Appl Biochem Biotechnol DOI 10.1007/s12010-015-1652-9

A Comprehensive Study on Chlorella pyrenoidosa for Phenol Degradation and its Potential Applicability as Biodiesel Feedstock and Animal Feed Bhaskar Das 1 & Tapas K. Mandal 2 & Sanjukta Patra 3

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 microalgae Chlorella pyrenoidosa. High-performance liquid chromatography (HPLC) analysis showed that C. pyrenoidosa degrades phenol completely up to 200 mg/l. It could also metabolize phenol in refinery wastewater. Biokinetic parameters obtained are the following: growth kinetics, μ max (media) > μ max (refinery wastewater), K s (media) < Ks(refinery wastewater), KI(media) >KI(refinery wastewater); degradation kinetics, qmax (media)>qmax (refinery wastewater), Ks(media) KI(refinery wastewater). The microalgae could cometabolize the alkane components present in refinery wastewater. Fourier transform infrared (FTIR) fingerprinting of biomass indicates intercellular phenol uptake and breakdown into its intermediates. Phenol was metabolized as an organic carbon source leading to higher specific growth rate of biomass. Phenol degradation pathway was elucidated using HPLC, liquid chromatography–mass spectrometry (LC-MS) and ultraviolet–visible (UV–visible) spectrophotometry. It involved both ortho- and meta-pathway with prominence of orthopathway. SEM analysis shows that cell membrane gets wrinkled on phenol exposure. Phenol degradation was growth and photodependent. Infrared analysis shows increased 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 Patra [email protected] Bhaskar Das [email protected] Tapas K. Mandal [email protected] 1

Centre for the Environment, Indian Institute of Technology Guwahati, Guwahati 781039, India

2

Department of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati 781039, India

3

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

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intracellular accumulation of neutral lipids opening possibility for utilization of spent biomass as biodiesel feedstock. The biomass after lipid extraction could be used as protein supplement in animal feed owing to enhanced protein content. The phenol remediation ability coupled with potential applicability of the spent biomass as biofuel feedstock and animal feed makes it a potential candidate for an environmentally sustainable process. Keywords Chlorella pyrenoidosa . Phenol degradation . Kinetic parameters . cis . cis-muconic acid . Ortho-pathway . FTIR fingerprinting . Lipid accumulation . Protein accumulation

Introduction Phenol widely used in industrial processes of petroleum refineries, resin plants, coking operations, etc. are released in wastewaters. The phenol concentration in the wastewater of petroleum refinery have been estimated to be 13–88 mg/l [1–4], 180 mg/l in coke wastewater from a steel facility [5], and 70 mg/l in resin industry wastewater [6]. Phenol is water soluble, so it can easily reach water sources downstream from discharges causing harmful effects to aquatic flora, fauna, and humans. Phenol released into the aquatic ecosystems is biodegraded by the naturally occurring microflora as bacteria, fungi, as well as algae. However, the studies related to phenol biodegradation by algae are much less than that available concerning bacteria and fungi. Phenol degradation by microalgae have been reported by strains of Chlorella sp., Scenedesmus obliqus and Spirulina maxima [7], Ochromonas danica [8], Ankistrodesmus braunii and Scenedesmus quadricauda [9], Chlorella vulgaris [10, 11], Chlorella VT-1 [10], Volvox aureus, Lyngba lagerlerimi, Nostoc linkia, and Oscillatoria rubescens [11]. However, none of the above studies have reported algal growth and degradation kinetics in phenol. Knowledge of growth and substrate utilization kinetics is essential to better understand the role played by microalgae during the natural biodegradation process in phenol-polluted aquatic ecosystems. The microbial phenol mineralization capability is completely dependent on activity of metabolic pathway involving a cascade of phenol metabolizing enzymes. The metabolic pathways involved in phenol biodegradation have been well studied in bacteria [12–14] and fungi [15–17]. The only complete pathway of algal phenol mineralization has 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 and O. rubescens [11] have been reported. Oxidation of catechol by C. vulgaris and V. aureus have also been reported; however, the product of oxidation was not determined [11]. The present research work provides evidence that green algae possess an enzymatic mechanism for phenol removal as in other microbial systems. Thus, the metabolic mechanism of phenol mineralization for green unicellular algae abundantly found in aquatic water bodies deserves adequate attention. To fulfill the lacuna of literature, we have undertaken this work with multi-fold objectives: (a) to evaluate the biomass growth and phenol degradation performance in nutrient media and refinery wastewater by kinetic modeling, (b) to understand the enzymatic mechanism and pathway elucidation, and (c) to evaluate the biochemical parameters of the algal biomass for further applications. Chlorella pyrenoidosa (NCIM 2738) was chosen as the model organism since it is one of the most predominant green microalgae in aquatic ecosystems and water treatment plants.

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Materials and Methods Chemicals MgSO 4 ,CaCl 2, K 2 HPO 4, FeSO 4, Na 2 EDTA, H 3 BO 3, MnCl2 .4H 2 O, ZnSO 4.7H 2O, Na2MoO4.2H2O, CuSO4.5H2O, and phenol were of analytical grade obtained from Merck, India. Catechol and cis,cis-muconic acid standards were of high-performance liquid chromatography (HPLC) grade obtained from Sigma-Aldrich, India. Fourier transform infrared (FTIR) grade KBR was obtained from Spectrochem, India. Glutaraldehyde 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 cultured in Fog’s 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 solution is composed of 2.86 mg H3BO3, 181.0 mg MnCl2.4H20, 22.0 mg ZnSO4.7H2O, 39 mg Na2MoO4.2H2O, and 8 mg CuSO4.5H2O dissolved in a final volume of 100 ml distilled water. Cultures were maintained on an orbital shaker operated at 140 rpm with illumination of 3500 lx for a photoperiod of 14 h light/10 h dark in order to simulate the natural light/dark cycle. For growth of microorganisms in refinery wastewater, samples were collected in sterile sample bottles from Indian petroleum refinery and then transported in ice packs to the laboratory. The oil components in refinery wastewater were determined by China National Standard Method [18]. The amount of phenol was determined in 0.2-μm filtered water sample by HPLC as described below. The pH of refinery wastewater was not adjusted for the degradation experiments. All other growth and 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 concentration range of 25–200 mg/l. The lowest phenol concentration was chosen to be 25 mg/l as it is lethal to aquatic organisms like fishes [19]. The upper level of phenol concentration was chosen to be 200 mg/l, keeping in mind the phenol concentration found in refinery wastewater [1–4]. A control media without phenol was maintained with all other conditions similar. To account for any abiotic loss of phenol, phenol media without algal cells was incubated under the same culture conditions. To determine photodependency of phenol metabolism, a similar experiment was carried out in the dark. Samples were collected at regular intervals of 24 h, and growth analysis was carried out by dry weight analysis. For measurement of residual phenol concentration, the samples were filtered using a 0.2-μm membrane filter. The filtrate was quantified for phenol concentration by HPLC (Prostar, Varian, USA) equipped with an ultraviolet–visible (UV–visible) detector operating at 270 nm and a C-18 column. HPLC analysis was performed using mobile phase of acetonitrile/water (70:30) at a flow rate of 0.5 ml/min. To verify differences in total chlorophyll concentration between phenol-degrading and control biomass,

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extraction was performed as per Cuaresma et al. [20]. The total chlorophyll in extracts was determined following the modified Arnon’s equation [21] as follows: Chlb ¼ ð16:72A665 – 9:16A652 Þ dilution factor ðmg=l Þ Chla ¼ ð34:9A652 – 15:28A665 Þ dilution factor ðmg=l Þ Chltot ¼ Chla þ Chlb ðmg=l Þ All experiments were carried in triplicates, and the mean values and standard error were calculated using Origin Pro8 and reported in the respective plots The biomass growth at various initial concentrations of phenol was utilized for calculating the specific growth rates, μ (day−1) according to the following equations: μ¼

ðInN 2 −InN 1 Þ t1 −t1

ð1Þ

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 growth pattern of C. pyrenoidosa. From the experimental data of specific growth rate (μ, day−1) with respect to various initial concentration of phenol (S0), the model equations were solved using nonlinear regression method and the values of kinetic parameters of different models were determined based on highest regression coefficient and least standard deviation value. The experimental data on the substrate degradation were utilized for calculating the specific degradation rate, q (day−1) according to the following equations: q¼−

1 dS o x dt

ð2Þ

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 present experimental data. The model equations were solved to determine the degradation kinetic parameters using present experimental values of q (day−1) for various phenol concentrations (So) [29]. For growth and degradation kinetics study in refinery wastewater log phase C. pyrenoidosa cells at the concentration of 220 mg/l was inoculated to refinery wastewater (0.2 μm filtered). Biomass growth was determined by dry cell weight analysis. The changes in the nature of other oil components of the wastewater were characterized after treating the sample with the microalgae. The specific growth and degradation rates were calculated according to Eqs. 1 and 2 as mentioned above. Experimental growth and degradation kinetic data were fitted to the kinetic 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 scanning electron microscope (1430vp, Leo, Germany). The surface of the algal biomass treated with 200 mg/l phenol for a period of 48 h were observed by SEM and also compared with control cells. Sample preparation was carried out as per protocol given by Sadiq et al. [30].

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Whole Cell Finger Printing of Biomass The whole cell fingerprint of the biomass was analyzed by FTIR with two objectives: (a) to determine whether phenol is bioaccumulated in the cell or biodegraded and (b) to analyze the biomolecular changes in the algal cell during the phenol removal process. Algal cells actively degrading 125 mg/l phenol were taken for this study. The harvested biomass was washed and dried in vacuum desiccator. One volume of dried algal sample was blended with 100 volume of dried KBr powder and pressed into tablets before measurement. The spectral acquisition was performed using a FTIR spectrometer (IR Affinity, Shimadzu, Japan) by means of 500 scans with 4 cm−1 of spectral resolution over the wave number range of 400–4000 cm−1. The spectrum was submitted to a 15-point smoothing filter for noise reduction. The characteristic peak areas were obtained using IR Solution FTIR software (Shimadzu, Japan).

Characterization of the Phenol Degradation Pathway Log phase algal biomass growing in Fog’s media supplemented with 125 mg/l phenol was harvested, washed thrice, and grounded in potassium phosphate buffer solution (50 mM) using a mortar–pestle 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 the supernatant was determined as per procedure given by Lowry et al. [31]. Phenol hydroxylase activity was analyzed in a assay mixture containing 50 mM potassium phosphate buffer solution (pH 7.2), 88 μg protein, 1.5 μM phenol, and 1.5 μM NADPH. Heatinactivated enzyme extract served as control. The incubation was stopped at equal intervals with 20 μl of 0.6 M perchloric acid. Samples were analyzed for phenol utilization and concomitant accumulation of the reaction product catechol by HPLC. The ortho-cleavage of catechol (hydroxylation product of phenol) to cis,cis-muconic acid is carried out by catechol-1,2-dioxygenase. Catechol-1,2-dioxygenase activity was analyzed in a reaction mixture containing 50 mM potassium phosphate buffer pH 7.2, 88 μg protein, and 1.5 μM catechol. Catechol cleavage to cis,cis-muconic acid was analyzed by HPLC. Catechol2,3-dioxygenase is responsible for meta cleavage of catechol to 2-hydroxymuconic semialdehyde (2-HMS). Catechol-2,3-dioxygenase activity was determined by increase in absorbance at 375 nm due to accumulation of the reaction product 2-HMS (E375 =14,700 mol L−1 cm−1). The breakdown products of cis,cis-muconic acid and 2-HMS were identified by electrospray ionization liquid chromatography–mass spectrometry (LC-MS) (Make: Agilent, Model: Infinity LC system) in negative charge mode. To characterize the metabolites, the catechol dioxygenase assay was carried out as mentioned above. The LC-MS was operated using acetonitrile/water (60:40) mixture as solvent at a flow rate of 0.5 ml/min with detector at 270 nm. The m/z signals corresponding to the metabolites were identified using the Tandem Mass Spectrum database (open source—Central Drug Research Institute, India).

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

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multimode microplate reader (Infinity, Tecan, Switzerland) at excitation and emission wavelengths at 490 and 580 nm, respectively. The excitation and emission standards were determined based on pre-scan characteristics of neutral lipid standard, triloein (Himedia, India). A standard curve of triloein (R2 =0.99) in the concentration range of 5–100 μg/ml was used for quantification of neutral lipids. The Nile red stained cells were also observed under a fluorescence microscope (CX41, Olympus, Japan) as per Greenspan et al. [34] and imaged at ×100 magnification.

Results and Discussion Biomass Growth and Phenol Degradation The effect of phenol concentration on biomass growth profile and experimental specific growth rate of C. pyrenoidosa have been shown in Fig. 1a and b, respectively. The growth curves 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 concentrations preceding it as seen from Fig. 1a. Lag phase is followed by exponential growth phase, which is simultaneously followed by phenol utilization by the biomass. After the exponential growth period, phenol is depleted, and the microalgae enters the stationary phase. The biomass growth even when phenol is depleted may be explained by biotransformation of phenol into its metabolic 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 results of appearance of lag phase with increased phenol concentration, simultaneous phenol transformation in the exponential phase, appearance of stationary phase concomitant with phenol depletion, and increase in biomass even after complete phenol utilization. From the growth curve, the specific growth rate of 0.16 day−1 of control culture was found to be comparatively lower as compared to specific growth rate achieved in presence of phenol (Fig. 1b). This higher specific growth rate is probably because of phenol utilization as an organic carbon source by C. pyrenoidosa. The specific growth rate was found to increase with increase in substrate concentration until the highest value of 0.65 day−1 was attained at phenol concentration of 125 mg/l (Fig. 1b). The highest total chlorophyll content of 27.03 mg/l in 125 mg/l phenol cultures is 26.07 % higher than that of 21.44 mg/l total chlorophyll in control cultures further 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 beyond 125 mg/l, suggesting growth inhibition effect of phenol. Utilization of phenol as an organic carbon 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 pyruvate and CO2, which can contribute to biomass growth. To determine the phenol degradation profile, the residual phenol was estimated by HPLC with a retention time of 19.4 min. HPLC data shows complete phenol degradation as shown in Fig. 2a. The specific degradation rate increases with phenol concentration until a maximum rate of 0.29 day−1, which was achieved at 125 mg/l phenol (Fig. 2b.). It is due to the highest specific 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 by inhibited biomass growth. Mathur and Mazumdar [37] observed similar phenomena during phenol degradation by P. putida. They reported that both specific growth and degradation rate

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Fig. 1 a Biomass growth profile of Chlorella pyrenoidosa in various initial phenol concentrations. b Variation in specific growth rate of Chlorella pyrenoidosa in various initial phenol concentrations

increases with increase in phenol concentration until a maximum value of 100 mg/l. They also reported that both growth and degradation rate declines due to substrate inhibition of phenol concentration beyond 100 mg/l. To negate the effect of any abiotic factors in phenol removal, the loss of phenol from culture media without C. pyrenoidosa was determined, and a 1 % abiotic loss of phenol was found within 4 days as compared to complete removal of phenol in C. pyrenoidosa inoculated cultures. This proves that C. pyrenoidosa is solely responsible for phenol removal from the sample. To determine if the process of phenol degradation in C. pyrenoidosa is photodependent, microalgal cells were incubated in phenol under dark condition. During this process, the biomass growth and phenol degradation have been found to be negligible as compared 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/dark cycle (81.56 % phenol removed within 3 days). It resembles the observation of Papazi and Kotzabasis [38]. They reported that phenol biodegradation is a photoregulated response in the algae Scenedesmus obliquus. They showed that phenol biodegradation by S. obliquus was

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

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reduced to 5 % in dark. Scraag [10] reported that there is no growth as well as phenol degradation by C. vulgaris under dark condition, which is in accord with the present observations. Attempts were also made to understand the dynamics of biomass growth and phenol degradation of C. pyrenoidosa in refinery wastewater. The refinery wastewater was quantified to contain 23.33 mg/l phenol. The pH of the refinery wastewater was noted as 7.9. Intending to understand the natural growth and phenol degradation profile of C. pyrenoidosa, the experiment in refinery wastewater was carried out without attempting to meet the nutritional requirement 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 refinery wastewater. The biomass growth profile indicates an initial lag phase of 2 days unlike that in 25 mg/l phenol supplemented Fog’s media where no lag phase was observed. After the lag phase, the biomass grows exponentially on the fourth day and then enters the stationary phase on the 5th day with a final biomass of 339 mg/l. There is no phenol removal by C. pyrenoidosa when it is in the lag phase of growth (till second day). After the second day, the microalgae uptakes phenol, which is coincident with exponential growth of the algal biomass. C. pyrenoidosa mineralized 38.32 % of phenol in refinery wastewater by 7 days unlike complete mineralization of 25 mg/l phenol concentration by third day in Fog’s media. Agarry et al. [39] reported inhibition of complete mineralization of 30 mg/l phenol in refinery wastewater by Pseudomonas aeruginosa and Pseudomonas fluorescens, which correlates well with present finding. P. aeruginosa mineralized 94.5 %, while P. fluorescens mineralized 69.4 % of initial phenol concentration. However, contrary to the present work, they added mineral salt medium to refinery wastewater to meet the nutritional requirement of the microorganism for proper growth. Refinery wastewater may contain other constituents that may prove inhibitory to the phenol degradation potential of the microorganisms [39]. Characterization of the nature of oil present in refinery wastewater by UV-spectophotometry shown in Fig. 3b indicates that the refinery wastewater before treatment (day 0) consists of both alkane (absorbance at 215–230 nm) and aromatic compounds (absorbance at 250–260 nm). Since the peak of maximum absorbance is around 215 nm, the nature of oil in refinery wastewater is clean oil. Dotted line (Fig. 3b) represents the oil component characteristics in wastewater after treatment with C. pyrenoidosa for 8 days, indicating the degradation of both alkanes and aromatic compounds. This adds to the potential of the algal candidate. Cometabolism of other substrates along with phenol may have slowed the phenol degradation rate.

Growth and Degradation Kinetic Modeling: Biokinetic Parameter Evaluation and Performance Assessment The behavior of biodegradation rate is a strong function of biomass growth rate. Any growth medium where the microbial population can double itself faster will potentially result in higher biodegradation rate [39]. Understanding of a microorganism’s degradation and growth kinetics will bring out its potential for phenol biodegradation [40]. Thus, an attempt has been made to find out the mathematical relationship between (a) growth rate of biomass and substrate concentration and (b) phenol degradation rate and its initial concentration. The results obtained by solving the various growth kinetic models have been tabulated in Table 1. From this table, it is clear that Yano model yielded comparatively high R2 value and least SD value confirming that Yano model best fitted the experimental data. A comparative plot of experimental and model predicted specific growth rates has been shown in Fig. 4a. The value of KS (half

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

saturation coefficient) indicates the affinity of the microorganism to the substrate. The value of KI (substrate inhibition constant) signifies the degree of resistance of microorganism to the toxic effect of the substrate [41]. The substrate consumption rate is the most important parameter for denoting microbial degradative performance [42]. Initial phenol concentration has strong influence on specific degradation rate making kinetic analysis of substrate consumption essential [39]. The value of kinetic parameters obtained by solving the various degradation kinetic models has been shown in Table 2. The specific degradation rate predicted by the various kinetic models at different initial concentration of phenol has also been plotted graphically in Fig. 4b. Table 2 indicates that Yano model yielded the highest correlation coefficient (R2) and the least standard deviation (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 the applicability of C. pyrenoidosa for phenol removal from refinery wastewater. The kinetic parameters obtained for various growth kinetic models have been shown in Table 3. Table 3 shows that Haldane model yielded the highest correlation coefficient (R2) and the least standard deviation (SD) and thus best fitted the experimental data. Table 4 describes the kinetic parameters of degradation kinetic models, and the Haldane model shows both highest correlation coefficient (R2) and least standard deviation (SD). Thus, the Haldane model represents appropriately the phenol degradation behavior of the microalgae in refinery wastewater used in the present study.

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

μmax (day−1)

Ks (mg/l)

KI (mg/l)

Haldane

5.572

444.1

24.46

Yano

4.344

410.5

214.5

32.26 519.7

Webb

5.394

480.74

15.44

Aiba Edward

7.15 28.1

472.5 111.5

132.7 105.3

K (mg/l)

R2

SD

0.94

0.049

0.97

0.035

0.92

0.055

0.95 0.95

0.045 0.044

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Fig. 4 a Growth kinetic model fitted to experimental batch growth data of C. pyrenoidosa. b Degradation kinetic model fitted to experimental batch degradation data of C. pyrenoidosa

Comparison of the biokinetic parameters may give the indication of how C. pyrenoidosa behaved under two significantly different culture conditions of nutrient sufficient media and refinery wastewater. For this reason, a comparison was made between the respective biokinetic parameters of best fit kinetic models representing the biomass growth and phenol degradation behavior. Kinetic modeling of the experimental biomass growth data suggests that μmax (0.017 day−1) and KI (10.46 mg/l) is lower in refinery wastewater (first row in Table 3) as compared to that in nutrient media (second row in Table 2). The Ks value (600.1 mg/l) in refinery wastewater (first row in Table 3) is higher than that in nutrient media (second row in Table 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 nutrient media (second row in Table 2). The Ks value (300.99 mg/l) in refinery wastewater (third column of first row in Table 4) is higher compared to that in nutrient media (thirrd column of second row in Table 2). Lower μmax values in refinery wastewater is probably due to the lack of optimal nutrient factors for growth as well as other growth inhibitory constituents, which may be present in refinery wastewater. Maximum degradation rate (qmax) is also lower in refinery 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 media containing optimal biomass growth conditions. Secondly, cometabolism of alkanes in refinery wastewater along with phenol (aromatic) as discussed in “Biomass growth and phenol degradation” may lead to decrease in phenol degradation rate (qmax). Ks being inversely related to affinity of microbial system for substrate, a higher Ks value indicates its lower affinity to the Table 2 Estimated value of degradation kinetic parameters during phenol biodegradation by C. pyrenoidosa (NCIM 2738) in phenol-containing nutrient media Model

qmax (day−1)

Ks (mg/l)

KI (mg/l)

Haldane

0.55

89.99

100.24

–

0.73

0.05

Yano

0.76

170.60

250.6

86.54

0.81

0.04

K (mg/l)

R2

SD

Webb

0.30

77.93

100.9

350.06

0.70

0.07

Aiba Edward

0.71 2.78

58.13 77.94

200.40 100.9

– –

0.65 0.76

0.08 0.06

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

μmax (day−1)

Ks (mg/l)

KI (mg/l)

K (mg/l)

R2

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 Edward

0.019 0.03

572.5 50.94

70.7 70.9

– –

0.84 0.34

0.023 0.05

substrate [43]. Higher Ks value suggests a decreased affinity for phenol of C. pyrenoidosa in refinery wastewater compared to that in nutrient media. Higher Ks value in refinery wastewater explains inhibition of complete phenol mineralization unlike complete mineralization in media. C. pyrenoidosa utilizes phenol less efficiently in refinery wastewater due to decreased affinity for phenol. KI value is involved in quantification of the effect of toxicity of a compound during the biodegradation process. A higher KI value implies less sensitivity of the microbe to substrate inhibition. Lower KI value in refinery wastewater suggests high sensitivity 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 the microalgae counter the inhibitory effect of phenol in a better way. On the other hand, lack of optimal biomass growth factors and possible presence of other additional inhibitory constituents in refinery wastewater compromises the ability of C.pyrenoidosa to resist the inhibitory effect of phenol.

Characteristics of Cell Surface Morphology on Phenol Treatment The SEM micrograph (Supp. Fig. 3a and b.) shows that phenol exposure affects the membrane morphology of cells. When cells were exposed to 200 mg/l phenol for 48 h, the cell surface was found to be wrinkled. Accumulation of phenol in the hydrophobic part of the membrane leads to disturbance in the interactions between the acyl chain of phospholipids. This causes modification 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 biochemical changes in response to phenol. Adsorption at 3444–3419 cm−1 is due to stretching of OH groups of alcohol, phenol, or carboxyl OH and hydrogen vibration of the amide N–H [45]. Table 4 Estimated degradation kinetic parameters for Chlorella pyrenoidosa (NCIM 2738) during phenol degradation in refinery wastewater Model

qmax (day−1)

Ks (mg/l)

KI (mg/l)

Haldane

0.012

300.99

53.24

–

0.99

0.02

Yano

0.018

220.6

200.6

40.54

0.48

0.04

K (mg/l)

R2

SD

Webb

0.107

350.93

100.9

400.06

0.11

0.09

Aiba Edward

0.054 0.058

208.13 77.94

30.4 100.9

– –

0.33 0.19

0.05 0.08

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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 the third 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 increased intracellular phenol accumulation with incubation time. Since the microalgae completely removed 125 mg/l phenol from the culture medium by the second day (Fig. 2a), its high intracellular accumulation is quite evident. An increased peak area indicates increase in concentration of functional groups whose stretching/bending is responsible for the peak. Thus, peak area differences have been successfully used to monitor change in concentration of different biomolecules as monosaccharides in Enterobacter cloacae [46], amide I and II, cellulosic compounds, nonstructural carbohydrates in different barley varieties [47], as well as erythromycin quantification in pharmaceutical formulations [48]. Similarly, comparatively higher percent transmittance as well as low peak area due to low intracellular phenol uptake on day 1 is explained by lower phenol removal rate till day 1. The first metabolic intermediate of phenol degradation pathway is catechol. The metabolic intermediates formed by breakdown of catechol contains carboxylic acid group (−COOH) [49]. The infrared spectra region 1754– 1710 cm−1 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 with phenol incubation, which is not evident in control cells. This shows that the accumulated intracellular phenol is metabolized into intermediate products downstream of catechol. Wharfe et al. [49] reported similar findings of an increased infrared absorbance due to carbonyl group vibration of intermediate products of phenol metabolism in a microbial consortium. The infrared region 1440–1380 cm−1 is attributed to CH bending of aliphatic groups. Decreased percent transmittance (Supp. Fig. 4a) and an increased peak area (Supp. Fig 4d) in this region suggest an increased accumulation of aliphatics in phenol-incubated cells. Intracellular accumulation of aliphatic intermediates of phenol metabolism may be associated to the decreased transmittance and increased peak area. Thus, infrared analysis suggests intracellular uptake of phenol by C. pyrenoidosa, and then phenol is broken down into intermediate products. This breakdown of intracellular uptaken phenol into its intermediate metabolites confirms the process of phenol removal to be biodegradation.

Elucidation of Phenol Degradation Pathway The phenol metabolic pathway was characterized by identifying the different intermediates produced during phenol degradation using HPLC, UV–visible spectrophotometry, and LCMS. Supplementary Fig. 5a and b suggests appearance of catechol peak (11.9 min) with progressive decrease in phenol peak (10.69 min), which confirms catechol accumulation in media (extra cellular). The present results also accord with the observation reported by ElSheekh et al. [11] for different algal species as V. aureus, N. linkia, and O. rubescens. It proves that phenol is degraded through an enzymatic pathway in C. pyrenoidosa. Intracellular phenol hydroxylase 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 to„ cis,cis-muconic acid by catechol-1,2-dioxygenase activity. c Meta cleavage of catechol to 2-hydroxymuconic semialdehyde (2-HMS) by catechol-2,3-dioxygenase activity. d LC-MS analysis of catechol dioxygenase assay mixture at 0 min (before incubation). e LC-MS analysis of catechol dioxygenase assay mixture at 20 min (after incubation). f Proposed pathway of phenol degradation in Chlorella pyrenoidosa (NCIM 2738)

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a

b

c

d

e

f

Phenol Phenol hydroxylase

Catechol

Catechol-1,2-dioxygenase

cis,cis-muconate

Catechol-2,3-dioxygenase

2-hydroxymuconic semialdehyde 2-hydroxymuconate semialdehyde hydrolase

Cis-2-hydroxypenta-2,4-dienoate β-ketoadipate

Citrate cycle

Acetaldehyde

Pyruvate

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heat-killed enzyme extract showed no phenol hydroxylation activity (no catechol and no phenol 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 orthopathway) or meta-cleaved (if it follows meta-pathway) by catechol-1,2-dioxygenase and catechol-2,3-dioxygenase, respectively. Catechol-1,2-dioxygenase activity was characterized by identifying its ortho-cleavage product namely cis,cis-muconic acid using HPLC as shown in Fig. 5b. The reaction product was identified to be cis,cis-muconic based on identical retention time of 4.2 min with that of standard cis,cis-muconic acid. Control incubations carried out by heat-killed enzyme extract showed no catechol-1,2-dioxygenase activity. Catechol-2,3-dioxygenase activity was also determined by identifying 2-hydroxymuconic semialdehyde as the meta-cleavage product of catechol, using UV-visible spectrophotometry as shown in Fig. 5c. Control incubations by heat-killed enzyme extract showed no accumulation of meta-cleavage product 2-hydroxymuconic semialdehyde, indicating no catechol-2,3dioxygenase activity. Both catechol-1,2-dioxygenase and catechol-2,3-dioxygenase activity suggests that phenol metabolism involves both ortho- as well as meta-pathway. The catabolic efficiencies of phenol hydroxylase, catechol-1,2-dioxygenase, and catechol-2,3-dioxygenase were estimated on basis of specific enzyme activities, which are tabulated in Table 5. Comparatively higher activity of catechol-1,2-dioxygenase against that of catechol-2,3-dioxygenase suggests efficiency of ortho- over meta-pathway in C. pyrenoidosa. Similar results about simultaneous 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 reported similar findings of coexistence of both ortho- and meta-pathway in Fusarium species. Semple and Cain [8] reported involvement of meta pathway in golden brown chrysophyte alga O. danica, whereas most eukaryotes generally utilize ortho-pathway [53]. Evidence of ortho-activity in other eukaryotes as Trichosporon cutaneum [15], Penicillium sp. [54], Fusarium 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-hydroxymuconic semialdehyde (meta-pathway) were identified by LC-MS analysis. In order to identify the target metabolites, we are analyzing the catechol dioxygenase assay mixture both at the start and 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 of 131 is consistent with the molecular ion mass of [M+Na–2H]− adduct of catechol. Two more m/z signals of low abundance at 177 (abundance=2989) and 195 (abundance=3060) are also noted. The m/z signal at 177 corresponds the molecular ion mass of [M+K–2H]− adduct of cis,cis-muconic acid (ortho-cleavage product of catechol). Similarly, the m/z signal at 195 is consistent with the molecular ion mass of [M+Cl]− adduct of β-ketoadipate, a metabolite of the 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

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salts may be added during sample preparation. This justifies the high probability of formation of adduct ions during LC-MS of biological samples [56]. One probable source of potassium leading to formation of potassium ion adduct in our samples is possibly the utilization of potassium phosphate buffer during cell free extract preparation. The chloride ion adduct formed is one of the commonly formed metal adduct ion during negative ion electrospray analysis [56]. Figure 5e shows that the abundance of the m/z signal 131 decreases (abundance=3236) after 20-min incubation of the reaction mixture, which confirms utilization of catechol. The increase in abundance of m/z signal 177 (abundance=263,655) shows increase in accumulation of ortho-cleavage product of catechol, i.e., cis,cis-muconic acid. This indicates active ortho-pathway for phenol metabolism. Similarly, the increase in abundance of m/z signal at 195 (abundance=373,154) shows increase in accumulation of ortho-pathway intermediate, β-ketoadipate. Figure 5e shows an additional abundance at m/z 338 (abundance= 35,223) with a molecular ion mass identical to [3M–H]− adduct of cis-2-hydroxypent-2,4dienoate, a metabolite from the meta-cleavage pathway. Identification of adduct ion of cis-2hydroxypent-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 at m/z 163 corresponding to molar mass of sodium adduct of cis,cis-muconic acid in the enzyme activity 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 for phenol degradation in C. pyrenoidosa has been shown in Fig. 5f. In the pathway, phenol hydroxylase is involved in initial attack on phenol hydroxylating phenol to catechol. The resulting catechol is ortho-cleaved by catechol-2,3-dioxygenase as well as meta-cleaved by catechol-1,2-dioxygenase. Catechol-1,2-dioxygenase ortho-cleaves catechol into its reaction product cis,cis-muconic acid. Determination of 3-oxoadipate, a metabolite downstream of cis,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. 2Hydroxymuconate semialdehyde hydrolase causes hydrolysis of 2-HMS to cis-2Hydroxypenta-2,4-dienoate in the meta-pathway. Thus, both ortho- as well as meta-pathway is involved in phenol degradation in C. pyrenoidosa. However, ortho-pathway is significantly active 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 potential feedstock for applications as biodiesel and animal feed. Microalgal biomass with high lipid and protein content could serve as biodiesel feedstock [57] and protein supplement in animal diet, respectively [58]. Liu et al. [59] characterized lipid content in algal strains in a bid to identify high lipid producing strains. They reported C. pyrenoidosa to be one of the best oil producers whose total lipid content varies between 18.67 % to as high as 52.08 % of dry biomass. 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]. The biochemical characteristic of the algal biomass was analyzed using FTIR. FTIR analysis shown in Supp. Fig. 4a depicts a snapshot of the changes in biomolecular level in response to phenol stress. The infrared region 2875–2850 cm−1 corresponds to symmetric stretching of CH2 and CH3 of lipids [61]. With phenol incubation, there is prominent decrease in transmittance (Supp. Fig. 4a) and increase in peak area (Supp. Fig. 6a) in this region indicating higher

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cellular lipid accumulation compared to control biomass. Gracia et al.[57] reported increased lipid production in Phaeodactylum tricornutum UTEX-640 when cultured mixotrophically with carbon sources glycerol and fructose. Kong et al. [62] reported stimulation of lipid biosynthesis in C. vulgaris when mixotrophically cultured in glucose and glycerol. These findings accords with present finding of increased lipid accumulation in the presence of phenol as additional carbon source. However, high neutral lipid accumulation in algal biomass is necessary for its commercial applicability as biodiesel feedstock [32]. The profile for neutral lipid biosynthesis in C. pyrenoidosa cells (Fig. 6a) shows 50 % increased neutral lipid accumulation in phenol-degrading biomass as compared to control on fourth day of incubation. Hamed and Klock [63] reported similar results of enhanced neutral lipid accumulation in Chlorella sorokiniana during mixotrophic culture on glycerol. Fluorescence microscopy of phenol-degrading cells (Fig. 6b) showed enhanced yellow gold fluorescence of neutral lipid bodies in cell cytoplasm compared to that from control cells (Fig. 6c). This further supports the finding of enhanced neutral lipid accumulation in phenol-degrading biomass. Therefore, the algal biomass (after phenol biodegradation) could serve as potential raw material for biodiesel production. Although mixotrophic cultivation allows microalgae to accumulate higher proportion 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 algal cultivation. This makes the process of production of algal biomass feedstock for biodiesel costly [64]. Thus, cheap alternative carbon sources for mixotrophic cultivation of algae could help commercialize algal biodiesel by reducing the cost of production of algal biomass feedstock. The present study shows that C. pyrenoidosa accumulates high proportion of lipids while metabolizing phenol, which is a waste product of various industrial processes. Thus, mixotrophic cultivation of C. pyrenoidosa using industrial waste phenol as a carbon source could provide exciting possibility to decrease the production cost of algal biomass feedstock for biodiesel.

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

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The infrared region 1200–900 cm−1 corresponds to symmetric stretching of C–O–C of polysaccharides [61]. The infrared region 1610–1685 cm−1 is associated to C=O stretching of proteins [61]. Phenol incubation causes increased intracellular protein synthesis compared to control as evident from decreased transmittance (Supp. Fig. 4a) as well as increased peak area (Supp. Fig. 6b) in this region (1610–1685 cm−1) in phenol-degrading biomass. Thus, the protein rich algal biomass (after phenol degradation) could be used as an animal feed supplement. Since the amino acid profiles of microalgal protein is comparable to other food proteins, it could serve as a protein supplement in animal feed [65]. Although microalgae have been widely used as protein source to supplement animal diets, recent research trend in this area is supplementation of animal diets with defatted (lipid extracted) microalgal biomass from biodiesel production processes [66]. Since phenol-degrading biomass of C. pyrenoidosa is lipid as well as protein rich (increased accumulation of lipid and protein during the phenol degradation process), the biomass after lipid extraction (for biodiesel production) could serve as a protein supplement in animal diets.

Conclusion This study showed photodependent phenol degradation capability of C. pyrenoidosa with complete degradation till 200 mg/l phenol concentration under the optimal nutrient conditions of Fog’s medium. The maximum specific rate of degradation was achieved at 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 of aliphatics from petroleum refinery wastewater. Biokinetic parameters obtained by kinetic modeling shows the differences in growth and phenol degradation dynamics of C. pyrenoidosa in nutrient media and petroleum refinery wastewater. Low μmax values obtained by kinetic modeling in refinery wastewater is probably related to lack of optimal growth factors as well as other inhibitory constituents, which may be present in refinery wastewater. Cometabolism of alkanes along with decreased μmax values may be responsible for decreased phenol degradation rates (low qmax value) as well as decreased phenol affinity (high Ks value)in refinery wastewater. SEM analysis indicates that the cellular membrane morphology gets wrinkled on phenol exposure. C. pyrenoidosa metabolizes phenol simultaneously by both ortho- as well as metapathway. The ortho-pathway is significantly predominant over the meta pathway. The phenol-degrading biomass has 50 % higher neutral lipid accumulation compared to control cells, suggesting exciting possibility to utilize the spent biomass as biodiesel feedstock. The defatted biomass could additionally serve as animal feed owing to its enhanced protein content. Thus, the mixotrophic growth of C. pyrenoidosa on industrial waste phenol could prove to be an environmentally sustainable process as it will cause remediation of the toxic waste phenol along with generation of biodiesel feedstock with decreased production costs solving a major bottleneck in commercialization of algal biodiesel. Acknowledgments Bhaskar Das acknowledges Indian Institute of Technology, Guwahati, for providing research 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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