation Kinetic Modeling: Biokinetic Parameter Evaluation and Performance AssessmentCharacteristics of Cell Surface Morphology on Phenol TreatmentFTIR Fingerprinting AnalysisElucidation of Phenol Degradation PathwayBiomolecular Characterization of the Biomass for Potential Applications

ConclusionReferences

phenol degradation by chlorella

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