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Effect of cresols treatment by microalgae on the cells’ composition
Journal of Water Process Engineering 26 (2018) 250–256
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Effect of cresols treatment by microalgae on the cells’ composition Riham Surkatti, Sulaiman Al-Zuhair
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T
Chemical Engineering Department, UAE University, 15551 Al Ain, United Arab Emirates
A R T I C LE I N FO
A B S T R A C T
Keywords: Microalgae Cresols Chemical composition Wastewater treatment
Microalgae is considered the most promising source of oils for biodiesel production. Beside microalgae contain proteins and pigments, which have large applications in the food and pharmaceutical industries. Combining the cultivation of microalgae for the production of these compounds with wastewater treatment renders the overall process very attractive and economically feasible. However, the selection of the most suitable application to be coupled with the wastewater treatment depends on the composition of the harvested microalgae. In the present work, the effectiveness of Chlorella sp. for the degradation of p-cresol have been evaluated at a concentration ranging from 35 to 330 mg/L. The effect of initial concentration on the contaminants removal, biomass productivity and the chemical composition of microalgae has been determined. Results shows that, Chlorella sp. can degrade p-cresol at concentration up to 330 mg/L, and use it for biomass growth. Biochemical assay showed an improvement in the lipid productivity at higher concentrations, combined with the reduction in the protein content. The highest pigment composition was obtained at the optimum biomass growth, at 150 mg/L. These results give deep understanding of the factors that must be considered when integrating wastewater treatment with using the harvested biomass in other applications.
1. Introduction Wastewater discharged from refineries contain high concentrations of cresols in the range of 99–130 mg/L (Table 1) [1]. Several techniques have been tested for the removal of cresols from refinery wastewater, including physical, chemical, and biological methods [2,3]. Compared to other techniques, biological treatment is less expensive, more environmental friendly, and leads to complete mineralization of the toxic compounds [4]. Due to their higher contaminants biodegradation rates, bacteria has been the most commonly used microorganism in biological treatment units [5], despite the successful use of fungi [6] and yeasts [7]. However, the bacteria commonly used for cresols removal, such as Pseudomonas putida, are pathogenic. In addition, the collected biomass after the treatment does not have a clear economic value. Some strains of microalgae on the other hand, have shown good capacity to utilize toxic compounds, including phenols, as a source of carbon and energy. Among these were fresh water strains, such as Chlorella sp. [9] and Scenedesmus obliquus and [10], and marine strains Nannochloropsis sp. [11]. Using microalgae for removing phenols allows the utilization of the produced biomass for producing valuable compounds [12], including lipids for biodiesel production [12–14] and proteins and pigments for food and pharmaceutical applications. Due to their abundance and amino acid profile, microalgae proteins have long
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been considered as an alternative protein source in foods. More importantly, these proteins have been tested as antitumor agents, and shown promising results [15]. Microalgae pigments also have large applications in the pharmaceutical industries as antioxidant, anticancer, immune regulation and immune suppressive. For example, carotenoids, can be used as pro-vitamin A source, antioxidant and anticancer [16]. Cresols are known to be more harmful compounds than phenol [17]. In addition, as shown in Table 1 cresols are found in amounts higher (5–6 times) than that of phenol in refinery wastewaters. Nevertheless, most studies reported on the treatment of phenols using microalgae were done on phenol. Furthermore, the study of the effect of the concentration of the cresols, in the wastewater to be treated, on the composition of the microalgae is lacking in literature. This work provides a deep understanding of this effect, which allows integrating the wastewater treatment with using the harvested biomass in other applications. 2. Materials and methods 2.1. Chemicals and culture medium Two strains of microalgae, namely freshwater, Chlorella sp. and marine Nannochloropsis sp., were obtained from a local marine
Corresponding author. E-mail address: [email protected] (S. Al-Zuhair).
https://doi.org/10.1016/j.jwpe.2018.10.022 Received 18 July 2018; Received in revised form 22 October 2018; Accepted 31 October 2018 2214-7144/ © 2018 Elsevier Ltd. All rights reserved.
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R. Surkatti, S. Al-Zuhair
carbohydrates and pigments contents.
Table 1 Characterization of real refinery wastewater [8]. Parameter
Value
pH TSS TDS Phenol o-Cresol p-Cresol COD SO4 n-Hexane
8.3–8.9 0.03–0.04 g/L 3.8–6.2 g/L 8–10 mg/L 55–68 mg/L 44–61 mg/L 3970–4745 mg/L 14.5–16 mg/L 1.8–1.85 mg/L
2.3. Analysis methods 2.3.1. Cresols analysis For measurement of residual cresols concentration, the collected supernatant after centrifugation were filtered using a 0.45-μm syringe filter and analyzed using HPLC (Prostar, Varian, USA) equipped with UV–vis detector operating at 280 nm and a C-18 column maintained at 45 °C. The analysis was performed using two mobile phases; the gradient profile started with mobile phase consists of 30% Methanol: 70% water, the ratio of the methanol was increased until reaching 100%. The run time was about 30 min, with flow rate of 1 mL/min for the mobile phase mixture. The instrument was calibrated using samples of standard cresols of known concentrations.
environment research center in Umm Al-Quwain, UAE. The freshwater strain was cultivated in the modified bold bassel medium (3N-BBM) and the marine strain was grown in Guillard F/2 medium, as described in our previous publication [18]. Chemicals required for the preparation of the microalgae media, synthetic wastewater, analysis reagents and solvents were all obtained from Sigma–Aldrich, USA and included: MgSO4, CaCl2, K2HPO4, FeSO, Na2(EDTA), H3BO3, MnCl2.4H2O, ZnSO4.7H2O, Na2MoO4.2H2O, CuSO4.5H2O, H2SO4, triethanolamine, CuSO4, phosphate buffer, o-cresol, p-cresol, KOH, NaOH, phenols standard, pigment standard methanol, acetone, acetonitrile, chloroform and ethanol.
2.3.2. Biomass analysis The harvested cells were re-suspended in distilled water, and the biomass cell count concentration was determined using UV-spectrophotometer (UVe1800, Shimadzu, Japan) at 680 nm. The concentration (cells/mL) was calculated from a pre-prepared calibration curves of optical density vs. cell concentration determined using Neubauer Hemocytometer, placed on a Nikon, Eclipse LV100 Pol microscope. 2.3.3. Cells composition analysis The analysis of the carbohydrate, protein and lipid were assayed as previously reported [19]. The total carbohydrates were determined using the Anthrone method [20]. An aliquot of 0.2 mL were withdrawn and centrifuged. The harvested cells were suspended in 0.2 mL of distilled and 0.4 mL of 40% (w/v) KOH were added. The mixture was heated at 90 °C for 1 h, and after cooling to room temperature, 1.2 mL ethanol was added and kept overnight at −20 °C. The sample was then centrifuged again, and the harvested cells were re-suspended in 1.5 mL distilled water and mixed with 0.8 mL of 75% H2SO and 0.4 mL of anthrone reagent (2 g L in 75% H2SO4) and then boiled at 100 °C for 15 min. After cooling, the absorbance at 578 nm was recorded using a spectrophotometer. A blank absorbance for each sample was read without the anthrone reagent. The instrument was calibrated using serial dilutions of known concentrations of D-glucose. The proteins were determined using the microbiuret method [21]. Two aliquots of 0.1 ml of sample was centrifuged, one as sample and one as blank. The harvested cells were suspended in 1 mL 0.5 N NaOH and kept at 80 °C for 10 min. The sample was then centrifuged, and the supernatant was collected. The process was repeated three times to make sure that all the proteins were extracted. 0.05 mL of copper sulphate (0.21% CuSO4·5H2O in 30% NaOH) was added to the 3 ml of the samples and optical density was measured at 310 nm. The instrument was calibrated using serial dilutions of known concentrations of Bovine serum albumin. For lipids assay, the harvested cells were re-suspended in 20 μL phosphate buffer (0.05 M, pH 7.4) and 0.48 mL solution of 25% methanol in 1 N NaOH. The mixture was ultrasonicated (Branson Sonifier 450, USA) for 10 min. Another 1 mL of the methanol in NaOH solution was added, and saponified by heating at 100 °C for 30 min. An aliquot of 0.5 mL was added to 0.75 mL of a solvent mixture chloroform/methanol (2:1, v/v) and vortexed for 2 min. The mixture was then centrifuged to get two phases. The lower organic layer was collected and reacted with 1 M triethanolamine:1 N acetic acid, 9:1 (v/v) solution, the sample were vortexed for 2 min and centrifuged to separate the sample in two layers, the organic layer was collected and the absorptivity was measured at 260 nm. The concentrations were determined by comparing the optical density to that of serial dilutions of known concentrations of standard fatty acids (of carbon length C7–C18).
2.2. Experimental set-up 2.2.1. Screening experiments Screening experiments were first carried out on the biodegradation of p- and o-cresol to compare the general performances of freshwater and marine strains. In these experiments, two sets of 10 mL culture media (3N-BBM and F/2, for freshwater and marine cultures, respectively) were prepared with initial biomass concentration of (1.18 ± 0.16) ×106 cell/mL containing Chlorella sp or Nannochloropsis sp, with p- and o- cresols, at an initial concentration of 95 mg/L, were used as a sole carbon source. The concentration of the cresol used was similar to that of total cresols found in real refinery wastewater, which was reported to be 99 mg/L [8]. The cultures mixtures were covered with cotton, and placed in a water bath (Daihan Labtech, Korea) maintained at 30 °C and110 rpm for five days. The vials were subjected to sufficient light with a photoperiod of 12 h light/12 h dark. At the end of the experiment, mixtures were centrifugation using an IEC-CL Multispeed centrifuge (Model No. 11210913, France) at 6000 rpm for 5 min. The supernatants were sent for cresols analysis, whereas the harvested biomass were suspended in 10 ml distilled water to determine its concentration. 2.2.2. Biodegradation experiments In this section, experiments were carried out on the treatment of pcresol using with Chlorella sp. cultures in 200 mL volume. The cultures consisted of the 3N-BBM medium with different concentrations of pcresol ranging from 95 to 330 mg/L, and inoculated with Chlorella sp., at an initial concentration of (1.18 ± 0.16) x106 cell/mL, which corresponds to an optical density of 1.2771. The flasks were covered with cotton and placed in the same water bath, at the same condition, used in the screening experiment. The flasks were subjected to sufficient light with a photoperiod of 12 h light/12 h dark. Control experiments were carried out using the same mixtures, but without microalgae, subjected to similar conditions. On a daily basis, 5 mL samples were collected and the microalgae cells were harvested by centrifugation at 6000 rpm for 5 min. The supernatants were sent for cresols analysis, whereas the harvested biomass were suspended in 5 ml distilled water to determine its concentration. At the end of the experiment (in day 5), the biomass was harvested by centrifugation and was analyzed for its proteins, lipids,
2.3.4. Pigments analysis The pigments content was determined using HPLC equipped with 251
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the same column used for the cresols analysis. The mobile phase consisted of two solvents; Solvent A [Acetonitrile/Methanol (60:40)] and Solvent B [Methanol/Ethyl Acetate (70:30)]. The flow rate through the column was 1.3 mL/ min. A gradient flow composition of mobile phase was used for best chromatographic results. The mobile phase gradient started with 100% solvent A, and changed to 100% solvent B in 8 min, and kept in that composition for 7 min, then returned to 100% A in 1 min. It was then left at this composition for another 25 min to stabilize the column for next injection. The sample injection volume was 20 μl, and the wavelength was 436 nm. The instrument was calibrated using serial dilution of standard pigments (chlorophyll A and B, and total carotenoids) in acetone. The calibration curve ranged between 0.005 μg/ml to 5 μg/ml was used. Fig. 2. Changes in p-cresol concentration (S) with time at initial Chorella sp. concertation (Xo) of 1.18 × 106 cell/mL, 30 °C, 110 rpm and different initial pcresol concentrations (So).
2.4. Statistical analysis Each experiment was performed in duplicate and the average values of the results were determined. The accuracy of the experimental results was evaluated from the standard deviations, shown as error bars in the figures. To determine the significance of a factor, the regression pvalue, was calculated using ANOVA test by Minitab 16 statistical software (MiniTab Inc.). Second order regression was selected for data that shows an increase then decrease behavior, whereas linear regression was selected for data that has single trend.
cresol. Therefore, subsequent deeper investigations were selected to be conducted on Chlorella sp. using p-cresol. The work can be extended in the future to do deeper analyses on the other strain and the other cresol. 3.2. Biomass growth and cresols degradation The effect of cultivation of Chlorella sp. in 3N-BBM medium of different initial p-cresol concentrations, ranging from 35 to 330 mg/L, was tested at initial biomass concertation, Xo, of 1.18 × 106 cell/mL, temperature of 30 °C, and agitation of 110 rpm. The changes in the cresol and the biomass growth (presented as ln X/Xo) over a period of five days are shown in Figs. 2 and 3, respectively. Running a parallel experiment without microalgae, showed minimal drop in the concentration over the five days, which did not exceed 1.5% at the highest concentration of 330 mg/L. This proves that the drop on p-cresol was mainly due to the bioactivity of the microalgae. The biodegradation procedure of p-cresol was proposed to go through two step process [22]. In the first step, the methyl group is split, which could be converted to methanol, and in the second the intermediately produced phenol is broken. It should be noted that the data shown in Figs. 2 and 3 are average values of two runs. The reproducibility of the data is confirmed from the small error bars shown in Fig. 2. The error bars in Fig. 3 were not shown to avoid overcrowding the graph, but the average relative error was 7.6%. The error bars for the growth can still be seen from the specific growth rate results shown in Fig. 4. It was clearly seen from Fig. 2, that except for the highest concentration of 330 mg/L, Chlorella sp. was capable of removing most of the p-cresol within 4 days. For concentrations up to 260 mg/L, Chlorella sp. was able to completely remove p-cresol. However, concentrations higher than 260 mg/L appeared to be inhibiting the microalgae capacity to degrade p-cresols, and at 330 mg/L only 20% removal percentage was achieved within 5 days. Fig. 3 shows that the microalgae was
3. Results and discussion 3.1. Bioremoval of p- and o-cresols The work started with a screening test to compare the effectiveness of marine and freshwater strains in degrading p- and o- cresols. The objective of this screening test was to find out whether one of the two strains may not grow in the cresols or show superior performance compared to the other. As mentioned earlier, the selected concentration was similar to that of total cresols found in real refinery wastewater [8]. Fig. 1a and b show the percentage removal of cresols and the concentration of the biomass after seven days, X, over the initial biomass concentration, Xo, which was 1.18 × 106 cell/mL, respectively. Both strains were found to degrade almost the same percentage of pcresol within the 5 days. The marine strain, Nannochloropsis sp., showed a slightly better growth rate than the freshwater strain, Chlorella sp. With o-cresol, the marine stain showed slightly better biodegradation performance, but surprisingly its growth was lower. As the cells use the degraded substrates for two functions, namely, production of new cells and maintenance of existing cells. Therefore, these results suggest that with o-cresol a smaller portion was directed towards Nannochloropsis sp cells growth, as compared to Chlorella sp. Both strains performed almost equally well. However, Chlorella sp growth and biodegradation were both better with p-cresol than with ocresol all, whereas with Nannochloropsis sp. the degradation of both types of cresols were similar, but the growth was much better in p-
Fig. 1. Percentage removal of cresols (a) and biomass growth (b) after growing at 30 °C and 110 rpm for 5 days in media containing Chlorella sp. and Nannochloropsis sp. at initial cresols (So) and biomass (Xo) concentrations of 95 mg/L and of 1.18 × 106 cell/mL, respectively. 252
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cresol biodegradation. [25]. Results showed high applicability at initial concentration of p-cresol biodegradation of 750 mg/L within 100 h. In addition, a bacterial strain, Pseudomonas putida, immobilized in PVA gel, was also used for the biodegradation of p-cresol, at concentrations range similar to the one used in this study (25 to 200 mg/L) [26]. Complete removal was achieved within only 40 min. On the other hand, using aerobic granulates, a longer time was required to remove p-cresol even at a low concentration (100 mg/L) [27]. To better understand the behavior, the biodegradation and specific growth rates were determined, and the results are shown in Fig. 4. The biodegradation rate was determined from the slope of the straight line in the initial part of the degradation. Whereas, the specific growth rate was determined from the slope of the logarithmic of X/Xo vs time in the exponential part, after the lag period. As expected it was found that both rates (biodegradation and specific growth) had similar trends. When the growth rate increased, the biodegradation rate increased and vice versa. It was found that the growth rate of Chlorella sp. increased with increasing the initial concentration of p-cresol, up to 140 mg/L, at which the optimum growth rate of 2.17 ± 0.17 day−1 was reached. Beyond that concentration, the growth started to slightly drop at 210 mg/L, and then sharply dropped after that, due to substrate inhibition. Similar behavior was observed for the p-cresol degradation, but the optimum drop was 51.51 mg/L.day at 210 mg/L. The effect of initial concentration on both the biodegradation rate and growth rate was significant, with a p-value of 0.05. The effect however, was less significant on the growth rate, with a p-value of 0.085. This results agree with previous study done using the same strain for phenol degradation [29]. The maximum biodegradation and growth rates were also obtained at an initial phenol concentration of 200 mg/L. The optimum drop rate was 54.5 mg/L.day, which were very close, but slightly higher than the one found in this work. However, a much lower growth rate of only 0.25 day−1, was reported. In addition to using a different substrate, the lower rate was due to the limited light intensity used in the previous study, suggested to enhance the heterotrophic growth. When p-cresol biodegradation was tested using a green microalgae Scenedesmus obliquus, he highest bioremoval of 100% was achieved at initial concentration of 0.162 mg/L, at which the biomass concentration increased by 20% of the initial value. [22]. At higher concretions the growth was inhibited. The results shown in Fig. 3, were used to determine the parameters of three growth models that incorporate limiting substrate-inhibition kinetics. The models used were Haldane, Aiba and Andrews, given in Eqs. (1)–(3), respectively
Fig. 3. Changes in the logarithmic of biomass concentration over initial concentration (ln X/Xo) with time at initial Chorella sp. concertation (Xo) of 1.18 × 106 cell/mL, 30 °C, 110 rpm and different initial p-cresol concentrations (So).
Fig. 4. p-Cresol degradation rate (dS/dt) and Chlorella sp. specific growth rate (μ) at initial Chorella sp. concertation (Xo) of 1.18 × 106 cell/mL, 30 °C, 110 rpm and different initial p-cresol concentrations (So).
able to grow in all concentrations of p-cresols, but with different growth rates. It was also noticed that as the initial concentration of p-cresol increased, the lag phase increased. This was expected, as the cells needed more time to adapt to the high substrate concentration. As mentioned earlier, the investigation of p-cresol removal using microalgae was rarely studied, and even in these limited studies, the concentrations tested were much lower than those found in real refinery wastewater. For example, eukaryotic algae, Ochromonas Danica, was tested for the biodegradation of p-cresol at initial concentrations in the range of 0.54 to 4.3 mg/L. The study showed complete removal of the p-cresol in the concentration range within a maximum period of 12 days [23]. Green microalgae, Scenedesmus obliquus, was also tested for the biodegradation but at even lower concentrations in the range 0.162 to 2.7 mg/L. [22]. The effect of the initial substrate concentration on the percentage removal was shown to be significant, dropping from 100% to 29%, when the initial concentration was increased from 0.162 to 2.7 mg/L [22]. As the concentrations of p-cresol in real refinery wastewater are order of magnitude higher than these tested ranges, the importance of the results found in this study, at the higher concentrations, becomes more obvious. Table 2 shows comparison in p-cresol biodegradation using several microorganisms at different initial concentration range. Filamentous fungal strain, Gliomastix indicus, showed higher performance for p-cresol removal than that of the tested microalgae in this work [24]. At high concentrations of up to 700 mg/L, more than 90% p-cresol was removed within about 4 days. In a more recent study, isolated bacteria from activated sludge were tested for p-
μ=
μ m So KS + So + S2o Ki
(1)
μ=
μ m So exp(−So Ki) K S + So
(2)
μ=
μm [(KS So) + 1]⋅[1 + (So Ki )]
(3)
Where, μ and μm are the specific and maximum specific growth rates (day−1), respectively, So is the initial substrate concentration (mg/L), and Ks and Ki are the substrate and inhibition constants (mg/L), respectively. The estimated values for the model kinetic parameters were determined by fitting non-linear regression model, using Excel solver with an objective function (O.F.) given by Eq. (4), and shown in Table 3. n
O.F. = ∑ (μ exp − μ pred.)2 i
(4)
Where, μ exp and μ pred are the experimental and model predicted specific growth rates. From the values of the coefficient of determination, R2, it can be 253
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Table 2 Biodegradation of p-cresol using different microorganisms. Microorganism type
strain
Reactor
Concentration range
Removal
Time
Ref.
Microalgae Microalgae fungus Bacteria Bacteria Bacteria Bacteria
Scenedesmus obliquus Ochromonas danica Gliomastix indicus Advenella sp Pseudomonas putida Mixed culture Aerobic granules
Shaking flasks Shaking flasks Shaking flasks Flasks Spouted bed bioreactor Batch reactor Sequencing batch reactor
0.54 to 2.7 mg/L 0.54 to 4.3 mg/L 10-700 mg/L 750 mg/l 25-200 mg/L 100 mg/L Up to 800 mg/L
29-100% 100% 90% 100% 100% 100% 88%
5 days 12 h 108h 100h 40 min 49 h 2 months
[22] [23] [24] [25] [26] [28] [27]
Table 3 Estimated value of microalgae growth kinetic parameters in p-cresol containing medium. Parameter
Haldane
Aiba
Andrews
μm Ks Ki R2
8.3 194.8 88.9 0.914
11.0 250.3 227.0 0.953
9.0 148.9 117.6 0.905
Fig. 6. Changes in proteins, lipids and carbohydrates contents of microalgae after growing for 5 days in media containing initial Chorella sp. concertation (Xo) of 1.18 × 106 cell/mL, 30 °C, 110 rpm and different initial p-cresol concentrations (So).
the wastewater treatment. Microalga biomass with high lipid content could serve as a good feedstock for biodiesel production. Whereas, the biomass with high protein content may be more suitable for pharmaceutical applications. Generally, the composition of the growth medium affects the biochemical composition of microalgae cells, by alternating of metabolic pathways in response to stress conditions [30]. Therefore, at the end of the 5 days, the biomass was collected and analyzed for proteins, carbohydrates, lipid and pigments content. The percentage compositions of proteins, carbohydrates and lipids are in Fig. 6, and those of the pigments, chlorophyll-a, chlorophyll-b and carotenoids are shown in Fig. 7. It was found that the lipids content increased with the increase in the p-cresol concentration. Growing in medium containing no p-cresol, the lipids content was 8.9%. This amount increased by 3.7 folds to 33%,
Fig. 5. Comparison between experimental data of Chlorella sp. specific growth rate (μ) and models predictions.
seen that all three models fitted the data well, with the Aiba model being the best, followed by the Haldane model. The goodness of the fitting can also be seen in Fig. 5, which shows the comparison between experimental data and models predictions. The results show that the values of μm for all models were similar, with that of the Aiba model being relatively the highest. The values of Ks and Ki of the Haldane and Andrews models were similar, whereas those of the Aiba model were higher. No previous studies on the kinetics of microalgae biodegradation of p-cresol are reported in open literature. The only available ones are with other microorganisms, such as bacteria and fungus. For example, kinetic of p-cresol degradation by fungal strains, Gliomastix indicus, have been studied using five several models [24]. The values of μm of Haldane, Aiba and Andrews were 9.5, 7.02 and 14.2 day−1, respectively, which are close to the ones found in this work. In addition, similar to this work, the highest value of μm was for Aiba model. However, with the fungal strains, the R2 values suggested that the Haldane and Andrews models represented the data better than Aiba model.
3.3. Effect of p-cresol on biochemical composition The most important aspect of this work was to determine the effect of the concentration of p-cresol on the chemical composition of the harvested microalgae. As mentioned earlier, this is important information for the determination of the application to be coupled with
Fig. 7. Changes in pigments contents of microalgae after growing for 5 days in media containing initial Chorella sp. concertation (Xo) of 1.18 × 106 cell/mL, 30 °C, 110 rpm and different initial p-cresol concentrations (So). 254
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and pigments composition, the experiment was repeated at the initial pcresol concentration of 190 mg/L that showed the optimum degradation and growth rates. In this experiment, the cells composition was measured twice a day for a period of two days, and the rate of the change was measured from the slope of the changes with time. The results show that the drop rate of proteins was -15.4% per day. A drop was also observed for carbohydrates, but at a much lower rate of 7.3% per day. The lipids show an increase with a rate of 19.1% per day.
by growing for 5 days in medium containing an initial concentration of 330 mg/L. The effect of the initial concentration of p-cresol on the lipid content was significant, with a p-value of 0.041. In contrast, the proteins content decreased with the increase in p-cresol concentration, from 58.7% with no p-cresol to 31.3 at 330 mg/L. The effect was also significant, with a similar extent to that on of lipids, with a p-value of 0.044. No significant change was observed in the carbohydrate level. This insignificant effect was also confirmed from the p-value, which was found to be 0.146. This finding suggests that for coupling wastewater treatment with biodiesel production, it would be preferable to cultivate the microalgae in wastewaters containing high concentrations of p-cresol. Whereas, cultivation in low concentrations may be more suitable for coupling with proteins extraction. The results found in this work agree with those found when the concentrations of nonylphenols, bisphenol, 17alpha-ethynylestradiol, and estradiol were increased in the media used for the cultivation of the marine diatom Navicula incerta [31]. It was also found that, the proteins content declined with the increasing in all compounds. The behavior of the lipids content was also similar, showing an increase in the lipid contents, with the increase in the concertation of the compounds in the media. An increase in the lipids content in diatom Skeletonema costatum was also observed by increasing the concentration of 2,4-dichlorophenol in the cultivating medium [32]. However, this was coupled with a decrease in carbohydrates content and the proteins showed no changes. Under environmental stress, microalgae direct their pathways towards storing energy in the form of lipid instead of using it as source of growth [33]. This was shown when Chlorella maxicana was cultivated under stress of salinity, showing an increase in lipids content from 23% to 37% as the NaCl concentration increased from 0.43 to 25 mM [34]. An increase in lipids was also observed when the cultures were stressed with nitrogen and phosphorus limitations [21,25,26]. In addition, previous studies confirmed the accumulation of lipids in Chlorella pyrenoidosa, when the cells are exposed to high phenol concentration, and the lipid accumulation was increased with increasing the incubation period [12]. Increasing of lipid biosynthesis was also reported during phenol degradation in diatom BD1IITG cells [35]. As shown in Fig. 7, chlorophyll A and chlorophyll B and carotenoids composition were found to be highly dependent on the initial p-cresol concentration. It was found that the pigments concentration increased with the increase in p-cresol concentration, to reach a maximum at 140 mg/L, then stated to drop after that. At low p-cresol concentrations, the contents of both chlorophylls a and b, were higher than that of carotenoids. The drop in chlorophylls content with the increase in pcresol concentration was more drastic that of carotenoids, and above 250 mg/L, the carotenoids content started to exceed that of chlorophyll b. chlorophyll A remained the main pigment under all tested concentrations of p-cresols. The effect of the initial p-cresol was most significant on chlorophyll A, with a p-value of 0.093, followed by carotenoids, with a p-value of 0.118 and least significant on chlorophyll b, with p-value of 0.203. These results are related to the enhancement in the biomass growth that encourage algal cells to produce more pigments at concentration with highest biomass growth rate. Similar trend was also obtained when Chlorella kessleri was cultivated in different concentrations of crude oils [36], with the maximum chlorophyll a and b contents achieved at the initial concentration that resulted in the highest biomass growth [36]. However, with Scenedesmus obliquus, the pigments production was found to decline with the increase in the initial concentration of crude oil, having the highest chlorophyll a content at the lowest oil concentration [37]. On the other hand, no changes in chlorophylls content of diatoms Navicula incerta [31] and Skeletonema costatum [32] was observed with increasing the initial concentration of the endocrine disrupting chemicals. To study the initial rate of the changes in proteins, carbohydrates
4. Conclusion This study demonstrates the capacity of Chlorella sp. for the biological treatment of wastewater contaminated with p-cresol. The biodegradability and cell growth were found to be affected by the initial cresol concentration. However the highest biomass productivity and biodegradation rate were obtained at initial p-cresol of 150 and 200 mg/L, respectively. The biodegradation kinetics were presented well by Haldane, Aiba and Andrews models, with Aiba model showing the best fit. The biomass composition of Chlorella sp. was also found to be affected by the initial concentration of p-cresol in the growth medium. As the initial concentration increased, the proteins reduced and the lipids increased, whereas the carbohydrates remains constant. The pigments content increased with the increase in p-cresol concentration to reach a maximum at 150 mg/L p-cresol, but then dropped at higher concentrations. Acknowledgement This work was supported by the Emirates Center for Energy and Environment Research [grant number 31R070] References [1] M.H. El-Naas, S. Al-Zuhair, A. Al-Lobaney, Treatment of petroleum refinery wastewater by electrochemical methods, Int. J. Eng. Res. Technol. 2 (10) (2013) 2144–2150. [2] A.H. Al-Muhtaseb, M. Khraisheh, Photocatalytic removal of phenol from refinery wastewater: catalytic activity of Cu-doped titanium dioxide, J. Water Process Eng. 8 (2015) 82–90. [3] L. Damjanović, V. Rakić, V. Rac, D. Stošić, A. Auroux, The investigation of phenol removal from aqueous solutions by zeolites as solid adsorbents, J. Hazard. Mater. 184 (1–3) (2010) 477–484. [4] T. Al-Khalid, M.H. El-Naas, Aerobic biodegradation of phenols: a comprehensive review, Crit. Rev. Environ. Sci. Technol. 42 (16) (2012) 1631–1690. [5] L. Levén, K. Nyberg, L. Korkea-aho, A. Schnürer, Phenols in anaerobic digestion processes and inhibition of ammonia oxidising bacteria (AOB) in soil, Sci. Total Environ. 364 (1–3) (2006) 229–238. [6] Y. Lu, L. Yan, Y. Wang, S. Zhou, J. Fu, J. Zhang, Biodegradation of phenolic compounds from coking wastewater by immobilized white rot fungus Phanerochaete chrysosporium, J. Hazard. Mater. 165 (1–3) (2009) 1091–1097. [7] J. Yan, W. Jianping, B. Jing, W. Daoquan, H. Zongding, Phenol biodegradation by the yeast Candida tropicalis in the presence of m-cresol, Biochem. Eng. J. 29 (3) (2006) 227–234. [8] M.H. El-Naas, R. Surkatti, S. Al-Zuhair, Petroleum refinery wastewater treatment: a pilot scale study, J. Water Process Eng. 14 (2016) 71–76. [9] J. Doucha, F. Straka, K. Lívanský, Utilization of flue gas for cultivation of microalgae (Chlorella sp.) in an outdoor open thin-layer photobioreactor, J. Appl. Phycol. 17 (5) (2005) 403–412. [10] A.P. Batista, P. Moura, P.A.S.S. Marques, J. Ortigueira, L. Alves, L. Gouveia, Scenedesmus obliquus as feedstock for biohydrogen production by Enterobacter aerogenes and Clostridium butyricum, Fuel 117 (PART A) (2014) 537–543. [11] S. Wahidin, A. Idris, S.R.M. Shaleh, The influence of light intensity and photoperiod on the growth and lipid content of microalgae Nannochloropsis sp, Bioresour. Technol. 129 (2013) 7–11. [12] B. Das, T.K. Mandal, S. Patra, A comprehensive study on Chlorella pyrenoidosa for phenol degradation and its potential applicability as biodiesel feedstock and animal feed, Appl. Biochem. Biotechnol. 176 (5) (2015) 1382–1401. [13] A. Demirbas, Importance of biodiesel as transportation fuel, Energy Policy 35 (9) (2007) 4661–4670. [14] T.M. Mata, A.A. Martins, N.S. Caetano, Microalgae for biodiesel production and other applications: a review, Renew. Sustain. Energy Rev. 14 (1) (2010) 217–232. [15] A. Jabeen, et al., Effect of Enzymatic pre-treatment of microalgae extracts on their anti-tumor activity, Biomed. J. 40 (6) (2017) 339–346. [16] I. Deniz, M. García-Vaquero, E. Imamoglu, Trends in Red Biotechnology: Microalgae for Pharmaceutical Applications, (2017).
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[17] M. Mahdavianpour, G. Moussavi, M. Farrokhi, Biodegradation and COD removal of p-Cresol in a denitrification baffled reactor: performance evaluation and microbial community, Process Biochem. 69 (March) (2018) 153–160. [18] H. Taher, S. Al-Zuhair, A. Al-Marzouqi, Y. Haik, M. Farid, Growth of microalgae using CO2enriched air for biodiesel production in supercritical CO2, Renew. Energy 82 (2015) 61–70. [19] Y. Chen, S. Vaidyanathan, Simultaneous assay of pigments, carbohydrates, proteins and lipids in microalgae, Anal. Chim. Acta 776 (2013) 31–40. [20] B. Kamlage, Methods for general and molecular bacteriology, Food / Nahrung 40 (2) (1996) pp. 103–103. [21] R.F. Itzhaki, D.M. Gill, A micro-biuret method for estimating proteins, Anal. Biochem. 9 (4) (1964) 401–410. [22] A. Papazi, K. Assimakopoulos, K. Kotzabasis, Bioenergetic strategy for the biodegradation of p-Cresol by the unicellular green alga Scenedesmus obliquus, PLoS One 7 (12) (2012). [23] K.T. Semple, Biodegradation of phenols by a eukaryotic alga, Res. Microbiol. 148 (4) (1997) 365–367. [24] R. Kant, S. Kumar, S. Kumar, A. Kumar, Biodegradation Kinetic Studies for the Removal of P -Cresol from Wastewater Using Gliomastix indicus MTCC 3869 vol. 40, (2008), pp. 293–303. [25] E. Xenofontos, A.M. Tanase, I. Stoica, I. Vyrides, Newly isolated alkalophilic Advenella species bioaugmented in activated sludge for high p-cresol removal, N. Biotechnol. 33 (2) (2016) 305–310. [26] R. Surkatti, M.H. El-Naas, Biological treatment of wastewater contaminated with pcresol using Pseudomonas putida immobilized in polyvinyl alcohol (PVA) gel, J. Water Process Eng. 1 (2014) 84–90. [27] F. Basheer, I.H. Farooqi, Biodegradation of p -cresol by aerobic granules in sequencing batch reactor, J. Environ. Sci. 24 (11) (2018) 2012–2018. [28] A. Hamitouche, Z. Bendjama, A. Amrane, Procedia Engineering Biodegradation of
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Effect of cresols treatment by microalgae on the cells’ composition Riham Surkatti, Sulaiman Al-Zuhair
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Chemical Engineering Department, UAE University, 15551 Al Ain, United Arab Emirates
A R T I C LE I N FO
A B S T R A C T
Keywords: Microalgae Cresols Chemical composition Wastewater treatment
Microalgae is considered the most promising source of oils for biodiesel production. Beside microalgae contain proteins and pigments, which have large applications in the food and pharmaceutical industries. Combining the cultivation of microalgae for the production of these compounds with wastewater treatment renders the overall process very attractive and economically feasible. However, the selection of the most suitable application to be coupled with the wastewater treatment depends on the composition of the harvested microalgae. In the present work, the effectiveness of Chlorella sp. for the degradation of p-cresol have been evaluated at a concentration ranging from 35 to 330 mg/L. The effect of initial concentration on the contaminants removal, biomass productivity and the chemical composition of microalgae has been determined. Results shows that, Chlorella sp. can degrade p-cresol at concentration up to 330 mg/L, and use it for biomass growth. Biochemical assay showed an improvement in the lipid productivity at higher concentrations, combined with the reduction in the protein content. The highest pigment composition was obtained at the optimum biomass growth, at 150 mg/L. These results give deep understanding of the factors that must be considered when integrating wastewater treatment with using the harvested biomass in other applications.
1. Introduction Wastewater discharged from refineries contain high concentrations of cresols in the range of 99–130 mg/L (Table 1) [1]. Several techniques have been tested for the removal of cresols from refinery wastewater, including physical, chemical, and biological methods [2,3]. Compared to other techniques, biological treatment is less expensive, more environmental friendly, and leads to complete mineralization of the toxic compounds [4]. Due to their higher contaminants biodegradation rates, bacteria has been the most commonly used microorganism in biological treatment units [5], despite the successful use of fungi [6] and yeasts [7]. However, the bacteria commonly used for cresols removal, such as Pseudomonas putida, are pathogenic. In addition, the collected biomass after the treatment does not have a clear economic value. Some strains of microalgae on the other hand, have shown good capacity to utilize toxic compounds, including phenols, as a source of carbon and energy. Among these were fresh water strains, such as Chlorella sp. [9] and Scenedesmus obliquus and [10], and marine strains Nannochloropsis sp. [11]. Using microalgae for removing phenols allows the utilization of the produced biomass for producing valuable compounds [12], including lipids for biodiesel production [12–14] and proteins and pigments for food and pharmaceutical applications. Due to their abundance and amino acid profile, microalgae proteins have long
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been considered as an alternative protein source in foods. More importantly, these proteins have been tested as antitumor agents, and shown promising results [15]. Microalgae pigments also have large applications in the pharmaceutical industries as antioxidant, anticancer, immune regulation and immune suppressive. For example, carotenoids, can be used as pro-vitamin A source, antioxidant and anticancer [16]. Cresols are known to be more harmful compounds than phenol [17]. In addition, as shown in Table 1 cresols are found in amounts higher (5–6 times) than that of phenol in refinery wastewaters. Nevertheless, most studies reported on the treatment of phenols using microalgae were done on phenol. Furthermore, the study of the effect of the concentration of the cresols, in the wastewater to be treated, on the composition of the microalgae is lacking in literature. This work provides a deep understanding of this effect, which allows integrating the wastewater treatment with using the harvested biomass in other applications. 2. Materials and methods 2.1. Chemicals and culture medium Two strains of microalgae, namely freshwater, Chlorella sp. and marine Nannochloropsis sp., were obtained from a local marine
Corresponding author. E-mail address: [email protected] (S. Al-Zuhair).
https://doi.org/10.1016/j.jwpe.2018.10.022 Received 18 July 2018; Received in revised form 22 October 2018; Accepted 31 October 2018 2214-7144/ © 2018 Elsevier Ltd. All rights reserved.
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carbohydrates and pigments contents.
Table 1 Characterization of real refinery wastewater [8]. Parameter
Value
pH TSS TDS Phenol o-Cresol p-Cresol COD SO4 n-Hexane
8.3–8.9 0.03–0.04 g/L 3.8–6.2 g/L 8–10 mg/L 55–68 mg/L 44–61 mg/L 3970–4745 mg/L 14.5–16 mg/L 1.8–1.85 mg/L
2.3. Analysis methods 2.3.1. Cresols analysis For measurement of residual cresols concentration, the collected supernatant after centrifugation were filtered using a 0.45-μm syringe filter and analyzed using HPLC (Prostar, Varian, USA) equipped with UV–vis detector operating at 280 nm and a C-18 column maintained at 45 °C. The analysis was performed using two mobile phases; the gradient profile started with mobile phase consists of 30% Methanol: 70% water, the ratio of the methanol was increased until reaching 100%. The run time was about 30 min, with flow rate of 1 mL/min for the mobile phase mixture. The instrument was calibrated using samples of standard cresols of known concentrations.
environment research center in Umm Al-Quwain, UAE. The freshwater strain was cultivated in the modified bold bassel medium (3N-BBM) and the marine strain was grown in Guillard F/2 medium, as described in our previous publication [18]. Chemicals required for the preparation of the microalgae media, synthetic wastewater, analysis reagents and solvents were all obtained from Sigma–Aldrich, USA and included: MgSO4, CaCl2, K2HPO4, FeSO, Na2(EDTA), H3BO3, MnCl2.4H2O, ZnSO4.7H2O, Na2MoO4.2H2O, CuSO4.5H2O, H2SO4, triethanolamine, CuSO4, phosphate buffer, o-cresol, p-cresol, KOH, NaOH, phenols standard, pigment standard methanol, acetone, acetonitrile, chloroform and ethanol.
2.3.2. Biomass analysis The harvested cells were re-suspended in distilled water, and the biomass cell count concentration was determined using UV-spectrophotometer (UVe1800, Shimadzu, Japan) at 680 nm. The concentration (cells/mL) was calculated from a pre-prepared calibration curves of optical density vs. cell concentration determined using Neubauer Hemocytometer, placed on a Nikon, Eclipse LV100 Pol microscope. 2.3.3. Cells composition analysis The analysis of the carbohydrate, protein and lipid were assayed as previously reported [19]. The total carbohydrates were determined using the Anthrone method [20]. An aliquot of 0.2 mL were withdrawn and centrifuged. The harvested cells were suspended in 0.2 mL of distilled and 0.4 mL of 40% (w/v) KOH were added. The mixture was heated at 90 °C for 1 h, and after cooling to room temperature, 1.2 mL ethanol was added and kept overnight at −20 °C. The sample was then centrifuged again, and the harvested cells were re-suspended in 1.5 mL distilled water and mixed with 0.8 mL of 75% H2SO and 0.4 mL of anthrone reagent (2 g L in 75% H2SO4) and then boiled at 100 °C for 15 min. After cooling, the absorbance at 578 nm was recorded using a spectrophotometer. A blank absorbance for each sample was read without the anthrone reagent. The instrument was calibrated using serial dilutions of known concentrations of D-glucose. The proteins were determined using the microbiuret method [21]. Two aliquots of 0.1 ml of sample was centrifuged, one as sample and one as blank. The harvested cells were suspended in 1 mL 0.5 N NaOH and kept at 80 °C for 10 min. The sample was then centrifuged, and the supernatant was collected. The process was repeated three times to make sure that all the proteins were extracted. 0.05 mL of copper sulphate (0.21% CuSO4·5H2O in 30% NaOH) was added to the 3 ml of the samples and optical density was measured at 310 nm. The instrument was calibrated using serial dilutions of known concentrations of Bovine serum albumin. For lipids assay, the harvested cells were re-suspended in 20 μL phosphate buffer (0.05 M, pH 7.4) and 0.48 mL solution of 25% methanol in 1 N NaOH. The mixture was ultrasonicated (Branson Sonifier 450, USA) for 10 min. Another 1 mL of the methanol in NaOH solution was added, and saponified by heating at 100 °C for 30 min. An aliquot of 0.5 mL was added to 0.75 mL of a solvent mixture chloroform/methanol (2:1, v/v) and vortexed for 2 min. The mixture was then centrifuged to get two phases. The lower organic layer was collected and reacted with 1 M triethanolamine:1 N acetic acid, 9:1 (v/v) solution, the sample were vortexed for 2 min and centrifuged to separate the sample in two layers, the organic layer was collected and the absorptivity was measured at 260 nm. The concentrations were determined by comparing the optical density to that of serial dilutions of known concentrations of standard fatty acids (of carbon length C7–C18).
2.2. Experimental set-up 2.2.1. Screening experiments Screening experiments were first carried out on the biodegradation of p- and o-cresol to compare the general performances of freshwater and marine strains. In these experiments, two sets of 10 mL culture media (3N-BBM and F/2, for freshwater and marine cultures, respectively) were prepared with initial biomass concentration of (1.18 ± 0.16) ×106 cell/mL containing Chlorella sp or Nannochloropsis sp, with p- and o- cresols, at an initial concentration of 95 mg/L, were used as a sole carbon source. The concentration of the cresol used was similar to that of total cresols found in real refinery wastewater, which was reported to be 99 mg/L [8]. The cultures mixtures were covered with cotton, and placed in a water bath (Daihan Labtech, Korea) maintained at 30 °C and110 rpm for five days. The vials were subjected to sufficient light with a photoperiod of 12 h light/12 h dark. At the end of the experiment, mixtures were centrifugation using an IEC-CL Multispeed centrifuge (Model No. 11210913, France) at 6000 rpm for 5 min. The supernatants were sent for cresols analysis, whereas the harvested biomass were suspended in 10 ml distilled water to determine its concentration. 2.2.2. Biodegradation experiments In this section, experiments were carried out on the treatment of pcresol using with Chlorella sp. cultures in 200 mL volume. The cultures consisted of the 3N-BBM medium with different concentrations of pcresol ranging from 95 to 330 mg/L, and inoculated with Chlorella sp., at an initial concentration of (1.18 ± 0.16) x106 cell/mL, which corresponds to an optical density of 1.2771. The flasks were covered with cotton and placed in the same water bath, at the same condition, used in the screening experiment. The flasks were subjected to sufficient light with a photoperiod of 12 h light/12 h dark. Control experiments were carried out using the same mixtures, but without microalgae, subjected to similar conditions. On a daily basis, 5 mL samples were collected and the microalgae cells were harvested by centrifugation at 6000 rpm for 5 min. The supernatants were sent for cresols analysis, whereas the harvested biomass were suspended in 5 ml distilled water to determine its concentration. At the end of the experiment (in day 5), the biomass was harvested by centrifugation and was analyzed for its proteins, lipids,
2.3.4. Pigments analysis The pigments content was determined using HPLC equipped with 251
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the same column used for the cresols analysis. The mobile phase consisted of two solvents; Solvent A [Acetonitrile/Methanol (60:40)] and Solvent B [Methanol/Ethyl Acetate (70:30)]. The flow rate through the column was 1.3 mL/ min. A gradient flow composition of mobile phase was used for best chromatographic results. The mobile phase gradient started with 100% solvent A, and changed to 100% solvent B in 8 min, and kept in that composition for 7 min, then returned to 100% A in 1 min. It was then left at this composition for another 25 min to stabilize the column for next injection. The sample injection volume was 20 μl, and the wavelength was 436 nm. The instrument was calibrated using serial dilution of standard pigments (chlorophyll A and B, and total carotenoids) in acetone. The calibration curve ranged between 0.005 μg/ml to 5 μg/ml was used. Fig. 2. Changes in p-cresol concentration (S) with time at initial Chorella sp. concertation (Xo) of 1.18 × 106 cell/mL, 30 °C, 110 rpm and different initial pcresol concentrations (So).
2.4. Statistical analysis Each experiment was performed in duplicate and the average values of the results were determined. The accuracy of the experimental results was evaluated from the standard deviations, shown as error bars in the figures. To determine the significance of a factor, the regression pvalue, was calculated using ANOVA test by Minitab 16 statistical software (MiniTab Inc.). Second order regression was selected for data that shows an increase then decrease behavior, whereas linear regression was selected for data that has single trend.
cresol. Therefore, subsequent deeper investigations were selected to be conducted on Chlorella sp. using p-cresol. The work can be extended in the future to do deeper analyses on the other strain and the other cresol. 3.2. Biomass growth and cresols degradation The effect of cultivation of Chlorella sp. in 3N-BBM medium of different initial p-cresol concentrations, ranging from 35 to 330 mg/L, was tested at initial biomass concertation, Xo, of 1.18 × 106 cell/mL, temperature of 30 °C, and agitation of 110 rpm. The changes in the cresol and the biomass growth (presented as ln X/Xo) over a period of five days are shown in Figs. 2 and 3, respectively. Running a parallel experiment without microalgae, showed minimal drop in the concentration over the five days, which did not exceed 1.5% at the highest concentration of 330 mg/L. This proves that the drop on p-cresol was mainly due to the bioactivity of the microalgae. The biodegradation procedure of p-cresol was proposed to go through two step process [22]. In the first step, the methyl group is split, which could be converted to methanol, and in the second the intermediately produced phenol is broken. It should be noted that the data shown in Figs. 2 and 3 are average values of two runs. The reproducibility of the data is confirmed from the small error bars shown in Fig. 2. The error bars in Fig. 3 were not shown to avoid overcrowding the graph, but the average relative error was 7.6%. The error bars for the growth can still be seen from the specific growth rate results shown in Fig. 4. It was clearly seen from Fig. 2, that except for the highest concentration of 330 mg/L, Chlorella sp. was capable of removing most of the p-cresol within 4 days. For concentrations up to 260 mg/L, Chlorella sp. was able to completely remove p-cresol. However, concentrations higher than 260 mg/L appeared to be inhibiting the microalgae capacity to degrade p-cresols, and at 330 mg/L only 20% removal percentage was achieved within 5 days. Fig. 3 shows that the microalgae was
3. Results and discussion 3.1. Bioremoval of p- and o-cresols The work started with a screening test to compare the effectiveness of marine and freshwater strains in degrading p- and o- cresols. The objective of this screening test was to find out whether one of the two strains may not grow in the cresols or show superior performance compared to the other. As mentioned earlier, the selected concentration was similar to that of total cresols found in real refinery wastewater [8]. Fig. 1a and b show the percentage removal of cresols and the concentration of the biomass after seven days, X, over the initial biomass concentration, Xo, which was 1.18 × 106 cell/mL, respectively. Both strains were found to degrade almost the same percentage of pcresol within the 5 days. The marine strain, Nannochloropsis sp., showed a slightly better growth rate than the freshwater strain, Chlorella sp. With o-cresol, the marine stain showed slightly better biodegradation performance, but surprisingly its growth was lower. As the cells use the degraded substrates for two functions, namely, production of new cells and maintenance of existing cells. Therefore, these results suggest that with o-cresol a smaller portion was directed towards Nannochloropsis sp cells growth, as compared to Chlorella sp. Both strains performed almost equally well. However, Chlorella sp growth and biodegradation were both better with p-cresol than with ocresol all, whereas with Nannochloropsis sp. the degradation of both types of cresols were similar, but the growth was much better in p-
Fig. 1. Percentage removal of cresols (a) and biomass growth (b) after growing at 30 °C and 110 rpm for 5 days in media containing Chlorella sp. and Nannochloropsis sp. at initial cresols (So) and biomass (Xo) concentrations of 95 mg/L and of 1.18 × 106 cell/mL, respectively. 252
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cresol biodegradation. [25]. Results showed high applicability at initial concentration of p-cresol biodegradation of 750 mg/L within 100 h. In addition, a bacterial strain, Pseudomonas putida, immobilized in PVA gel, was also used for the biodegradation of p-cresol, at concentrations range similar to the one used in this study (25 to 200 mg/L) [26]. Complete removal was achieved within only 40 min. On the other hand, using aerobic granulates, a longer time was required to remove p-cresol even at a low concentration (100 mg/L) [27]. To better understand the behavior, the biodegradation and specific growth rates were determined, and the results are shown in Fig. 4. The biodegradation rate was determined from the slope of the straight line in the initial part of the degradation. Whereas, the specific growth rate was determined from the slope of the logarithmic of X/Xo vs time in the exponential part, after the lag period. As expected it was found that both rates (biodegradation and specific growth) had similar trends. When the growth rate increased, the biodegradation rate increased and vice versa. It was found that the growth rate of Chlorella sp. increased with increasing the initial concentration of p-cresol, up to 140 mg/L, at which the optimum growth rate of 2.17 ± 0.17 day−1 was reached. Beyond that concentration, the growth started to slightly drop at 210 mg/L, and then sharply dropped after that, due to substrate inhibition. Similar behavior was observed for the p-cresol degradation, but the optimum drop was 51.51 mg/L.day at 210 mg/L. The effect of initial concentration on both the biodegradation rate and growth rate was significant, with a p-value of 0.05. The effect however, was less significant on the growth rate, with a p-value of 0.085. This results agree with previous study done using the same strain for phenol degradation [29]. The maximum biodegradation and growth rates were also obtained at an initial phenol concentration of 200 mg/L. The optimum drop rate was 54.5 mg/L.day, which were very close, but slightly higher than the one found in this work. However, a much lower growth rate of only 0.25 day−1, was reported. In addition to using a different substrate, the lower rate was due to the limited light intensity used in the previous study, suggested to enhance the heterotrophic growth. When p-cresol biodegradation was tested using a green microalgae Scenedesmus obliquus, he highest bioremoval of 100% was achieved at initial concentration of 0.162 mg/L, at which the biomass concentration increased by 20% of the initial value. [22]. At higher concretions the growth was inhibited. The results shown in Fig. 3, were used to determine the parameters of three growth models that incorporate limiting substrate-inhibition kinetics. The models used were Haldane, Aiba and Andrews, given in Eqs. (1)–(3), respectively
Fig. 3. Changes in the logarithmic of biomass concentration over initial concentration (ln X/Xo) with time at initial Chorella sp. concertation (Xo) of 1.18 × 106 cell/mL, 30 °C, 110 rpm and different initial p-cresol concentrations (So).
Fig. 4. p-Cresol degradation rate (dS/dt) and Chlorella sp. specific growth rate (μ) at initial Chorella sp. concertation (Xo) of 1.18 × 106 cell/mL, 30 °C, 110 rpm and different initial p-cresol concentrations (So).
able to grow in all concentrations of p-cresols, but with different growth rates. It was also noticed that as the initial concentration of p-cresol increased, the lag phase increased. This was expected, as the cells needed more time to adapt to the high substrate concentration. As mentioned earlier, the investigation of p-cresol removal using microalgae was rarely studied, and even in these limited studies, the concentrations tested were much lower than those found in real refinery wastewater. For example, eukaryotic algae, Ochromonas Danica, was tested for the biodegradation of p-cresol at initial concentrations in the range of 0.54 to 4.3 mg/L. The study showed complete removal of the p-cresol in the concentration range within a maximum period of 12 days [23]. Green microalgae, Scenedesmus obliquus, was also tested for the biodegradation but at even lower concentrations in the range 0.162 to 2.7 mg/L. [22]. The effect of the initial substrate concentration on the percentage removal was shown to be significant, dropping from 100% to 29%, when the initial concentration was increased from 0.162 to 2.7 mg/L [22]. As the concentrations of p-cresol in real refinery wastewater are order of magnitude higher than these tested ranges, the importance of the results found in this study, at the higher concentrations, becomes more obvious. Table 2 shows comparison in p-cresol biodegradation using several microorganisms at different initial concentration range. Filamentous fungal strain, Gliomastix indicus, showed higher performance for p-cresol removal than that of the tested microalgae in this work [24]. At high concentrations of up to 700 mg/L, more than 90% p-cresol was removed within about 4 days. In a more recent study, isolated bacteria from activated sludge were tested for p-
μ=
μ m So KS + So + S2o Ki
(1)
μ=
μ m So exp(−So Ki) K S + So
(2)
μ=
μm [(KS So) + 1]⋅[1 + (So Ki )]
(3)
Where, μ and μm are the specific and maximum specific growth rates (day−1), respectively, So is the initial substrate concentration (mg/L), and Ks and Ki are the substrate and inhibition constants (mg/L), respectively. The estimated values for the model kinetic parameters were determined by fitting non-linear regression model, using Excel solver with an objective function (O.F.) given by Eq. (4), and shown in Table 3. n
O.F. = ∑ (μ exp − μ pred.)2 i
(4)
Where, μ exp and μ pred are the experimental and model predicted specific growth rates. From the values of the coefficient of determination, R2, it can be 253
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Table 2 Biodegradation of p-cresol using different microorganisms. Microorganism type
strain
Reactor
Concentration range
Removal
Time
Ref.
Microalgae Microalgae fungus Bacteria Bacteria Bacteria Bacteria
Scenedesmus obliquus Ochromonas danica Gliomastix indicus Advenella sp Pseudomonas putida Mixed culture Aerobic granules
Shaking flasks Shaking flasks Shaking flasks Flasks Spouted bed bioreactor Batch reactor Sequencing batch reactor
0.54 to 2.7 mg/L 0.54 to 4.3 mg/L 10-700 mg/L 750 mg/l 25-200 mg/L 100 mg/L Up to 800 mg/L
29-100% 100% 90% 100% 100% 100% 88%
5 days 12 h 108h 100h 40 min 49 h 2 months
[22] [23] [24] [25] [26] [28] [27]
Table 3 Estimated value of microalgae growth kinetic parameters in p-cresol containing medium. Parameter
Haldane
Aiba
Andrews
μm Ks Ki R2
8.3 194.8 88.9 0.914
11.0 250.3 227.0 0.953
9.0 148.9 117.6 0.905
Fig. 6. Changes in proteins, lipids and carbohydrates contents of microalgae after growing for 5 days in media containing initial Chorella sp. concertation (Xo) of 1.18 × 106 cell/mL, 30 °C, 110 rpm and different initial p-cresol concentrations (So).
the wastewater treatment. Microalga biomass with high lipid content could serve as a good feedstock for biodiesel production. Whereas, the biomass with high protein content may be more suitable for pharmaceutical applications. Generally, the composition of the growth medium affects the biochemical composition of microalgae cells, by alternating of metabolic pathways in response to stress conditions [30]. Therefore, at the end of the 5 days, the biomass was collected and analyzed for proteins, carbohydrates, lipid and pigments content. The percentage compositions of proteins, carbohydrates and lipids are in Fig. 6, and those of the pigments, chlorophyll-a, chlorophyll-b and carotenoids are shown in Fig. 7. It was found that the lipids content increased with the increase in the p-cresol concentration. Growing in medium containing no p-cresol, the lipids content was 8.9%. This amount increased by 3.7 folds to 33%,
Fig. 5. Comparison between experimental data of Chlorella sp. specific growth rate (μ) and models predictions.
seen that all three models fitted the data well, with the Aiba model being the best, followed by the Haldane model. The goodness of the fitting can also be seen in Fig. 5, which shows the comparison between experimental data and models predictions. The results show that the values of μm for all models were similar, with that of the Aiba model being relatively the highest. The values of Ks and Ki of the Haldane and Andrews models were similar, whereas those of the Aiba model were higher. No previous studies on the kinetics of microalgae biodegradation of p-cresol are reported in open literature. The only available ones are with other microorganisms, such as bacteria and fungus. For example, kinetic of p-cresol degradation by fungal strains, Gliomastix indicus, have been studied using five several models [24]. The values of μm of Haldane, Aiba and Andrews were 9.5, 7.02 and 14.2 day−1, respectively, which are close to the ones found in this work. In addition, similar to this work, the highest value of μm was for Aiba model. However, with the fungal strains, the R2 values suggested that the Haldane and Andrews models represented the data better than Aiba model.
3.3. Effect of p-cresol on biochemical composition The most important aspect of this work was to determine the effect of the concentration of p-cresol on the chemical composition of the harvested microalgae. As mentioned earlier, this is important information for the determination of the application to be coupled with
Fig. 7. Changes in pigments contents of microalgae after growing for 5 days in media containing initial Chorella sp. concertation (Xo) of 1.18 × 106 cell/mL, 30 °C, 110 rpm and different initial p-cresol concentrations (So). 254
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and pigments composition, the experiment was repeated at the initial pcresol concentration of 190 mg/L that showed the optimum degradation and growth rates. In this experiment, the cells composition was measured twice a day for a period of two days, and the rate of the change was measured from the slope of the changes with time. The results show that the drop rate of proteins was -15.4% per day. A drop was also observed for carbohydrates, but at a much lower rate of 7.3% per day. The lipids show an increase with a rate of 19.1% per day.
by growing for 5 days in medium containing an initial concentration of 330 mg/L. The effect of the initial concentration of p-cresol on the lipid content was significant, with a p-value of 0.041. In contrast, the proteins content decreased with the increase in p-cresol concentration, from 58.7% with no p-cresol to 31.3 at 330 mg/L. The effect was also significant, with a similar extent to that on of lipids, with a p-value of 0.044. No significant change was observed in the carbohydrate level. This insignificant effect was also confirmed from the p-value, which was found to be 0.146. This finding suggests that for coupling wastewater treatment with biodiesel production, it would be preferable to cultivate the microalgae in wastewaters containing high concentrations of p-cresol. Whereas, cultivation in low concentrations may be more suitable for coupling with proteins extraction. The results found in this work agree with those found when the concentrations of nonylphenols, bisphenol, 17alpha-ethynylestradiol, and estradiol were increased in the media used for the cultivation of the marine diatom Navicula incerta [31]. It was also found that, the proteins content declined with the increasing in all compounds. The behavior of the lipids content was also similar, showing an increase in the lipid contents, with the increase in the concertation of the compounds in the media. An increase in the lipids content in diatom Skeletonema costatum was also observed by increasing the concentration of 2,4-dichlorophenol in the cultivating medium [32]. However, this was coupled with a decrease in carbohydrates content and the proteins showed no changes. Under environmental stress, microalgae direct their pathways towards storing energy in the form of lipid instead of using it as source of growth [33]. This was shown when Chlorella maxicana was cultivated under stress of salinity, showing an increase in lipids content from 23% to 37% as the NaCl concentration increased from 0.43 to 25 mM [34]. An increase in lipids was also observed when the cultures were stressed with nitrogen and phosphorus limitations [21,25,26]. In addition, previous studies confirmed the accumulation of lipids in Chlorella pyrenoidosa, when the cells are exposed to high phenol concentration, and the lipid accumulation was increased with increasing the incubation period [12]. Increasing of lipid biosynthesis was also reported during phenol degradation in diatom BD1IITG cells [35]. As shown in Fig. 7, chlorophyll A and chlorophyll B and carotenoids composition were found to be highly dependent on the initial p-cresol concentration. It was found that the pigments concentration increased with the increase in p-cresol concentration, to reach a maximum at 140 mg/L, then stated to drop after that. At low p-cresol concentrations, the contents of both chlorophylls a and b, were higher than that of carotenoids. The drop in chlorophylls content with the increase in pcresol concentration was more drastic that of carotenoids, and above 250 mg/L, the carotenoids content started to exceed that of chlorophyll b. chlorophyll A remained the main pigment under all tested concentrations of p-cresols. The effect of the initial p-cresol was most significant on chlorophyll A, with a p-value of 0.093, followed by carotenoids, with a p-value of 0.118 and least significant on chlorophyll b, with p-value of 0.203. These results are related to the enhancement in the biomass growth that encourage algal cells to produce more pigments at concentration with highest biomass growth rate. Similar trend was also obtained when Chlorella kessleri was cultivated in different concentrations of crude oils [36], with the maximum chlorophyll a and b contents achieved at the initial concentration that resulted in the highest biomass growth [36]. However, with Scenedesmus obliquus, the pigments production was found to decline with the increase in the initial concentration of crude oil, having the highest chlorophyll a content at the lowest oil concentration [37]. On the other hand, no changes in chlorophylls content of diatoms Navicula incerta [31] and Skeletonema costatum [32] was observed with increasing the initial concentration of the endocrine disrupting chemicals. To study the initial rate of the changes in proteins, carbohydrates
4. Conclusion This study demonstrates the capacity of Chlorella sp. for the biological treatment of wastewater contaminated with p-cresol. The biodegradability and cell growth were found to be affected by the initial cresol concentration. However the highest biomass productivity and biodegradation rate were obtained at initial p-cresol of 150 and 200 mg/L, respectively. The biodegradation kinetics were presented well by Haldane, Aiba and Andrews models, with Aiba model showing the best fit. The biomass composition of Chlorella sp. was also found to be affected by the initial concentration of p-cresol in the growth medium. As the initial concentration increased, the proteins reduced and the lipids increased, whereas the carbohydrates remains constant. The pigments content increased with the increase in p-cresol concentration to reach a maximum at 150 mg/L p-cresol, but then dropped at higher concentrations. Acknowledgement This work was supported by the Emirates Center for Energy and Environment Research [grant number 31R070] References [1] M.H. El-Naas, S. Al-Zuhair, A. Al-Lobaney, Treatment of petroleum refinery wastewater by electrochemical methods, Int. J. Eng. Res. Technol. 2 (10) (2013) 2144–2150. [2] A.H. Al-Muhtaseb, M. Khraisheh, Photocatalytic removal of phenol from refinery wastewater: catalytic activity of Cu-doped titanium dioxide, J. Water Process Eng. 8 (2015) 82–90. [3] L. Damjanović, V. Rakić, V. Rac, D. Stošić, A. Auroux, The investigation of phenol removal from aqueous solutions by zeolites as solid adsorbents, J. Hazard. Mater. 184 (1–3) (2010) 477–484. [4] T. Al-Khalid, M.H. El-Naas, Aerobic biodegradation of phenols: a comprehensive review, Crit. Rev. Environ. Sci. Technol. 42 (16) (2012) 1631–1690. [5] L. Levén, K. Nyberg, L. Korkea-aho, A. Schnürer, Phenols in anaerobic digestion processes and inhibition of ammonia oxidising bacteria (AOB) in soil, Sci. Total Environ. 364 (1–3) (2006) 229–238. [6] Y. Lu, L. Yan, Y. Wang, S. Zhou, J. Fu, J. Zhang, Biodegradation of phenolic compounds from coking wastewater by immobilized white rot fungus Phanerochaete chrysosporium, J. Hazard. Mater. 165 (1–3) (2009) 1091–1097. [7] J. Yan, W. Jianping, B. Jing, W. Daoquan, H. Zongding, Phenol biodegradation by the yeast Candida tropicalis in the presence of m-cresol, Biochem. Eng. J. 29 (3) (2006) 227–234. [8] M.H. El-Naas, R. Surkatti, S. Al-Zuhair, Petroleum refinery wastewater treatment: a pilot scale study, J. Water Process Eng. 14 (2016) 71–76. [9] J. Doucha, F. Straka, K. Lívanský, Utilization of flue gas for cultivation of microalgae (Chlorella sp.) in an outdoor open thin-layer photobioreactor, J. Appl. Phycol. 17 (5) (2005) 403–412. [10] A.P. Batista, P. Moura, P.A.S.S. Marques, J. Ortigueira, L. Alves, L. Gouveia, Scenedesmus obliquus as feedstock for biohydrogen production by Enterobacter aerogenes and Clostridium butyricum, Fuel 117 (PART A) (2014) 537–543. [11] S. Wahidin, A. Idris, S.R.M. Shaleh, The influence of light intensity and photoperiod on the growth and lipid content of microalgae Nannochloropsis sp, Bioresour. Technol. 129 (2013) 7–11. [12] B. Das, T.K. Mandal, S. Patra, A comprehensive study on Chlorella pyrenoidosa for phenol degradation and its potential applicability as biodiesel feedstock and animal feed, Appl. Biochem. Biotechnol. 176 (5) (2015) 1382–1401. [13] A. Demirbas, Importance of biodiesel as transportation fuel, Energy Policy 35 (9) (2007) 4661–4670. [14] T.M. Mata, A.A. Martins, N.S. Caetano, Microalgae for biodiesel production and other applications: a review, Renew. Sustain. Energy Rev. 14 (1) (2010) 217–232. [15] A. Jabeen, et al., Effect of Enzymatic pre-treatment of microalgae extracts on their anti-tumor activity, Biomed. J. 40 (6) (2017) 339–346. [16] I. Deniz, M. García-Vaquero, E. Imamoglu, Trends in Red Biotechnology: Microalgae for Pharmaceutical Applications, (2017).
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