Kinetic growth and biochemical composition variability of Chlorella pyrenoidosa in olive oil washing wastewater cultures enriched with urban wastewater

Journal of Water Process Engineering 35 (2020) 101197

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Kinetic growth and biochemical composition variability of Chlorella pyrenoidosa in olive oil washing wastewater cultures enriched with urban wastewater

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Murad Maaitaha, Gassan Hodaifab,c,*, Ana Malvisb, Sebastián Sáncheza,c a

Chemical, Environmental and Materials Engineering Department, University of Jaen, ES-23071 Jaen, Spain Molecular Biology and Biochemical Engineering Department, Pablo de Olavide University, ES-14013 Seville, Spain c Center for Advanced Studies in Olive Grove and Olive Oils, University of Jaen, ES-23071 Jaen, Spain b

ARTICLE INFO

ABSTRACT

Keywords: Bioprocess Chlorella pyrenoidosa Olive oil mill wastewater Lipids Biofules

Olive mills generates wastewaters (OMWs) characterized by high organic and inorganic load, which includes sugars, phenolic compounds, polyalcohols, pectins, lipids, Na, K, Ca ..., but deficient in nitrogen and phosphorus. Urban wastewater treatment plants with primary (natural sedimentation) and secondary (biological removal) treatments are unable to remove the total nitrogen and phosphorus from these wastewaters, which can be therefore considered as a sustainable source of both nutrients. The enrichment of OMW with urban wastewater from secondary treatment (UW) can provide optimal nutrient concentrations to produce an algal biomass with high added value. Chlorella pyrenoidosa is a green unicellular alga that can eliminate nutrients and produce biomass with high lipids content. Experiments have been carried out in photobioreactors of 0.5 L useful capacity. Different enriched dilutions of olive oil washing wastewater (OOWW) with ultrapure water (%OOWWenriched = 5–100% v/v) were prepared as culture media. Common operating conditions were pH 8, aeration level 1 v/v/ min, initial illumination intensity 126.2 μE/(m2 s) under 12 h light/12 h dark cycles and temperature of 25 °C. Results obtained showed that the highest values of maximum specific growth rate and volumetric biomass productivity were μm = 0.0203 h−1 and Pb = 1.73 × 10-3 g/(L h), respectively. Maximum percentages of chlorophylls (0.96 %) and carotenoids (0.24 %) were obtained in the final biomass of the culture formed by 30 % (v/v) of OOWWenriched. The highest proteins (43.7 %) and lipids (51.5 %) contents were obtained in the biomass from the culture medium with 100 % (v/v) of OOWWenriched, which is suitable for biofuel production. Final treated water could be discharged into water public channels, used in irrigation or as drinking water if other operation units such as ultrafiltration and reverse osmosis are added to the bioprocess.

Abbreviations: a, Parameter in Equation (1); AOPs, Advanced oxidation processes; BOD5, Biological Oxygen Demand (g O2/L); Ca, Carotenoids content (g/L); CaTotal, Total carotenoids content (g/L); CHL, Chlorophyll content (g/L); CHLTotal, Total chlorophyll content (g/L); COD, Chemical Oxygen Demand (g O2/L); c, Parameter in Equation (3); DHA, Docosahexaenoic acid, 22:6 n3, (%); DMP, 2,6-dimethylphenol; DO, Dissolved oxygen in water (mg/L); EC, Electric conductivity (mS/cm); EFA, Percentage of essential fatty acids (%); EPA, Eicosapentaenoic acid, 20:5 n3, (%); Fas, Fatty acids; IC, Inorganic carbon (mg/L); KI, Empirical inhibition constant in Equation (2) (%); Ks, Empirical saturation constant in Equation (2) (%); MUFAs, Monounsaturated fatty acids (%); NO3−+NO2−, NitrateNitrite; n3/n6, Polyunsaturated fatty acids n3 and n6 ratio; OMW-2, Olive mill wastewater from olive oil extraction that operates with two outlets decanter; OMW-3, Olive mill wastewater from olive oil extraction that operates with three outlets decanter; OMWs, All types of olive mill wastewaters; OOWW, Olive oil washing wastewater from the vertical centrifuge in olive oil extraction that operates with two outlets decanter; OOWWenriched, OOWW enriched with 10 % (v/v) of C. pyrenoidosa inoculum; Pb, Volumetric biomass productivity (g/(L h)); Pb,max, Highest value of volumetric biomass productivity in absence of inhibition (g/(L h)); PUFAs, Polyunsaturated fatty acids (%); RL, Rodriguez-Lopez mineral culture medium; SFAs, Saturated fatty acids (%); TC, Total carbon (mg/L); TN, Total nitrogen (mg/L); TOC, Total organic carbon (mg/L); TP, Total phosphorous (mg/L); TPCs, Total phenolic compounds (mg/L); UW, Urban wastewater from secondary treatment; WOW, Wastewater from olives washing machines; x, Biomass concentration (g/L); x0, Initial biomass concentration (g/L); μm, Maximum specific growth rate (1/h); μm,max, Highest value of maximum specific growth rate in absence of inhibition (1/h) ⁎ Corresponding author. E-mail address: [email protected] (G. Hodaifa). https://doi.org/10.1016/j.jwpe.2020.101197 Received 13 September 2019; Received in revised form 12 February 2020; Accepted 15 February 2020 2214-7144/ © 2020 Elsevier Ltd. All rights reserved.

Journal of Water Process Engineering 35 (2020) 101197

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1. Introduction

the production of biomass with desirable characteristics. The relationship between the nutrients concentration and the composition of the biomass is well studied [22]. On the other hand, the culture medium has a significant effect on the specific growth rate and the maximum level of biomass production. The deficiency in an essential nutrient in the culture medium entails the adaptation of the metabolism of the microalgae in response to new external conditions. In general, changes in the culture medium results in the variation of the biochemical composition of the biomass, fundamentally proteins, lipids, carbohydrates and pigments. Furthermore, various physical and chemical parameters can affect algal growth including light irradiance, temperature, CO2 concentration in the gas phase, pH of the culture medium, mechanical agitation, aeration and salinity. This study examines the growth of C. pyrenoidosa on culture media based on olive oil washing wastewater (OOWW) with the dual objectives: i) OOWW treatment and ii) providing a low-cost substrate for algal biomass production, which can be used for biofuels production. For this purpose, different dilutions of OOWW (from 5% to 100 %, v/v) with ultrapure water enriched with urban wastewater from secondary treatment (by the addition of the inoculum) were used for culture media formation. Kinetic growth, biochemical composition of the final biomass and final water treated quality were evaluated.

In modern society, wastewaters constitutes a critical problem in organization of the city infrastructure and also in planning of industrial activities; there is an increasing concern over the climate change and energy crisis, caused by the globally increasing energy demand while depletion of fossil resources [1]. On the other hand, scarcity and limited access to water resources worldwide require the preservation of existing resources from possible contamination. Different human activities contribute to this problem, including industrial and agricultural activities. In this sense, one of the major source of pollution across Mediterranean countries are olive mill wastewaters (OMWs), generated seasonally during the olive oil extraction process. The OMWs are normally discharged into natural water reservoirs without any pre-treatment, thus contaminating groundwater resources and causing serious environmental and health problems to humans, flora and fauna, mainly due to its high organic load content (specifically, phenolic compounds). For this reason, the OMWs pollution is a major concern among the main olive oils producing countries where 30 million m3 of OMWs are generated annually [2–7]. The extraction process of olive oils has evolved over the years from discontinuous to continuous methods. In the first method, olive oil was obtained by applying hydraulic pressure (press method). Nevertheless, the olive oil industry was modernized with the appearance of continuous methods using centrifugal separators. At first, a process with decanter of three outlets (olive oil, pomace and wastewater) was used. From the 1990s in order to reduce the environmental impact generated by this process, the number of outlets in the horizontal centrifuge (decanter) was reduced from three to two, one for olive oil and the other for olive pomace and vegetable water (plus addition water of process). It is important to indicate that in the later process two different types of wastewater are generated: wastewater from olives washing (WOW) and wastewater from olive oil washing (OOWW) in the vertical centrifuge. Currently, Spain is the world’s largest olive oil producer in the world and practically uses the later process. MartínezNieto et al., [8] and Hodaifa et al., [9] have demonstrated that wastewater from olives washing (WOW) does not present an environmental problem (COD < 1 g O2/L) as it can be directly used for irrigation or reused after simple treatment by physical and chemical methods. Whereas, olive oil washing wastewater (OOWW) constitute a serious environmental problem due to its high organic matter content (COD =3 g O2/L up to 15 g O2/L [10,11]). Chemical, biological and integrated technologies were used for the treatment of olive oil industry’s wastewater [12]. Biological method (anaerobic digestion) appeared to be unsuccessful due to the presence of phenolic compounds and residual oil. However, processes based on the use of different physicochemical operations can reduced the concentration of phenolic compounds and residual olive oil in wastewater. Membrane technology and biological treatment were studied for mills in which high volumes and heavy OMW-3 were produced, whereas chemical oxidation or advanced oxidation processes (AOPs) as Fenton and photo-Fenton were recommended for OMW-2 (WOW and OOWW) [8,9,13]. Supercritical hydrothermal gasification was also studied due to its suitability to convert into gases all kind of olive mill wastewater [14]. Currently, the use of evaporation ponds is the most widespread practice used for olive mill wastewaters management. The microalgae cultivation for sustainable biofuel production has recently gained much interest due to its easy culture, rapid growth rate and the production of high added value compounds including non-polar triacylglycerol, which is the main substrate to produce biodiesel [15]. In addition, microalgae have great biotechnological potential in the pharmaceutical, nutraceutical, food and feed industries [16,17]. Moreover, these microorganisms have successfully performed numerous environmental applications such as CO2 mitigation, wastewater treatment and biofuels production [18–21]. The composition of the culture medium is a fundamental factor in

2. Materials and methods 2.1. Microorganism and photobioreactor All cultures were performed with the green microalga Chlorella pyrenoidosa Chick, strain 8H Emerson, from the Cambridge Botanical School Algae Culture Collection. Stirred batch photobioreactors with 0.5 L working volume (dimensions: 7 cm diameter, 20 cm height) in sterile conditions were used. The artificial illumination was provided under a light-dark cycle (12 h illumination per day) from one side of photobioreactors [36]. 2.2. Experimental conditions and procedure The olive oil washing wastewaters (OOWW) used in this work were obtained from an olive-oil extraction plant in the province of Jaen, which uses a continuous centrifugation process with two outlets decanter. Prior to C. pyrenoidosa culture, OOWW were pre-treated by centrifugation to separate the solid phase and filtered-sterilized through a glass wool pre-filter and cellulose nitrate membrane (0.45 μm pore diameter). Then, the culture media were formed through the preparation of different OOWW dilutions (5%, 10 %, 20 %, 30 %, 50 % and 100 % of OOWW, v/v). All cultures media were prepared with OOWW and ultrapure water and enriched with a constant quantity of 10 % (v/v) of C. pyrenoidosa inoculum to reactors. In this sense, no direct mixing between OOWW and UW was performed to control the inhibitory growth effects of OOWW (caused mainly by phenolic compounds and residual olive oil in OOWW) in order to determine the optimum mixing proportions for the formation of the culture medium. The inoculum was previously taken from a bubble column that used continuously and treated urban wastewater from secondary treatment as nutrient medium (Fig. 1). The average initial concentration, for all the experiments, in the photobioreactors at time zero after inoculum addition was 0.0238 g/L with a standard deviation equal to 0.0813 g/L. All experiments were performed in isothermal conditions by using a thermostat control system to maintain a constant temperature inside the photobioreactors. The difference of temperature between the inside of the photobioreactors and the thermostat circuit was less than 1 °C. The common culture conditions maintained over the course of the experiments were pH = 8, temperature = 25 °C, without mechanical stirring and aeration rate = 1 v/v/min supplied by sterilized air throughout filtration with fluoropore membrane filter, hydrophobic PTFE (0.2 μm pore diameter). The daily illumination regime was a 2

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using pH-meter Crison, mod. GLP 22C and a conductimeter Crison, mod. GLP31, respectively. Total solids and ashes were determined by drying and incinerating samples in a stove at temperature of 105 ± 1 °C and an oven at 575 ± 25 °C, respectively. The difference between both parameters corresponds to the organic matter percentage. The determination of chemical oxygen demand COD and total phenolic compounds TPCs were carried out according to norms ISO 8466-1, DIN 38402 A51 and DIN 38409-41 [27–29]. Fat matter was determined by a liquid-liquid extraction operation using n-hexane. Then, solvent separation was carried out by distillation and the fat matter was dried at 105 ± 1 °C. Dissolved oxygen (DO) was measured directly by using an oxymeter (Crison, mod, OX192), and the biological oxygen demand (BOD5) was determined by the difference between dissolved oxygen in sample before and after 5 days in hermetically sealed bottles at 20 °C. Total carbon, total organic carbon, inorganic carbon (TC, TOC and IC), total nitrogen and nitrate and nitrite ions (TN and NO3−+NO2−) were measured by a Total Carbon and Nitrogen Analyzer provided by Skalar Company (mod. FormacsHT and FormacsTN). Total phosphorus was determined photometrically by reacting orthophosphate ions in sulfuric acid solution with molybdate ions to form molybdophosphoric acid. Ascorbic acid reduces this compound to phosphormolybdenum blue. Nitrate was determined photometrically by reacting nitrate ions, in sulphuric and phosphoric solution, with 2,6-dimethylphenol (DMP) to form the compound 4-nitro-2,6-dimethylphenol. Nitrite ions in acid solution form with the sulphanilic acid a diazonium salt that reacts with the N-(1-naftill)-ethylenediamine dihydrochloride giving a reddish violet azo dye, which was determined photometrically. Ammonium ion (NH4+) in strong alkaline solution is present entirely as ammonia. This reacts with hypochlorite ions to form monochloramine, which in turn reacts with 2-chlorphenol or thymol to form indophenol blue. This is then determined photometrically at 690−712 nm [30,31]. Sulphates and chloride were determined photometrically at 420 nm and 450 nm, respectively [27,28]. Copper ions were determined photometrically by reacting ammoniacal medium copper (II) ions with cuprizone to form a blue complex. Magnesium ions, in neutral solution, form with phthaleins purple a violet dye, which is determined photometrically. On the other hand, biomass concentration (x, g/L) was indirectly measured according to Camacho et al., [30] after cell separation and washing. From all samples taken during the course of all experiments a straight-line calibration of A600-dry weight (g/L) was performed, Eq. (1).

Fig. 1. Bubbling column (Height 198 cm and diameter 20 cm) used to supply inoculum to experiments. Operating conditions: urban wastewater from secondary treatment as culture medium, aeration 0.5 v/v/min, environment temperature and natural light.

light-dark cycle (12 h/12 h), with an initial illumination intensity of 126.2 μE/(m2 s) (measured with QSL 2100, Bio Spherical Instruments, Inc.). During the experiments, the pH adjustment of the culture media was carried out with the addition of a 0.1 M HCl and 0.1 M NaOH solutions. The biomass concentration (x, g/L), chlorophylls A and B, total chlorophylls (CHLTotal) and carotenoid contents (CaTotal) were determined over the course of each experiment. At the end of each experiment, the algal biomass was separated and washed. Lipids (including fatty acids profile) and crude protein (Crude protein = total nitrogen×6.25, [22]) contents were determined. The carbohydrates content was calculated as 100 % minus the percentages of proteins, lipids, pigments and genetic material (in all experiments the percentage of genetic material was considered equal to 1%). In addition, an elemental analysis was carried out on the harvested biomass at the en d of the experiments.

x (g/L)=0.331×A600 + 0.001

(1)

The chlorophyll (CHL) and carotenoid contents (Ca) were determined by a photocolorimetric method prior to extraction with acetone at 90 % [31]. Crude protein concentration was quantified according to [32]. At the end of each experiment, the algal biomass harvested was separated, and washed twice with ultrapure water. Then elemental composition and fatty-acids profile in the lipid fraction were determined by gas chromatography [33]. Furthermore, the quality of the obtained water (treated wastewater) at the end of each culture was determined.

3. Analytical methods Wastewaters used in this work were characterized in terms of pH, electric conductivity, total solids, organic matter, phenolic compounds, fat matter, ash, dissolved oxygen, BOD5, COD, total Table 1nitrogen, total phosphorus, nitrates and nitrites, ammonium, magnesium, copper, and chloride (Table 1), [23–26]. The pH and electric conductivity values were measured directly

3.1. Calculation and statistical methods The experiments were repeated at least twice and the analytical methods were performed at least three time in order to check the reproducibility of the results. Mathematical equations and statistical analysis were obtained by using OriginPro 8.0 program. 3

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Table 1 Physico-chemical characterization of crude olive oil washing wastewater ‘OOWWcrude’, urban wastewater ‘UW’, inoculum and OOWW enriched with the inoculum used in this work. Parameter

OOWWcrude

UW

pH Electric conductivity (mS/cm) Total solids (%) Organic matter (%) Total phenolic compounds (mg/L) Fat matter (%) Ash (%) Dissolved O2 (mg/L) BOD5 (mg O2/L) COD (mg O2/L) Total carbon (mg/L) Total organic carbon (mg/L) Inorganic carbon (mg/L) Total nitrogen (mg/L) Total phosphorus (mg/L) NO3−+NO2− (mg/L) NH4+ (mg/L) SO42− (mg/L) Mg2+ (mg/L) Cu2+ (mg/L) Cl− (mg/L)

6.29 1.32 0.13 0.090 11.1 0.030 0.040 2.01 1358 1362 257.9 191.5 66.4 7.49 1.63 Not detect < 4.00 217 62.4 1.60 172

7.1 5.47 0.131 0.018 0.07 0.780 0.113 3.70 132 252

Inoculum (UW + RL)

265 17.1 66.2 205 162 14.8 < 0.05 1200

76.6 33.2 43.5 340.3 55 340.3

100 % OOWWenriched(90 %OMWW + 10 % Inoculum)

1235 1251 239.8 175.7 64.1 40.8 6.97 34.1 24.1

4. Results and discussion

4.2. Biomass growth and kinetic parameters

4.1. Characterization of wastewaters used as culture media

For cell growth monitoring, cells number and biomass concentration along the cultures were determined. The observation of the culture medium by optic microscope along the growth allowed the evaluation of the real state of the culture and the presence of microbial contamination, especially, with bacteria. In addition, the determination of cells number enabled to determine the average cell weight. Fig. 2 shows the representation of the biomass concentration versus the corresponding number of cells determined in all the experiments. The highest biomass concentration and cell number registered in the cultures were equal to 0.542 g/L and 3.65 × 1010 Cells/L. In addition, the average cell weight (value of dry-weight (g/L) to number of cells (cells/L)) was kept constant over the exponential and deceleration

Table 1 shows the characterization of crude OOWW, urban wastewater from secondary treatment (UW), inoculum and the mixture of OOWW with inoculum (OOWWenriched). The difference in the biochemical composition between OOWW and UW can be observed. More precisely for electric conductivity (EC), BOD5, COD, total nitrogen (TN), total phosphorus (TP) and chloride ions. High EC, TN, TP, and chloride values were obtained for the UW when compared to those for the OOWW: 4, 35, 10 and 7 times higher in UW, respectively. This fact support the theory of enriching OOWW with UW given its higher content in essential nutrients for microalgae growth such as nitrogen and phosphorus. The high EC value (5.47 mS/cm) in UW is due to the high concentration of inorganic ions such as chloride (1200 mg/L). Similar values of BOD5 (1358 mg O2/L) and COD (1362 mg O2/L) in OOWW were observed, which indicates the biodegradability of organic content. The OOWW has higher BOD5 and COD values when compared to the UW from secondary treatment, notwithstanding the OOWW has less organic load than indicated in the literature (COD from 3 to 15 g O2/L) [10,11]. This fact is due to the operation mode applied in the olive mill where samples were collected. The olive mill respects the cycles of cleaning and water change in the olive washing machines and in the vertical centrifuge where the OOWW is obtained. In fact, the OOWW is colourless as the olive mill performs the complete control of the operations throughout the process in order to avoid OOWW colouring. This allows no light limitation in algal cultures. In other words, this OOWW has physicochemical parameters (Table 1) far from the typical parameters values of OOWW from olive mills indicated in the literature with high COD (3−15 g O2/L) and total phenolic compounds values (up to 749.0 mg/L) [10,11]. The mixture of inoculum based on UW and OOWW allowed the culture media enrichment. In this sense, the preparation of a culture medium with 90 % OOWW and 10 % inoculum (v/v) allowed nitrogen and phosphorus increase from 7.49 mg/L to 40.8 mg/L and from 1.63 mg/L to 6.97 mg/L, respectively (Table 1). In other words, the content in macronutrients, among others, increased in the culture medium for microalga growth.

Fig. 2. Dry weight-cell number relationship obtained in all OOWW cultures of C. pyrenoidosa (Blue line corresponds to 95 % confidence intervals). Common culture conditions: pH = 8, mechanical stirring =0 rpm, aeration rate = 1 v/ v/min, initial illumination intensity = 126.2 μE/(m2 s), light/dark periods = 12 h/12 h and temperature = 25 °C.

4

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Table 2 A comparison between the average cell weights of C. pyrenoidosa obtained in this work and the average cell weights of Scenedesmus obliquus determined for different culture media. Microalga

Culture medium

Average cell weights (pg/cell)

C. pyrenoidosa S. obliquus

Different concentration of OOWWenriched from process with decanter of two outlets in ultrapure water (0-100 % v/v) Mineral synthetic medium of Rodríguez-López [32]. Urban wastewater from secondary treatment. Different concentration of OMW from process with decanter of two outlets in ultrapure water (0–100 % v/v). Mixture between 0–50 % of urban wastewater from secondary treatment plus 5 % of bleach OMW from process with decanter of three outlets. Mixture between 1–10 % of OMW from process with decanter of three outlets plus urban wastewater from secondary treatment.

13 19 19 20 33

phases at 13 pg/cell. This value is lower than that registered for Scenedesmus obliquus cells in different cultures media in which the highest value was determined in the culture medium formed by mixing OMW-2 from process with two outlets decanter and with urban wastewater from secondary treatment (Table 2). The determination of the maximum specific growth rate (μm, h−1) and biomass productivity (Pb, g/(L h)) was performed from the experimentally obtained growth curves (Fig. 3A). In this sense, μm was determined in the exponential growth phase according to Eq. (1), ln (x/x0) = μm t+a

continued at a lower rate (Fig. 3A). Biomass productivity (Pb) was determined in this phase according to Eq. (3) x=Pb t+c

(1)

µ m, max [%OOWWenriched] KS+ [%OOWWenriched] +

[%OOWW2enriched] Ki

(3)

This linear growth period is characteristic for bioprocesses controlled by nutrients availability. Its appearance is related to the limitation of any nutrient in the culture medium or limited availability of light or CO2 [37,[38]]. Fig. 3C shows that the maximum Pb value was determined in the 10 % OOWWenriched culture. Accordingly, a decrease in the Pb value with the increase of the OOWWenriched concentration in the culture medium can be observed, obtaining the lowest Pb value in the most concentrated medium (100 % OOWWenriched), similarly to the μm. The same light intensity and airflow (CO2) were supplied to the cultures. Thus, the appearance of this phase might be due to the increase of inhibitory compounds concentration in the culture media coupled by the increase of OOWWenriched concentration (mainly phenolic compounds). In fact, several researchers indicated the capacity of C. pyrenoidosa to remove phenolic compounds and other pollutants. Dayana and Bakthavatsalam [39,40] demonstrated the capacity of C. pyrenoidosa (KX686118) to remove phenolic compounds (> 90 %) from coal effluents of gasification plants. In a similar way, the variation in the volumetric biomass productivities versus the OOWWenriched was adjusted by the Haldane model [35], eq. (2), with an acceptable goodness of the fit (R2 = 0.975 and SSE = 5.96 × 10−8, Table 3) and the obtained data are consisted with that observed experimentally. In this case, there is a noticeable difference between the values of experimental Pb, max and those determined by the model. This fact might be due to the depletion of one or more nutrients, or to the accumulative effect of growth-inhibiting compounds in the biomass such as phenolic compounds, or due to the covering of cells with a layer of residual fat contained in wastewater, which prevents the exchange of nutrients between the cell and the culture medium [36]. The first assumption is not possible give that with the increase in the OOWW concentration in the culture medium, the decrease in the biomass productivity values was greater (Fig. 3C). Bearing in mind that both μm and Pb decreased with the increase in the OOWWenriched concentrations in the culture media, the accumulative effect of the growth-inhibiting compounds becomes more evident. In this sense, the value of KI in the case of Pb variation is lower than that determined for μm (Table 3).

where the slope ‘μm’ corresponds to the maximum specific growth rate determined in the exponential phase of growth and ‘a’ is the intercept when x = 0. Fig. 3B shows the variation of μm values when the OOWWenriched concentration was varied in the culture medium from 5% to 100 % of OOWWenriched (v/v). It can be observed that μm values showed a pronounced increase with the augment of the OOWWenriched concentration until reaching the maximum μm value, equal to 0.0203 (1/h) in the medium composed of 30 % OOWWenriched (v/v). Then, μm values decrease in the most concentrated culture media. These results were to be expected due to the presence in the OOWWenriched of harmful compounds, such as fat matter, organic acids and phenolic compounds, which are responsible for the toxic and inhibitory effect [34]. A number of empirical models have been proposed for the description of substrate limitation and inhibition of microbial growth. Haldane [35] proposed an equation for uncompetitive inhibition, which can be written as:

µm =

37

(2)

where ‘μm, max’ is the highest value of maximum specific growth rate in absence of inhibition, and ‘KS’ and ‘Ki’ are empirical saturation and inhibition constants, respectively. The adjustment of the experimental results to this mathematical model has made it possible to determine the different kinetic parameters (Table 3). The difference between the experimental maximum value of ‘μm’ and ‘μm, max’ calculated by the model was expected since the last value is a theoretical value in culture without inhibition effect. The values of ‘KS = 3.56 %’ and ‘Ki = 84.7 %’ are consistent with the experimental results. The parameters of the goodness of the fit were R2 = 0.990 and sum of squared errors (SSE) = 2.14 × 10−6. When comparing this behaviour with that obtained by Hodaifa et al., [36] for culture media formed in wastewater from the process using a decanter of two outlets (mixture of olives and olive oil washing wastewaters with initial COD =7.1 g O2/L), it is observed that a higher μm, max value (0.048 1/h) was obtained. However, the fall in μm values is more abrupt with the increase of the wastewater concentration in the culture media (μm = 0.015 1/h in cultures without dilution). In all experiments, exponential phase was followed by a linear behaviour during the deceleration growth phase in which cell growth

4.3. Biomass generation and biochemical composition The variation in the concentration of net biomass and its biochemical composition is dependent on the concentration of OOWWenriched in the culture medium (Fig. 4). The net biomass generation (x-x0 variation = 0.135-0.238 g/L) decrease with the increase of OOWWenriched concentrations in the culture media (Fig. 4A). This behaviour was expected considering that the increase of OOWWenriched concentration in the culture media involves higher values of phenolic compounds and fat matter in the medium that prevents microalgae growth because of their toxic effect. 5

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Table 3 Calculation of the kinetic parameters of the mathematical model of inhibition by substrate (Haldane model [26]) fitted to the variation of the maximum specific growth rates and volumetric biomass productivities. Common culture conditions: pH = 8, mechanical stirring =0 rpm, aeration rate = 1 v/v/min, initial illumination intensity = 126.2 μE/(m2 s), light/dark periods = 12 h/12 h and temperature = 25 °C. Parameters

Maximum specific growth rate

μm, max. experimental, (1/h) Pb, max. experimental, (1/h) μm, max. model, (1/h) Pb, max. model, (1/h) KS (%) KI (%) SSE R2

0.0203 0.0292 3.56 84.7 2.14 × 10−6 0.990

Volumetric biomass productivity 0.00173 0.00468 10.1 11.6 5.96 × 10−8 0.975

concentration in the culture medium until reaching the concentration of 30 % OOWW (v/v), (Fig. 4B). Then, these percentages decreased. This behaviour is similar to that registered for maximum specific growth rates and volumetric biomass productivities, which means that the generation of pigments is associated to C. pyrenoidosa growth. In general, the percentages of pigments (1.10 ± 0.07 %) were maintained constant in the final harvested biomass from all cultures. Nevertheless, total chlorophylls and total carotenoids percentages varied while the highest values registered for total chlorophylls (0.96 %) and total carotenoids (0.24 %) were determined in the culture with 30 % of OOWW (v/v). On the other hand, the total chlorophylls content in all the cultures was maintained 4.3 ± 0.2 times higher than the total carotenoids independently of the net biomass generation variation. In addition, Fig. 4C shows the decrease of total lipid percentages when the increase of net biomass generation. Opposite behaviour was registered for the carbohydrates percentages, while the proteins percentages were maintained constant (44.1 ± 0.9 %) with the increase of biomass generation. This fact was also confirmed by the biomass elemental analysis, in which the mean value of nitrogen percentage was equal to 7.1 % (Table 4). These results could be explained considering that the culture media enrichment by nitrogen addition with inoculum at constant percentage equal to 10 % (v/v) and the variation of the OOWW percentage in the culture media did not affect the final nitrogen content since OOWW only have 7.5 mg/L (Table 1). In other words, biomass generation was not limited by nitrogen when the OOWW percentage was varied. In any case, all cultures had a deficiency in the availability of nitrogen since the OOWWenriched contained only approximately around 40 mg/L of total nitrogen. This value is 4 times lower than that of the mineral synthetic medium of Rodríguez-López (RL, [41]) with 140 mg N available/L, which is normally used as control culture for this microalga. The highest values of lipids percentages (44.0–51.5 %) were determined in cultures containing more than 20 % of OOWWenriched (v/v) while the highest value of carbohydrates percentage (29.4 %) was determined in the culture with 5% of OOWWenriched. In this sense, Wu and Miao [42] highlighted that nitrogen is an essential nutrient in many macromolecules (such as proteins, chlorophylls, RNA, DNA) and it acts as one of the most essential nutrients for microalgae growth. Nitrogen limitation or starvation normally result in a decrease in photosynthesis, protein and lipid synthesis, as well as an increase in carbohydrate formation [42,43]. On the other hand, N and P are known algal growth limiting factors. The optimal value of N:P ratio for freshwater algae has been suggested to be in the range of 6.8–10 [44]. However, the N:P ratio in this study was approximately 5.9 in the case of the culture medium with 100 % OOWW enriched. Nitrogenous matter was mainly composed of inorganic NH4–N in UW. The initial NH4–N concentration was, approximately 24.1 mg/L (Table 1). Algae have a considerable intracellular capacity for storing soluble and organic N molecules. Nitrogen is rapidly

Fig. 3. A) Growth curves of C. pyrenoidosa in 5%OOWWenriched (v/v). Exponential phase (solid line): ln (x/x0) is plotted as a function of time. Deceleration phase (solid line): biomass concentration is plotted as a function of time. B) Behaviour of maximum specific growth rate and C) Volumetric biomass productivity of C. pyrenoidosa values with the variation of OOWWenriched concentrations in the culture media formed (Solid line in (B) and (C) corresponding to the inhibition by substrate Haldane model). Common culture conditions: 5% of OOWWenriched, pH = 8, mechanical stirring =0 rpm, aeration rate = 1 v/v/ min, initial illumination intensity = 126.2 μE/(m2 s), light/dark periods = 12 h/12 h and temperature = 25 °C.

Fig. 4 (B and C) shows the percentages variation of the cell components (pigments, proteins, carbohydrates and lipids) present in the biomass versus the net biomass generation obtained (x-x0, g/L) in the cultures. The percentages of total chlorophylls (CHL) and total carotenoids (Ca) were increasing with the rise of OOWWenriched 6

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Table 4 Elemental composition of the harvested biomass at the end of the experiments. Common culture conditions: pH = 8, mechanical stirring =0 rpm, aeration rate = 1 v/v/min, initial illumination intensity = 126.2 μE/(m2 s), light/dark periods = 12 h/12 h and temperature = 25 °C. %OOWW (v/v)

%N

%C

%H

%S

%O

N/C

5 10 20 30 50 100

7.12 6.92 6.89 7.10 7.30 6.99

42.4 40.9 43.3 42.2 44.1 40.1

6.81 6.73 6.63 6.84 6.61 6.34

0.98 0.86 0.84 0.88 0.77 0.75

42.7 44.6 42.3 43.0 41.3 45.8

0.167 0.169 0.159 0.168 0.166 0.174

Han et al., [45] indicated that nitrogen limitation and high pH conditions constitute an effective method for lipid accumulation in microalgae biomass. Lipids content in some microalgae such as Chlorella sp., Nannochloris sp. and Neochloris oleobundans, can significantly increase under nitrogen limitation conditions, commonly ranging from 30 % to 60 % [46]. Gardner et al. [47], found that the triacylglycerol contents of Chlorella sp. and Scenedesmus sp. accumulated faster when the cells were cultured in the controlled high pH. However, nitrogenlimitation or high pH induction had little contribution to the lipid productivity of the cells, since the cells grow decelerate under the stress conditions. Tan et al., [48] demonstrated that low molar ratio of N/C (nitrogen limitation) in the culture medium play an important role in lipid accumulation. Several researchers have indicated that Chlorella accumulates more lipids with decreased molar ratios of N/C, typically at N/C ratio of 0.02–0.0109 [49]. In this study, the initial molar ratio of N/C was only 0.17 in the 100 % OOWWenriched indicating no severe nitrogen limitation. Elemental composition of the harvested biomass at the end of each experiment is recorded in Table 4. Results indicate that oxygen (43.3 % ± 1.6) and carbon (42.2 % ± 1.5) were the most abundant elements in biomass, followed by nitrogen (7.05 % ± 0.15), hydrogen (6.66 % ± 0.18) and sulphur (0.84 % ± 0.08). As observed, no significant differences in the elemental composition of the harvested biomass from the experiments were registered. Khalid et al., [50] proved the ability of C. sorokiniana to change its internal elemental composition as a function of the nutrients concentration in culture medium. Furthermore, Whitton et al., [51], also demonstrated the relationship between microalgae elemental composition and nutrients concentration. It was proved, for five freshwater microalgae species, that those species with high N and low P internal composition were able to uptake ammonium and phosphate more efficiently. Lastly, Beuckels et al., [52] proved that Chlorella and Scenedesmus are able to adjust the internal concentration of N and P in biomass according to the N and P concentration in the wastewater medium. Table 5 lists the composition of fatty acids (FAs) of the lipid fraction of the C. pyrenoidosa biomass obtained at the end of each experiment, as a percentage (w/w) of the total lipid fraction. FAs were grouped into saturated (SFAs), monounsaturated (MUFAs) and polyunsaturated fatty acids (PUFAs). The most abundant group was PUFAs, with an average content of 44.5 % (SD = 2.47 %) followed by SFAs, 28.8 % on average (SD = 1.16 %) and MUFAs, 13.3 % on average (SD = 0.699 %). Within SFAs, palmitic (C16:0) and stearic (C18:0) were the most abundant, with values ranging from 19.2 % (100 % OOWWenriched) to 21.9 % (5% OOWWenriched) for palmitic, and from 6.54 % (100 % OOWWenriched) to 7.72 % (5%OOWWenriched) for stearic. Oleic acid (C18:1) was the major MUFAs (9.06 % on average) and the most abundant PUFAs identified were C18:3 (22.5 % on average) and C18:2n6 (15.9 % on average). Meanwhile, eicosapentaenoic acid or EPA (C20:5n3) was detected at low concentrations in all conditions, ranging from 0.185 %, when 100 % OOWWenriched was used, to 1.65 % in 10 % OOWWenriched culture

Fig. 4. Net biomass generation obtained versus different OOWWenriched percentages in culture media (A), total chlorophylls (CHLtotal) and total carotenoids (Catotal) percentages on final algal biomass versus generated biomass in each culture (B) and variation of proteins, lipids and carbohydrates on final algal biomass versus generated biomass in each culture (C). Common culture conditions: pH = 8, mechanical stirring =0 rpm, aeration rate = 1 v/v/min, initial illumination intensity = 126.2 μE/(m2 s), light/dark periods = 12 h/12 h and temperature = 25 °C.

consumed as being indispensable for the regulation of the metabolic pathways. It is used to produce amino acids, and other organic Ncontaining macromolecules. At too high concentrations, the NH4–N becomes toxic and can thus inhibit algal growth. 7

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C18:1 in various microalgae species [53]. The final biochemical composition of the harvested C. pyrenoidosa biomass at the end of the cultures can be seen in Fig. 5. No variation was observed in the total pigments (1.10 ± 0.07 %) and crude proteins contents (44.1 ± 0.8 %). Meanwhile, total lipids content increase with the increase of %OOWWenriched in the culture media and it was maintained constant (48.8 ± 3.4 %) in culture media containing from 30 % to 100 % of OOWWenriched (v/v). Opposite behaviour was observed for carbohydrates content, which decreased from 29.4 % to 2.81 % with the %OOWWenriched increase. These behaviours are to be expected given the virtually constant nitrogen availability in the culture media. The same UW added to culture media as inoculum and the global variation in nitrogen by the addition of OOWW is depreciated since the TN in OOWW is only 7.5 mg/L. Nitrogen limitation or starvation normally results in the decrease of photosynthesis, proteins and lipids synthesis; on the contrary, carbohydrate synthesis is increased [43,44]. The nitrogen availability in OOWWenriched was 4 times lower than that in the control mineral synthetic medium of Rodríguez-López [41] with 140 mg N available/L. Becker [54], determined a crude protein percentage equal to 57 % for C. pyrenoidosa in synthetic medium, which is higher than that obtained in the experiments of this work (44.1 %). Nevertheless, the same author obtained a carbohydrate content of 26 % and 2% of lipids, which are lower than those obtained in this work equal to 29.4 % and 51.5 %, respectively. The final harvested biomass could be used in combination with other substrates for biofuels production or maybe as supplementary substrate in the anaerobic digester for biogas production. Moreover, as the least favourable option, it could be used for domestic, commercial or industrial boilers and as a fuel for generators of electric energy production.

Table 5 Fatty-acids profile of lipid fraction of C. pyrenoidosa biomass obtained at the end of the experiments. Common culture conditions: pH = 8, mechanical stirring =0 rpm, aeration rate = 1 v/v/min, initial illumination intensity = 126.2 μE/ (m2 s), light/dark periods = 12 h/12 h and temperature = 25 °C. %Fatty acids

Myristic acid (C14:0) Myristoleic acid (C14:1) Palmitic acid (C16:0) Palmitoleic acid (C16:1) Hexadecadienoic acid (C16:2) C16:3 Stearic acid (C18:0) Oleic Acid (C18:1) Linolenic acid (C18:2n3) Linoleic acid (C18:2n6) Arachidic acid (C20:0) α-Linolenic acid (C18:3) Gadoleic acid (C20:1n9) Stearidonic acid (C18:4n3) All cis-11,14-eicosadienoic acid (C20:2n6) Docosanoic Acid (C22:0) Docosenoic Acid (C22:1n9) Eicosapentaenoic acid (C20:5n3) DPA. Clupanodonic acid (C22:5n3) %SFAs %MUFAs %PUFAs

%OOWWenriched (v/v) 5

10

20

30

50

100

0.707 0.782 21.9 2.90 3.74

0.517 0.778 20.7 3.30 5.19

0.489 0.779 20.6 3.17 4.37

0.678 0.825 19.8 3.65 3.26

0.605 1.24 21.0 4.58 5.68

0.521 1.04 19.2 1.67 4.35

0.351 7.72 8.85 0.124 14.3 0.116 24.1 – – 0.648

0.375 7.64 8.61 0.108 17.3 0.108 23.1 – 0.176 0.181

0.348 7.31 8.68 0.0809 17.1 0.116 22.0 – 0.0894 0.206

0.353 7.40 9.06 0.151 13.1 0.0981 24.6 0.230 0.0980 0.131

0.373 6.72 8.25 0.0875 15.5 0.120 18.5 – 0.0922 0.179

0.428 6.54 10.9 0.0808 18.1 0.127 22.6 – – 0.448

– – 0.289

0.180 – 1.65

0.316 – 0.284

0.572 0.140 0.470

1.08 0.251 0.447

0.260 – 0.185

–

0.185

1.11

0.294

–

0.146

30.4 12.5 43.6

29.1 12.7 48.3

28.8 12.6 45.6

28.5 13.9 42.5

29.5 14.3 40.9

26.6 13.6 46.3

4.4. Photo-bioremediation of urban and olive mill wastewaters media. No docosahexaenoic acid or DHA (C22:6n3) was found in any condition. Nutrient starvation, temperature, pH and light intensity are the main factors affecting FAs composition [53]. In this sense, as pH, light intensity and temperature were maintained constant along the cultures, the observed differences in the FAs composition should be attributed to the differences in nutrients concentration. Regarding nutrients, nitrogen limitation is the major critical compound affecting the lipid metabolism and the proportion between saturated and unsaturated FAs. Besides, phosphorous deprivation increase the content of C16:0 and

Table 6 shows the physicochemical characterization of enriched olive mill wastewater (without dilution) treated and the total removal percentages achieved after the bioprocess applied using C. pyrenoidosa. In general, high elimination percentages (> 86 %) were obtained in the main contamination parameters (total solid, total phenolic compounds and chemical oxygen demand). The proposed bioprocess (microfiltration plus C. pyrenoidosa growth) in this work improve the physiochemical characteristics of OOWW. In this sense, the highest elimination percentages registered after the bioprocess were obtained for total solids (98.8 %), COD (86.3 %) and TPCs (85.7 %). Table 6 Physico-chemical characterization of olive oil washing wastewater enriched with urban wastewater without dilution (100 % OOWWenriched) and total removal yield after microfiltration plus C. pyrenoidosa growth. Common culture conditions: pH = 8, mechanical stirring =0 rpm, aeration rate = 1 v/v/min, initial illumination intensity = 126.2 μE/(m2 s), light/dark periods = 12 h/12 h and temperature = 25 °C.

Fig. 5. Biochemical composition of the harvested C. purenoidosa biomass at the end of the cultures. Common culture conditions: pH = 8, mechanical stirring =0 rpm, aeration rate = 1 v/v/min, initial illumination intensity = 126.2 μE/ (m2 s), light/dark periods = 12 h/12 h and temperature = 25 °C. 8

Parameter

Treated OOWWenriched

Total removal yields

Electric conductivity (mS/cm) Total solids (%) Total phenolic compounds (mg/L) Dissolved O2 (mg/L) COD (mg/L) Total phosphorus (mg/L) NH4+ NO3− (mg/L) NO2− (mg/L) SO42− (mg/L) Mg2+ (mg/L) Cu2+ (mg/L) Cl− (mg/L)

1.01 0.00157 1.59 5.50 187 0.710

– 98.8 85.7 – 86.3 56.4

0.980 0.200 93.0 32.6 0.940 91.0

49.7 48.3 57.1 47.8 41.3 47.1

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Furthermore, removal percentages equal to 49.7 %, 57.1 %, 48.3 % and 47.8 % were obtained for NO3−, SO42-, NO2- and Mg2+, respectively. C. pyrenoidosa was also able to reduce Cu2+ (41.3 %), total phosphorus (56.4 %) and Cl- (47.1 %). COD and TPCs remediation was particularly relevant since high organic loads and the presence of antioxidant compounds (such as phenolic compounds) is not desirable in wastewaters intended to be reused for irrigation or discharged into water public channels. In this sense, other authors have obtained lower COD elimination levels when using OMWs as culture media for different microalgae strains. Markou et al., [55] achieved COD elimination percentages ranging from 28.8%–66.9% after the culture of Spirulina platensis in OMW-3. Furthermore, Travieso et al., [56] obtained 37 % of COD removal from OMW-2 after a process based on anaerobic digestion, sedimentation and Chlorella zofingiensis culture. Regarding TPCs, Markou et al., [55], demonstrated that the increase of OMW-3 concentration in the culture media results in a decrease in the capacity of microalgae to remove phenolic compounds, concluding that TPCs degradation depends on their concentration in culture media, obtaining removal percentages ranging from 41.9%–100%. In this sense, several studies have demonstrated the ability of microalgae to assimilate nitrogen in different forms, being ammonium ions the preferred source since less energy is required [57]. The presence of phosphorus in the medium is highly relevant for microalgae cell growth due to its role in metabolism and phosphorylation reactions. On the other hand, the increase in dissolved oxygen concentration after the microalgae culture indicated a photosynthetic activity instead of a carbon oxidation throughout heterotrophic growth [57]. The treated water obtained after the bioprocess was characterized by a high quality since it did not present any toxicity (heavy metal or phenolic compounds) considering that it comes from the agri-food industry. Quality parameters registered in Table 6 indicate that treated water could be used for irrigation, discharges to surface water and groundwater or for drinking water if it is passed through some additional operations unit such as ion exchange, reverse osmosis or ultrafiltration to adjust its organic or inorganic content. In the same way, Spanish standards for treated OMW-2 to use in irrigation, established the following parameters pH in the range 6.0–9.0, suspended solids < 500 mg/kg and COD < 1000 mg O2/L. In addition, the treated water after the process comply with European Directive 91/271/EEC concerning urban wastewater treatment where COD < 125 mg O2/L and TN = 10 mg/L for treated water discharge into water public channels. In contrast, European Drinking Water Directive 2015/1787 specifies that drinking water is all water used in any food-production process. This Directive establishes the chemical parameters that determine drinking water quality (electric conductivity < 2500 μS/cm, turbidity acceptable to consumers and no abnormal change, TOC = no abnormal change, iron =0.2 mg/L, sulphate =250 mg/L, sodium =200 mg/L and ammonium =0.5 mg/L).

sustainable proposal to explore the combination of different resources for its better utilization. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors are grateful to the Ministry of Economy and Competitiveness for granting funds for Project CTM2009-11613 “Tertiary wastewater treatment, carbon dioxide removal and production of biofuels”. In addition, the authors acknowledge the Andalusia Regional Government (Spain) for its financial support to our research group‘Bioprocesses TEP-138’ and the International Olive Council for supporting Murad Maaitah predoctoral scholarship. References [1] T. Cai, S.Y. Park, R. Racharaks, Y. Li, Cultivation of Nannochloropsis salina using anaerobic digestion effluent as a nutrient source for biofuel production, Appl. Energy 108 (2013) 486–492, https://doi.org/10.1016/j.apenergy.2013.03.056. [2] S. Hachicha, J. Cegarra, F. Sellami, R. Hachicha, N. Drira, K. Medhioub, E. Ammar, Elimination of polyphenols toxicity from olive mill wastewater sludge by its cocomposting with sesame bark, J. Hazard. Mater. 161 (2009) 1131–1139, https:// doi.org/10.1016/j.jhazmat.2008.04.066. [3] C. Amor, M.S. Lucas, J. García, J.R. Dominguez, J.B. De Heredia, J.A. Peres, Combined treatment of olive mill wastewater by Fenton’s reagent and anaerobic biological process, J. Environ. Sci. Health, Part A Toxic/Hazard. Substances Environ. Eng. 50 (2) (2015) 161–168, https://doi.org/10.1080/10934529.2015. 975065. [4] H.K. Obied, M.S. Allen, D.R. Bedgood, P.D. Prenzler, K. Robards, R. Stockmann, Bioactivity and analysis of biophenols recovered from olive mill waste, J. Agri. Food Chem. 53 (4) (2005) 823–837, https://doi.org/10.1021/jf048569x. [5] G. Aliotta, A. Fiorentino, A. Oliva, F. Temussi, Olive oil mill wastewater: isolation of polyphenols and their phytotoxicity in vitro, Allelopathy J. 9 (1) (2002) 9–17. [6] A. Fiorentino, A. Gentili, M. Isidori, Environmental effects caused by olive mill wastewaters: toxicity comparison of low-molecular-weight phenol components, J. Agri. Food Chem. 51 (4) (2003) 1005–1009, https://doi.org/10.1021/jf020887d. [7] M. Kotsou, I. Mari, K. Lasaridi, I. Chatzipavlidis, C. Balis, A. Kyriacou, The effect of olive oil mill wastewater (OMW) on soil microbial communities and suppressiveness against Rhizoctonia solani, Agric., Ecosyst. Environ., Appl. Soil Ecol. 26 (2) (2004) 113–121, https://doi.org/10.1016/j.apsoil.2003.12.001. [8] L. Martínez Nieto, G. Hodaifa, S. Rodríguez, J.A. Giménez, J.M. Ochando-Pulido, Degradation of organic matter in olive-oil mill wastewater through homogeneous Fenton-like reaction, Chem. Eng. J. 173 (2) (2011) 503–510, https://doi.org/10. 1016/j.cej.2011.08.022. [9] G. Hodaifa, J.M. Ochando-Pulido, S. Rodriguez, A. Martinez, Optimization of continuous reactor at pilot scale for olive-oil mill wastewater treatment by Fenton-like process, Chem. Eng. J. 220 (2013) 117–124, https://doi.org/10.1016/j.cej.2013. 01.065. [10] L.M. Nieto, G. Hodaifa, S.R. Vives, J.A.G. Casares, S.B. Driss, R. Grueso, Treatment of olive-mill wastewater from a two-phase process by chemical oxidation on an industrial scale, Water Sci. Technol. 59 (10) (2009) 2017–2027, https://doi.org/10. 2166/wst.2009.165. [11] J.M. Ochando-Pulido, J.R. Corpas-Martínez, A. Martinez-Ferez, About two-phase olive oil washing wastewater simultaneous phenols recovery and treatment by nanofiltration, Process Saf. Environ. Prot. 114 (2018) 159–168, https://doi.org/10. 1016/j.psep.2017.12.005. [12] S.S. Kontos, P.G. Koutsoukos, C.A. Paraskeva, Removal and recovery of phenolic compounds from olive mill wastewater by cooling crystallization, Chem. Eng. J. 251 (2014) 319–328, https://doi.org/10.1016/j.cej.2014.04.047. [13] C. Agabo García, G. Hodaifa, Real olive oil mill wastewater treatment by photoFenton system using artificial ultraviolet light lamps, J. Cleaner Prod. 162 (2017) 743–753, https://doi.org/10.1016/j.jclepro.2017.06.088. [14] P.C. Lanzat, B. García Jarana, X. Chen, C.M. Olmos Carreño, J. Sánchez Oneto, J.R. Portela, E.J. Martínez de la Ossa, Energy production by hydrothermal treatment of liquid and solid waste from industrial olive oil production, J. Appl. Solut. Chem. Model. 5 (2016) 103–116, https://doi.org/10.6000/1929-5030.2016.05. 03.1. [15] Y.C. Sharma, B. Singh, J. Korstad, A critical review on recent methods used for economically viable and eco-friendly development of microalgae as a potential feedstock for synthesis of biodiesel, Green Chem. 11 (2011) 2993, https://doi.org/ 10.1039/c1gc15535k. [16] P. Spolaore, C. Joannis-Cassan, E. Duran, A. Isambert, Commercial applications of microalgae, J. Biosci. Bioeng. 101 (2006) 87–96, https://doi.org/10.1263/jbb.

5. Conclusions Based on the experimental results obtained in this work, it can be concluded that C. pyrenoidosa has the capacity to grow in both wastewaters, OOWW and UW. The OOWW is characterized by its high organic load and low nitrogen and phosphorus contents, essential elements for microalgae growth. UW still has good inorganic fraction and low organic matter content. This wastewater could supply essential nutrients (N and P among others) to OOWW as a low cost substrate. The mixture of different urban and industrial wastewaters enables to design an optimal culture medium for microalgae growth in order to obtain an economic viability (biofuels production) and efficient wastewaters bioremediation method (irrigation or drinking water) for its recuperation and recirculation, which allows a good circular economy in industry and environment. The bioprocess studied in this work is a 9

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