Capacity of Marine Microalga Tetraselmis suecica to Biodegrade Phenols in Aqueous Media

Abstract

Phenolic compounds are toxic and dangerous to the environment and human health. Although the removal of phenols and their derivatives is very difficult, it has been achieved by applying some biological processes. The capacity of microalga to remove phenolic compounds has been demonstrated; however, few reports of the removal of these compounds in a mixture have been published. The removal of phenol, p-cresol and o-cresol was performed by batch kinetics at 50 and 100 mg L⁻¹, and the simultaneous degradation of phenol, p-cresol and o-cresol was carried out in a mixture at 40 mg L⁻¹ using the marine microalga Tetraselmis suecica. The kinetic study was carried out for 192 h. For concentrations of 50 mg L⁻¹ and 100 mg L⁻¹, phenolic compound consumption efficiencies greater than 100% and 85%, respectively, were obtained, and up to 73.6% removal in the mixture. The results obtained indicate that the marine microalga carries out a process of the oxidation of organic matter and phenolic compounds, mineralizing up to 31.4% to CO2 in the mixture. Biological treatments using the marine microalga T. suecica can be considered feasible to treat effluents with concentrations similar to those of the present study.

1. Introduction

Wastewater is a natural sub-product from any productive process. Phenol is one of the main contaminants in the effluents of chemical industries and petrochemicals, coke oven plants, coal mining and pharmaceuticals, with concentrations of 2 mg L⁻¹ to up to 6000 mg L⁻¹ [1].

Phenol and its derivatives are corrosive and toxic compounds that are highly dangerous, even at low levels, posing a risk to human health and the environment. Therefore, the management of phenol-polluted wastewater represents major environmental challenges, due to its chemical complexity [2]. Hence, strategies for its treatment and elimination have been studied. In this regard, physical and chemical methods are the most frequently applied processes to remove phenols from the environment and industrial wastewater. Among them, electro-oxidation, activated carbon adsorption and photocatalytic degradation are the most common [3].

Microalgae have gained attention for their wide applications in different areas, including the removal of polluting compounds in water [4], due to their ability to remove toxic compounds [5] such as phenol [6], as well as various phenolic compounds [7]. In particular, treatment with microalgae species such as Chlorella, Scenedesmus, Dunaliella and Porphyridium [8], which are freshwater or marine microalgae, has been studied for water treatment [9]. Few papers have mentioned the marine genus Tetraselmis [10]. In general, microalgae exhibit a low tolerance to high phenol concentration, and consequently, low degradation rates are achieved in this condition [11].

The main advantages of using microalgae as biological treatments to remove phenols from wastewater (freshwater or marine) are their characteristics in terms of growth and composition. Microalgae are diverse, fast-growing and adaptable to various environments, with cultivation options in either natural or artificial conditions; they use sunlight as an energy source and, in addition, some species have a mixotrophic metabolism [12]. Moreover, the cultures are low-cost and easy to carry out in comparison with electro-oxidation or activated carbon adsorption.

Degradation studies of phenolic compounds have been reported using Chlorella pyrenoidosa, at concentrations up to 800 mg L⁻¹, achieving a removal of up to 97% [2]. In addition, Chlorella vulgaris has been used in the degradation of a mixture of phenol and p-cresol in the treatment of coal gasification wastewater. The degradation was improved after the addition of NaHCO₃ [11]. The synergistic effect of glucose (as co-substrate) and phenol was studied using this microalga, increasing the tolerance of microalga to phenol up to 10 g L⁻¹ [13]. An interesting study revealed the metabolic pathways of Tetraselmis suecica when it is cultured under nitrogen depletion and starvation. This study highlighted that transcripts involved in signal transduction pathways, stress and antioxidant responses, and solute transport were strongly up-regulated when T. suecica was cultured under nitrogen starvation, with incidence in the biochemical composition and photosynthesis activity [14]. This can be a mechanism used by T. suecica to remove contaminants from water, although in some cases, the contaminant removal is associated with many other contributing factors. Among them, Forootanfar et al. [15] described how T. suecica was able to remove 67% of the p-chlorophenol at an initial concentration of 20 mg L⁻¹ from the medium within a 10-day period. However, the efficacy of the process was dependent on the p-chlorophenol concentration at 60 mg L⁻¹, which was inhibitory. The adaptation phase was p-chlorophenol-dependent; in this regard, at high concentration, the lag phase growth was the longest, suggesting that p-CP was not used as a primary growth substrate by the algae because p-CP elimination was increased by enhancing the cell numbers during the 10-day period. In this way, Forootanfar et al. [15] established that besides cell adsorption, absorption is another biological pathway for removing organic contaminants by bioaccumulation, biodegradation and biotransformation. In addition, these authors used the purified laccase enzyme for the degradation of substituted phenols. Moreover, Petroutsos et al. [16] discovered another mechanism in T. marina to eliminate 2,4-dichlorophenol, the conjugation of a phenolic compound with glucose (glucosidation) and the malonylation of phenols such as quercetin, which reduced the toxicity of this hazardous compound.

On the other hand, Pop et al. [17] studied the bisphenol effect at 50, 100 and 200 ppm in the aquatic plant Lemna minor. The results show chlorosis for the 200 and 100 ppm groups, and no budding formation at all concentrations evaluated. At 100 ppm, non-fermenting Gram-negative bacteria (Klebsiella aerogenes) and Escherichia coli were found in the root of the plants; it is maybe associated with the plant chlorosis. However, an interesting protective effect to oxidative stress was found in the 100 and 200 ppm groups. The above shows Lemna minor has the potential to withstand and metabolize Bisphenol A up to 100 ppm.

However, to date, there are just a few reports on the removal of phenolic compounds in a mixture of phenol, p-cresol and o-cresol, which is the way in which they are normally found in wastewater. Therefore, the aim of this study was to evaluate the ability of the marine microalgae Tetraselmis suecica to remove phenol, p-cresol and o-cresol separately and simultaneously in a mixture.

2. Materials and Methods

2.1. Tetraselmis suecica Culture

The T. suecica microalgae strain was acquired from the Northwest Biological Research Center (CIBNOR) of Baja California Sur, Mexico. The culture medium for biomass production was seawater with a salinity of 35‰, previously sterilized at 15 lb in⁻² for 15 min, adding a sterile nutrient solution, an “algal” medium composed of macro elements and trace elements [18].

The microalga was inoculated at an initial cell density of 1 × 10⁶ cells mL⁻¹ in glass serological culture bottles with 800 mL of medium. The microalga was placed in an incubation room with a light intensity of 180 µmol m⁻² s⁻¹, in cycles of 12 h of light–darkness at a temperature of 20 °C and an airflow of 0.55 vvm (air volume/volume of medium per minute), enriched with a pulse of CO₂ to maintain the pH between 7 and 8. Biomass harvesting was carried out once the exponential phase of growth was achieved.

2.2. Degradation Kinetics

Biological degradation kinetic studies show the consumption of a nutrient and/or contaminant over time, in order to establish the efficiency of removal and rate of consumption. The kinetics were performed in 160 mL serological bottles, using a working volume of 60 mL, consisting of 55 mL of algal medium, to which 1 g L⁻¹ NaHCO₃ was added as a carbon source. The bottles were inoculated, in duplicate, with 1 g of Total Suspended Solids (TSS) L⁻¹ of biomass. Subsequently, the phenolic compounds were added separately (phenol, p-cresol and o-cresol), each at concentrations of 50 and 100 mg L⁻¹. The experimental units remained in support with agitation at 200 rpm (Thermo Scientific Model No. 2345 Vernon Hills, IL, USA) and with an incidence of light (180 µmol m⁻² s⁻¹). The samples were taken at 0, 6, 12, 24, 48, 72, 96, 120, 144, 168 and 192 h after starting the degradation kinetics. Finally, degradation kinetics was carried out with the mixture of the three phenolic compounds at a concentration of 40 mg L⁻¹ each, under the same conditions of inoculation, agitation and light incidence as the previous tests.

Control kinetics were performed. First, abiotic kinetics in serological bottles with the algal medium enriched with NaHCO₃ were carried out under the same conditions as the biotic kinetics, testing concentrations of 50 and 100 mg L⁻¹ of each phenolic compound in order to rule out chemical oxidation. Measurement of the phenol concentration was at 0, 15, 24, 48, 72, 96, 120 and 144 h. Total organic carbon COD was measured at the beginning at 0, 74 and 144 h. In addition, a control test was carried out with inert biomass and 50 mg L⁻¹ of each phenolic compound to rule out the adsorption of contaminants in the cell wall of the microalgae. For this test, the biomass was exposed to a temperature of 180 °C for 24 h to cause the destruction of the microalgae. The other conditions of these kinetics are the same as those described for biotic kinetics. Measurement of the phenol concentration was at 0, 6, 24, 72, 120 and 192 h. COD was measured at 0, 24, 120 and 192 h.

2.3. Determination of Phenolic Compounds

For the quantification of the phenolic compounds, firstly, the wavelength of maximum absorbance for phenol and p-cresol was determined by spectrophotometric scanning, defined at 271 nm and 291 nm, respectively. A calibration curve was prepared in a concentration range of 0 to 200 mg L⁻¹ of each compound, separately. The samples were filtered through a 0.45 µm nylon membrane, before being read in the spectrophotometer. On the other hand, the wavelength of maximum absorbance for o-cresol was determined as proposed by APHA [19]. A total of 125 µL of 0.5 N NH₄OH was added to 5 mL of the sample and the pH was adjusted to 7.9 ± 0.1 with a phosphate buffer. Next, 50 µL of 4-amino-antipyridine was added, followed by 50 µL of potassium ferricyanide K₃[Fe(CN)₆]. After 15 min the absorbance was registered at 500 nm in a spectrophotometer. A calibration curve in the range of 0 to 5 mg L⁻¹ of o-cresol was used for quantification.

The determination of the phenolic compounds in the mixture was carried out by means of high-performance liquid chromatography (HPLC, Aligent Varian ProStar model) with the modified methodology of Meza-Escalante et al. [20]. A C-18 column was used at 40 °C with a mobile phase of acetonitrile water (30:70), a flow rate of 1.2 mL min⁻¹ and a run time of 15 min at a pressure of 80 atm. The measurement was performed at a wavelength of 291 nm with an injection volume of 20 µL.

The quantification was carried out by comparing the areas of the calibration curves (in the range of 15 to 150 mg L⁻¹) of the individual phenolic compounds.

From the concentrations of the phenolic compounds at different times, the removal efficiency was calculated. In addition, the specific consumption rate was determined based on the following equation [21]:

$$qs=\frac{ds}{dt} · \frac{1}{\mathrm{X}}=\frac{Ln So-Ln S}{\mathsf{∆}t}· \frac{1}{\mathrm{X}}$$

2.4. Determination of COD and Inorganic and Organic Carbon

Complete oxidation of the phenolic compounds in a mixture was analyzed through total carbon (TC), inorganic carbon (IC) measurements, and Chemical Oxygen Demand (COD). COD was determined with a spectrophotometer (Spectroquant Pharo 300, Swedesboro, NJ, USA), following the procedure described in standard methods [19]. For quantification, a calibration curve of potassium biphthalate from 0 to 500 mg L⁻¹ was performed. TC and IC were measured in a TOC-L Shimadzu total organic carbon analyzer (Shimadzu Co., Kyoto, Japan). Calibration curves were made with potassium biphthalate for TC and the IC curve was made with sodium carbonate and sodium bicarbonate, both from 0 to 500 mg L⁻¹. The samples were taken on the third and eighth day of incubation.

3. Results

In the present study, the capacity of the microalga T. suecica for the elimination of phenolic compounds (phenol, o-cresol, and p-cresol), individually and in a mixture, was evaluated. These compounds are mainly present in industrial wastewater of pharmaceutical, tannery and petrochemical origin.

3.1. Kinetic Study of Phenol, p-Cresol and o-Cresol at 50 and 100 mg L⁻¹

Firstly, the control kinetics carried out (biotic and with inert biomass) did not show removal of the compounds (coefficient of variation <10%), which rules out chemical oxidation processes and the presence of the adsorption of contaminants in the cell wall of the microalgae. The above suggests that any removal of the phenolic compounds present in the tests was a biological process carried out by microalgae.

The biotic kinetics of the separate phenolic compounds at 50 and 100 mg L⁻¹ are shown in Figure 1. In Figure 1A, the total removal of o-cresol was achieved at 75 h, phenol at 96 h and p-cresol at 120 h, when the phenolic compounds were added at 50 mg L⁻¹. However, it can be seen that o-cresol was immediately removed, achieving a concentration of 5 mg L⁻¹ at 24 h of bioassay, which is 90% removal, while phenol and p-cresol barely reached about 50% removal. A significant difference was observed between o-cresol and the other treatments (p ≤ 0.05). On the other hand, in Figure 1B, a similar behavior was observed in the consumption of the separate phenolic compounds at 100 mg L⁻¹, highlighting the removal of o-cresol at 24 h, which was similar to phenol and p-cresol removal at up to 48 h, with a non-significant difference observed between them. The maximal removal of 94.5% was reached at 192 h (

Table 1. Specific consumption rates (q_s) and removal efficiencies (RE) obtained by marine microalgae at two different concentrations of phenolic compounds (PC). The result is given as a percentage.

Compound	50 mg L−1		100 mg L−1
	qs	RE	qs	RE
Phenol	1.86 ± 0.22	100 ± 0	1.12 ± 0.01	91.69 ± 0.77
p-cresol	0.93 ± 0.05	100 ± 0	1.11 ± 0.05	85.20 ± 0.66
o-cresol	3.27 ± 0.03	100 ± 0	1.97± 0.31	94.50 ± 0.35

The q_s values are given in mg PC (g SST · h)⁻¹ ± the standard deviation. Data were calculated using at least three points. The removal efficiency at 100 ppm of the PC was determined for 192 h of incubation.

). The high removal of o-cresol was according to the highest consumer-specific rate (q_s) in both concentrations (Table 1).

These results suggest that the increase in the concentration of phenolic compounds inhibits the growth and metabolic activity of T. suecica; the same behavior was noticed in Chlorella vulgaris cultured in the presence of phenol [13] when its concentration was increased. In addition, Joseph and Joseph [8] showed that, although chlorophyte microalgae, such as T. suecica, are the most resistant to phenolic compounds, in some species, inhibition may occur at concentrations from 100 mg L⁻¹. Moreover, at a higher initial concentration of phenol, it takes more time to be degraded completely by Chlorella pyrenoidosa [2], which accords with the behavior shown by T. suecica in this study.

In this regard, the ability of an improved strain of Chlorella sp. was demonstrated, which was capable of removing 500 to 700 mg L⁻¹ of phenol completely within seven days at an initial biomass of 0.6 g/L⁻¹ and under continuous illumination (118 μmol m⁻² s⁻¹) [21]. These findings highlight the importance of the adaptability of strains to increase the removal capacity of phenolic compounds. On other hand, Wang et al. [22] showed that phenol, at concentrations of less than 100 mg L⁻¹, could be completely degraded in 4 days (96 h) using the microalga, Isochrysis galbana.

Regarding the elimination efficiencies (Table 1), it is observed that in the kinetics at 50 mg L⁻¹, 100% consumption efficiency was achieved for all the phenolic compounds. On the other hand, at a concentration of 100 mg L⁻¹, the consumption efficiencies achieved 85.2% for p-cresol and 94.5% for o-cresol.

Moreover, the specific rate of consumption (q_s) was calculated to both bioassays (Table 1). The qs at 50 mg L⁻¹ of each compound are indicated, where it can be seen that o-cresol shows the highest specific rate of consumption (q_s), with 3.27 ± 0.03 mg o-cresol g TSS ⁻¹ h⁻¹, followed by phenol and, finally, p-cresol, which obtain consumption rates of approximately 1.7 and 3.5 times lower. By increasing the concentration to 100 mg L⁻¹ of phenolic compounds, a higher rate of consumption was again obtained for o-cresol, with a q_s = 1.97 ± 0.31 mg o-cresol g TSS⁻¹ h⁻¹, followed by phenol and p-cresol, which obtain specific rates of consumption of approximately 1.75 and 1.77 times lower with respect to o-cresol.

There are no reports on the specific rates of consumption of phenolic compounds with microalgae, but there is information on the q_s of p-cresol in a denitrifying biological process, which are higher than those reported in the present study, with values of at least two and six times higher at 50 ppm [20,23] and three times for 100 ppm [24]. However, the effect of increasing the concentration of the phenolic compound caused, in all studies, a decrease in the rate of consumption.

The order of degradability observed in this work was o-cresol > phenol > p-cresol. This was contrary to that obtained by Papazi and Kotzabasis [7], who show a degradation of o-cresol < p-cresol, due to the position of the methyl group with respect to the hydroxyl group. In this regard, the inhibition was demonstrated by the decreases in qs when phenolic compounds were increased, except in the case of p-cresol (from 0.93 ± 0.05 to 1.11 ± 0.05 mg p-cresol g TSS⁻¹ h⁻¹). In the case of phenol and o-cresol, qs decreased 1.7 times when phenolic concentration was increased from 50 to 100 mg L⁻¹.

The degradation of phenolic compounds has been studied using marine algae, microalgae and bacteria. In addition, degradation was studied considering the coexistence or mixture of phenol and isomers of cresol, such as o-cresol, m-cresol and p-cresol. In this regard, the presence of p-cresol inhibited the degradation of phenol or o-cresol, or in other cases, m-cresol degradation was inhibited by the presence of phenol [25]. On the other hand, a marine alga, Ochromonas dánica, removed 100% of phenols at concentrations of 60 and 375 g L⁻¹ [26]. In some cases, bacteria have been used to degrade phenols and cresols in a mixture. In addition, Chlorella vulgaris was able to degrade phenol and cresol during co-metabolism; phenol at low concentrations (100 mg L⁻¹) significantly promoted the degradation of p-cresol. Moreover, phenol degradation was improved when NaHCO₃ was added to the culture media [11] or when a co-substrate such as glucose was used [13].

3.2. Biotic Removal Kinetics for a Mixture of Phenolic Compounds

The capacity of microalgae to remove phenolic compounds has been demonstrated. However, few reports of the removal of these compounds in a mixture have been published. In this regard, the capacity of T. suecica to simultaneously remove phenol, p-cresol and o-cresol was evaluated.

The simultaneous removal of phenol, p-cresol and o-cresol was evaluated in a mixture, as shown in Figure 2. The total time of the kinetics was 168 h. Up to 36% of o-cresol was removed at 6 h; from that time, there was no more removal, with a constant concentration of 25.5 mg L⁻¹ remaining. p-cresol was slowly and continuously removed over time, achieving a removal of 73.6% at 168 h. Phenol concentration was constant. No removal of phenol was observed until 120 h; from that time, only 12.5% was removed at 168 h. This behavior suggests that the presence of cresol isomers inhibits the microalga’s metabolism to remove phenol when it is in contact with the three phenolic compounds in a mixture. On the other hand, the most significantly consumed COD was at 12 h of kinetics (Figure 3). After this time, an inhibition of organic matter consumption was observed, achieving 31.4% of removal efficiency of COD. This result can be compared with TOC and IC quantification, in which TOC decreased, while IC increased within the time (data not shown). This finding could be due to phenol and cresols having been degraded and mineralized to CO₂. Comparing these results with those in Figure 2, it is possible to assume that p-cresol is the main phenolic compound degraded to CO₂, due to its removal of 73.6%.

Studies on the biodegradation of cresol isomers have rarely been compared with those about phenol [10]. In this regard, Surkatti and El-Naas [4] studied the degradation of phenol and cresol isomers by bacteria, finding that in the presence of p-cresol or m-cresol, the o-cresol degradation was inhibited. Moreover, when an autochthonous microbial mixture was produced to degrade phenolic compounds, in the presence of phenol, m-cresol degradation was inhibited [22]. In the present study, phenol presence affected the o-cresol degradation, because when o-cresol was studied separately, a removal of 100% was achieved in both evaluated concentrations (50 and 100 mg L⁻¹). According to this, phenol at low concentrations significantly promoted the degradation of p-cresol by Chlorella vulgaris during cometabolism [13], but in our case, p-cresol was the only phenolic compound in the kinetics. When the degradation of phenol, p-cresol and o-cresol was simultaneously evaluated, the degradation order was phenol < o-cresol < p-cresol. This behavior was similar to results obtained by Papazi and Kotzabasis [7], who demonstrated that the degradability of cresols by microalgae is affected by the position of the methyl group with respect to the hydroxyl.

Furthermore, adsorption and absorption (it can also involve bioaccumulation, biodegradation and biotransformation) are biological pathways for removing organic contaminants [16]. In this study, only the adsorption mechanism was determined, which was at its minimum in the control assay with an inert biomass of T. suecica. Therefore, we assume that absorption was the mechanism used by T. suecica to eliminate the phenolic compounds. However, it is possible that other mechanisms are involved in this process, such as the conjugation of phenolic compounds with sugars and conjugated carbohydrates with phenols such as quercetin, which reduce the toxicity of this hazardous compound. This proposed mechanism was discovered in T. suecica for the elimination of p-chlorophenol by Petroutsos et al. [16].

In

Table 2. Degradation of phenolic compounds by marine and freshwater microalgae.

Microalga	Phenolic Compound	Conditions	Initial Concentration (mg L−1)	Removal (%)	Reference
Tetraselmis suecica	phenol, p-cresol, o-cresol (separately) mixture of phenol, p-cresol and o-cresol	192 h	50 100 40 (each one in a mixture)	100 85 Up to 73.6	This work
Chlorella pyrenoidosa	phenol 1 p-cresol 2	coal gasification effluent, pH 8	800 1 400 2	97.4	[2]
Chlorella pyrenoidosa	phenol	refinery wastewater	200	100	[6]
Chlorella vulgaris	phenol 1 p-cresol 2	cometabolic NaHCO3	100 1 300 2	68.2 1 64 2	[11]
Chlorella vulgaris	phenol	mixotrophic with glucose addition (co-sustrate), 6 days.	Up to 400	Up to 30	[13]
Tetraselmis suecica	p-chlorophenol	10-day period in aqueous medium 1. Immobilized in alginate beads 2	20	67 1 94 2	[15]
Tetraselmis marina	2,4-dichlorophenol (2,4-DCP)	6 days glycosidation and malonylation	Not defined	Up to 1 mM	[16]
Chlorella sp	phenol	0.6 g/L−1 initial biomass, 7 days	500	100	[22]
Isochrysis galbana	phenol	96 h	<100	100	[23]
Ochromonas dánica	phenol	heterotrophic growth with 2 mM glucose, 2 days	94	100	[26]

Superscript in the same line indicates the phenolic compound with its respective concentration used.

, the capacity of microalgae, marine and freshwater, to remove or eliminate phenolic compounds is summarized, including the results of the present study. It is interesting to remark upon the diversity of phenolic derivates that microalgae can eliminate from different sources and matrices in a variety of concentrations, mainly presenting the mechanism of absorption in most of them, which includes biotransformation, bioaccumulation and biodegradation.

4. Conclusions

The marine microalgae Tetraselmis suecica turned out to be an alternative for the removal of the phenolic compounds studied (phenol, p-creosol and o-cresol), reaching removal efficiencies of 85% at concentrations of up to 100 ppm, which can be an alternative for the elimination of these compounds in the treatment of wastewater. Moreover, the present study constitutes a clear demonstration of the simultaneous elimination by microalgae of those pollutants generated mainly by the petrochemical industry.