Bioresource Technology 112 (2012) 234–241

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Bioresource Technology

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Cultivation of Arthrospira (Spirulina) platensis in olive-oil mill wastewatertreated with sodium hypochlorite

Giorgos Markou a,⇑, Iordanis Chatzipavlidis b, Dimitris Georgakakis a

a Department of Natural Resources Management and Agricultural Engineering, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greeceb Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 November 2011Received in revised form 16 February 2012Accepted 18 February 2012Available online 27 February 2012

Keywords:Arthrospira (Spirulina)BiomassCyanobacteriaOlive-oil mill wastewater

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.biortech.2012.02.098

⇑ Corresponding author. Tel.: +30 6937085422; faxE-mail address: markoug@aua.gr (G. Markou).

The subject of this paper is the cultivation of the cyanobacterium Arthrospira (Sprirulina) platensis in olive-oil mill wastewater (OMWW) treated with sodium hypochlorite (NaOCl). The main positive effect of NaO-Cl on the OMWW characteristics is the decrease of the phenol concentration and turbidity, rendering theOMWW suitable for A. platensis growth. Maximum biomass production (1696 mg/l) was obtained whenthe concentration of OMWW in the cultivation medium was 10% with the supplementation of 1 g/lNaNO3 and 5 g/l NaHCO3. However, the addition of NaHCO3 has no significant effect, indicating thatthe only limited nutrient in this wastewater is nitrogen, while carbon is provided by the organic com-pounds of the wastewater. The maximum of the removals of chemical oxygen demand (COD) and carbo-hydrates was 73.18% and 91.19%, respectively, while phenols, phosphorus and nitrates in some runs wascompletely removed.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Olive-oil, a valuable product from olive fruits, plays a veryimportant role in the Mediterranean diet (Visioli and Galli, 1998).However, olive-oil mill wastewater (OMWW), which is generatedby the olive-oil extraction process, is one of the most serious envi-ronmental pollutants in the Mediterranean countries, such asSpain, Italy, Greece and Turkey, which are the major olive-oil pro-ducing countries in the world (http://faostat.fao.org/). The OMWWgenerated at the three-phase olive-oil extraction process amountsto 1–1.6 m3 per ton of olive fruits processed (Paraskeva and Dia-madopoulos, 2006). The polluting potential of this wastewater ismainly related to its high chemical oxygen demand (COD; 50–150 g O2/l) and low biodegradation due to its antibacterial activity.The high polluting organic load of this wastewater is due to its highcontent of sugars, tannins, polyphenols, polyalcohols, pectins andlipids. Especially the polyphenolic compounds are supposed to beresponsible for the antibacterial activity of this wastewater (Dare-ioti et al., 2010).

The cultivation of microalgae in wastewater has been proposedsince the 1960s , but recently there is a raise in the interest of thistopic due to the potential of microalgae to be used as a substratefor biofuels production (Park et al., 2011). Nevertheless, microalgaebiomass products could be used in many applications, includinganimal nutrition and in the agricultural sector (Spolaore et al.,

ll rights reserved.

: +30 2105294015.

2006). In general, the cultivation of microalgae in wastewater hasa dual aim: on the one hand to produce valuable microalgal bio-mass and on the other hand to treat the wastewater by reducingits organic and inorganic (mainly N and P) load. OMWW derivedfrom the three-phase extraction olive-oil process has the potentialto be used as a cultivation medium for microalgae growth, since itincludes all the necessary nutrients. However, the OMWW afterdilution might be nitrogen deficient (Hodaifa et al., 2009; SánchezVillasclaras et al., 1996).

The cyanobacterium (blue-green alga) Arthrospira platensis hasbeen extendedly studied due to its potential commercial applica-tions as a source of proteins, vitamins, essential amino acids, fattyacids etc. (Rangel-Yagui et al., 2004). In addition, A. platensis isthought as one of the most appropriate microalgae for wastewatertreatment (Vonshak, 2002). It has the ability to utilize organic com-pounds as an energy and/or carbon source. This ability is calledmixotrophy. The mixotrophic growth of A. platensis is supposedto be advantageous over the photoautotrophic, in which theneeded energy derives from light energy and carbon from inor-ganic molecules (Andrade and Costa, 2007). The mixotrophy con-tributes to the removal of the organic pollutants from thewastewaters through the biodegradation and/or the utilization ofthe organic compounds. Moreover, cyanobacteria are capable tobiodegrade phenolic compounds through the mechanism of bio-transformation (Lika and Papadakis, 2009). However, OMWW con-tains high amounts of phenols which inhibit the algal growth (Pin-to et al., 2002). In addition, the suspended solids of the OMWWcontribute to its turbidity. Wastewaters with high turbidity might

G. Markou et al. / Bioresource Technology 112 (2012) 234–241 235

affect the photosynthetic potential of the microalgae, resulting inlow biomass production (Borowitzka, 1998).

Several studies exist, in which raw OMWW (Hodaifa et al.,2009; Sánchez et al., 2001; Sánchez Villasclaras et al., 1996) oranaerobic digested OMWW (Travieso Córdoba et al., 2008) wasused for the cultivation of micro-algae. In all of these studies, themicro-algae used were green algae. In the best knowledge of theauthors, no study exists dealing with the cultivation of cyanobac-teria using OMWW as a cultivation medium.

In the laboratory scale, the cultivation media is sterilizedmainly through autoclaving or filtration, while in practice, in theaquaculture hatcheries, bleach (sodium hypochlorite) is frequentlyused (Kawachi and Noël, 2005). Chlorination is a widely usedmethod for the disinfection of water and for wastewater treatment(Black and Veatch Corporation, 2010). Recently, post-chlorinateddomestic wastewater was used for Chlorella cultivation (Mutandaet al., 2011). In addition, hypochlorites (sodium or calcium) areused as oxidants for the OMWW purification (Niaounakis and Hal-vadakis, 2006). Thus, this work aims to study the effects of variousconcentrations of hypochlorites (sodium and calcium) on severalphysico-chemical characteristics of OMWW and to cultivateA. platensis in OMWW treated with hypochlorite and with theaddition or not of nitrogen and carbon as nutrients.

2. Methods

2.1. Olive-oil mill wastewater

The OMWW used in the study was obtained from an olive-oilmill in Korinthos, Northern Greece. The raw OMWW (OMWWRaw),was generated by the three-phase olive-oil extraction process andwas left to settle for 10 d (Markou et al., 2010). The light superna-tant after the sedimentation was kept and used as substrate for theexperiment. Some physico-chemical characteristics of the rawOMWW (OMWWRaw), of the suspended fraction of the OMWW(OMWWSusp) after centrifugation for 5 min at 5000 rpm and ofthe settled for 10 d OMWW (OMWW10) are shown in Table 1.

2.2. Microorganism and growth conditions

The cyanobacterium A. platensis SAG 21.99 used in the studywas obtained from SAG (Sammlung von Algenkulturen der Univer-sität Göttingen). A. platensis SAG 21.99 is a filamentous cyanobac-terium with brackish water grow habitat. The cultivation wascarried out in cylindrical Plexiglas photobioreactors (Ph) with aninner diameter of 67 mm. The working volume was set on 0.5l. Cul-tures were aerated with about 0.2 Vair/VPh min. The filtered air wasprovided by a membrane air pump, Sera Air 550. The cultivation

Table 1Physico-chemical characteristics of the OMWW.

Parameter OMWWRaw

pH 5.37Electrical conductivity [mS/cm at 20 �C] 6.25Total solids (TS) [%] 4.32 ± 0.03Volatile solids (VS) [%] 3.86 ± 0.1Total P [g P/l] 0.35 ± 0.02PO4

3- (reactive) [g PO43�/l] 0.36 ± 0,04

NH3–N [mg N/l] 2.3 ± 0.67Total Kjeldahl nitrogen (TKN) [g N/l] 2.90 ± 0.46NO3

�–N + NO2�–N [mg/l] 99.13 ± 5.13

Phenols [g/l] 3.71 ± 0.36Chemical oxygen demand (COD) [g O2/l] 56.74 ± 4.29Carbohydrates [g/l] 14.81 ± 0.97Alkalinity [g CaCO3/l at pH endpoint 4.3] 1.29

n.d. = not detected, n = 3; ± S.D.

medium was adjusted to pH 8.5 with 1 N NaOH, in order all theruns to have the same initial pH. The cultures were performed un-der 10 klx of light intensity with a photoperiod of 20:4 (light:dark)at 30 ± 2 �C.

The inoculum was prepared as follows: A. platensis cultivated inZarrouk medium (Markou et al., 2012) was centrifuged, washedseveral times to washout the medium’s salts and resuspended in1% NaCl. The inoculum used corresponded to an initial biomassconcentration of about 130 mg/l.

2.3. Hypochlorite treatment

The OMWW10 was first diluted 10fold and then treated with0.25, 0.5, 1, 2.5 and 5% (v/v) of 5% sodium hypochlorite (NaOCl)and 5% calcium hypochlorite Ca(OCl)2. The amounts of hypochlo-rite used corresponded to 1.25, 2.5, 5, 12.5 and 25 g NaOCl orCa(OCl)2 per liter of undiluted OMWW10. The treated OMWW10

(tOMWW10) stood for 1 d in the dark and then was neutralizedwith sodium thiosulfate (Kawachi and Noël, 2005). The neutralizedtOMWW10 samples were used for the determination of the hypo-chlorite treatment effects on the various physico-chemical charac-teristics of the OMWW10. The neutralized tOMWW10 was used alsoas the cultivation substrate for the cultivation of A. platensis. Thedilution that occurred by the addition of 5% of hypochlorite is in-cluded in the results of the effect of the hypochlorite treatment.

2.4. Experiment set-up

The OMWW10 was first treated with NaOCl (12.5 g/l of undi-luted OMWW10) and then was neutralized with sodium thiosulfate(Kawachi and Noël, 2005). The tOMWW10 was diluted with deion-ized water to make concentrations of 5%, 10% and 25%, in order tobe used as cultivation medium for the growth of A. platensis. Eachmedium concentration had four different levels, (1) without thesupplementation of nutrients, (2) supplementation with 1 g/lNaNO3, (3) supplementation with 5 g/l NaHCO3 and (4) supple-mentation with 1 g/l NaNO3 and 5 g/l NaHCO3. The overall experi-mental set-up is presented in Table 2.

2.5. Analytical methods

Dry biomass was measured indirectly by spectrophotometry at560 nm according to standard curves generated by plotting drybiomass against optical density. The samples for analyzes werecentrifuged and washed several times with 1% NaCl and 2 timeswith deionized water. Chlorophyll was determined according toVonshak (2002), proteins according to the Lowry method (Lowryet al., 1951) using bovine serum as a standard; total lipids were

OMWWSusp OMWW10

– 5.42– 6.443.47 ± 0.02 3.25 ± 0.023.00 ± 0.02 2.77 ± 0.020.30 ± 0.02 0.23 ± 0.030.25 ± 0,02 0.20 ± 0.02n.d. n.d.1.13 ± 0.1 1.67 ± 0.08– 36.57 ± 4.043.22 ± 0.26 3.12 ± 0.2942.36 ± 1.97 43.87 ± 1.0913.86 ± 0.59 13.40 ± 0.23– 1.27

Table 2Experimental set-up.

Run NaOCl g/la OMWW10 (%)b NaNO3 g/lc NaHCO3 g/lc

R5/0/0 12.5 5 – –R10/0/0 12.5 10 – –R25/0/0 12.5 25 – –R5/1/0 12.5 5 1 –R10/1/0 12.5 10 1 –R25/1/0 12.5 25 1 –R5/0/5 12.5 5 – 5R10/0/5 12.5 10 – 5R25/0/5 12.5 25 – 5R5/1/5 12.5 5 1 5R10/1/5 12.5 10 1 5R25/1/5 12.5 25 1 5

a (w/v) mass of NaOCl per volume of undiluted OMWW10.b (v/v) volume of OMWW10 per volume of deionized water.c (w/v) mass of nutrient per mass of cultivation medium.

236 G. Markou et al. / Bioresource Technology 112 (2012) 234–241

determined according to the sulfo-phospho-vanillin reactionmethod (Zöllner and Kirsch, 1962), using triolein as a standard;carbohydrates were determined by the phenol–sulfuric acidmethod (DuBois et al., 1956), using D-glucose as a standard.Total phenols was determined using the Folin–Ciocalteu reagent(Lesage-Meessen et al., 2001), using phenol as a standard.Phosphorus, ammonia nitrogen, total Kjeldahl nitrogen, COD, totalsolids (TS), volatile solids (VS) and alkalinity were measuredaccording to APHA (1995). Nitrates were analyzed by the cadmiumreduction method as it was modified by Gaugush and Heath,(1984). Turbidity was measured according to Application 24 ofHach-Lange (Hach-Lange. Application 24: Turbidity according toEN ISO 7027– Measurement of the attenuation of a radiantflux). pH and electrical conductivity (E.C.) was measured byHach HQ40 with the proper probes. All spectrophotometric deter-minations were carried out on a Dr. Lange, Cadas 30 (Germany)spectrophotometer. Light intensity was measured with DigitalLux Meter, Model 1010B, and the measurement was carried outin the middle of the photobioreactor.

3. Results and discussion

3.1. Effect of hypochlorites

In Fig. 1 the effects of hypochlorite treatment on various phys-ico-chemical characteristics of the OMWW10 are presented. Grad-ual increase of pH and electrical conductivity (E.C.) was observedas the mass of the applied hypochlorite increased. Both character-istics were higher using NaOCl. Turbidity decreased gradually asthe hypochlorite dosage increased. The NaOCl treatment had astronger turbidity decrease than the Ca(ClO)2 treatment and the re-mained turbidity was 25% and 70% of the initial turbidity, respec-tively. Phenols were strongly degraded even in a lowhypochlorite dosage of 0.5 g/l. The degradation was about 45%and 55% for the NaOCl and Ca(ClO)2 treatment, respectively. Inthe highest dosages of hypochlorite the remained phenols were15% and 5% of the initial amount for the NaOCl and Ca(ClO)2 treat-ment, respectively. The only study found, which discusses the ef-fect of hypochlorites (Ca(ClO)2) on the OMWW was the study ofBoukhoubza et al. (2009), in which, however, Ca(ClO)2 was addedafter the treatment of OMWW with lime and the adjustment ofpH to 12. Thus, direct comparison with the present study couldnot be made.

Although the phenols reduction was almost the same for bothhypochlorites, NaOCl was chosen as the treatment chemical be-cause of the higher turbidity decrease and the higher final pH. Tur-bidity decrease is significant because it affects the photosyntheticefficiency of the microalgae (Borowitzka, 1998). Also, higher pH fa-

vors the growth of A. platensis because this cyanobacterium is analkaliphile with optimum pH in the range of 9–10.5 and thrivesin pH even higher than 11. After the oxidation effect of NaOCland its neutralization with sodium thiosulfate, the NaOCl is re-duced to NaCl. A. platensis is known to be tolerant to high concen-trations of NaCl and capable to grow even in seawater (MaryLeema et al., 2010). Thus the higher electrical conductivity in thetOMWW10 treated with NaOCl than that of Ca(ClO)2 is assumedto affect not negatively the growth of A. platensis. Because of theabove mentioned effects of hypochlorites on the characteristicsof OMWW, the treatment with NaOCl was chosen for furtherinvestigation.

The dosage for treating the OMWW10, so that the tOMWW10

would be used as cultivation medium, were selected with the cri-terion of the decrease of phenols, which act toxic to the microor-ganisms. The dosage of 12.5 g/l NaOCl was chosen, for the reasonthat in higher dosages (25 g/l) great amounts of hypochlorite haveto be applied without a gain of any analogous results. The tOM-WW10 with 12.5 g/l NaOCl was used as a cultivation medium forthe growth of A. platensis. The supplementation of the tOMWW10

with NaNO3 and/or NaHCO3 was performed, so that the effect ofthe addition of nitrogen and carbon nutrients would be studied.

3.2. Biomass production

In Fig. 2 the biomass productions of A. platensis cultivated inmedia with various concentrations of tOMWW10 with or withoutthe addition of nutrients are presented. The highest biomass pro-duction was 1696 mg/l and obtained in R10/1/5 followed by1648 mg/l in R10/1/0. As shown in Fig. 2, A. platensis could not growin media with tOMWW10 without the addition of nitrogen.Although OMWW contains relative high amounts of organic nitro-gen (TKN, (Table 1)), its availability to algae is very low. Nitrogen inform of nitrates, which is available to algae, after the dilution of theOMWW become restricting in supporting efficient algal growth. Inruns, in which nitrogen was supplied, the growth was considerablyenhanced, a fact that shows that nitrogen is the limited nutrient incultures with diluted OMWW as the cultivation substrate. Analo-gous results were demonstrated by Sánchez Villasclaras et al.(1996), who reported that the supplementation of KNO3 in culturesof Chlorella pyrenoidosa and Scenedesmus obliquus enhanced theirgrowth rate. In the present study the highest biomass productivityof 4.4 mg/(l h) was obtained in R10/1/5, which is higher than that ofabout 2.5 mg/(l h) reported by Sánchez Villasclaras et al. (1996).

R25/1/0 had a peculiar behavior; A. platensis could not grow untilthe 8th day, after which the growth and the biomass accumulationstarted. In contrast with the other runs, R25/1/0 had a considerablyhigh lag phase. The significant difference between the R25/1/0 andR25/1/5 perhaps indicates that the addition of inorganic carbon(NaHCO3) favors the algal growth in milieus with high organic loadand decreases the time of the lag phase.

With the addition of carbon as NaHCO3 the growth of A. platen-sis was slightly enhanced in comparison with the runs with onlytOMWW10. However, the addition of carbon could not supportthe growth of A. platensis for more than few days along with lowbiomass concentration. In runs with the simultaneous nitrogenand carbon supplementation biomass production was slightlyhigher than in runs with only nitrogen addition, but no significantdifferences were observed, except in the runs with 25% tOMWW10.This indicates that the only limiting nutrient factor for the cultiva-tion of A. platensis in media supplemented with OMWW is nitrogenand that OMWW has a sufficient carbon content to allow algae togrow.

In general, in all cases studied, the maximum biomass concen-tration was obtained in media with 10% OMWW10. The same resultwas reported by Sánchez et al. (2001) in cultures of Chlorella

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Fig. 1. Effect of the treatment of OMWW10 with hypochlorite in various dosages. The dosages correspond to the mass of hypochlorite used per volume of undiluted OMWW10.NaOCl (s) and Ca(ClO)2 (j). n = 6, ± S.D.

G. Markou et al. / Bioresource Technology 112 (2012) 234–241 237

pyrenoidosa. Nevertheless, the OMWW concentration in the culti-vation media might vary strongly due to the high variation of thecharacteristics of the OMWW.

In preliminary experiments with untreated OMWW10, the max-imum OMWW concentration, in which A. platensis could grow(450 mg/l), was 2.5%. Thus, the treatment of OMWW in order todecrease the content of phenols is essential for the cultivation ofA. platensis in OMWW with acceptable biomass production.

3.3. Biomass composition

In Table 3 the biomass composition of A. platensis cultivatedwith tOMWW10 are listed. The carbohydrates content varied from16.52% to 63.75%. The highest percentage was obtained in R5/1/5

and the lowest in R25/1/5. The proteins content varied from22.04% to 38.13%. The lowest percentage was obtained in R5/1/5

and the highest in R25/1/5. Proteins were considerably low inR5/1/0 and R5/1/5 and increased as the tOMWW10 concentration in-creased. The lipids content varied from 7.37% to 16.91%. The chlo-rophyll content varied from 0.39% to 1.40%. The lowest percentagewas obtained in R5/1/5 and the highest in R25/1/5.

Based on our calculations the cultures R5/1/0 and R5/1/5 werephosphorus limited, while R10/1/0 and R10/1/5 were at least phospho-rus and possibly also nitrogen limited. In contrast in R25/1/0 andR25/1/5 both nutrients were in excess. Under phosphorus or nitrogenlimitation microalgae change their metabolic pathways and switch

the formation of proteins and other compounds into the formationof carbohydrates or lipids. The carbohydrates or lipids accumulationis proportional to the degree of the nutrient deficiency (Dean et al.,2008; Healey and Hendzel, 1975; Hu, 2004). This fact explains theobservation that as the concentration of tOMWW10 is reducedthe carbohydrate content is increased and the protein contentdecreased. Although the phosphorus concentration in R5/1/0 andR5/1/5 was equal, the difference of the carbohydrate contentbetween these runs indicates that the addition of NaHCO3 resultedin phosphorus precipitation and consequently in a higher degreeof phosphorus limitation effect. Nevertheless, no clear effect oftOMWW10 concentration on the lipids content was observed.

In R5/1/0 and R5/1/5 the phosphorus limitation affected also thechlorophyll content, causing the phenomenon of chlorosis. In theother runs, the chlorophyll content was higher as the tOMWW10

supplementation was higher. This could be explained by the factthat, as the tOMWW10 concentration in the cultivation media in-creased, the turbidity of the cultivation media also increased andin combination with the biomass density the low light penetrationresulted to a higher chlorophyll synthesis (Rangel-Yagui et al.,2004).

As is shown in Table 3, the fraction of the biomass that corre-sponds to other compounds, such as minerals (ash), nucleic acids(DNA and RNA), pigments other than chlorophyll, vitamins etc. itis not fixed and varies in the different tOMWW10 concentrations.This biomass fraction was higher in R25/1/0 and R25/1/5 and lower

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Fig. 2. Dry biomass production of Arthrospira platensis cultivated in media containing various concentrations of olive oil mill wastewater with and without thesupplementation of various nutrients. (a) tOMWW10, (b) tOMWW10 + 1 g/l NaNO3, (c) tOMWW10 + 5 g/l NaHCO3 and (d) tOMWW10 + 1 g/l NaNO3 + 5 g/l NaHCO3. 5%tOMWW10 (s), 10% tOMWW10 (h), 25% tOMWW10 (D). n = 6, ± S.D.

Table 3Biomass composition of A. platensis cultivated in media supplemented with tOMWW10 with or without nutrient additions.

Run Carbohydrates (%) Lipids (%) Proteins (%) Chlorophyll (%) Other (%)

R5/1/0 47.74 ± 2.53 7.37 ± 0.02 23.29 ± 3.96 0.89 ± 0.15 20.71R10/1/0 34.58 ± 1.83 7.52 ± 0.04 32.27 ± 0.39 1.03 ± 0.01 24.60R25/1/0 23.44 ± 2.36 7.12 ± 0.14 36.47 ± 1.04 1.02 ± 0.05 31.95R5/1/5 63.75 ± 3.03 8.96 ± 0.21 22.04 ± 0.42 0.39 ± 0.10 4.86R10/1/5 33.64 ± 2.44 16.91 ± 1.86 31.52 ± 0.09 1.02 ± 0.04 16.91R25/1/5 16.52 ± 0.52 11.79 ± 0.83 38.13 ± 3.07 1.40 ± 0.28 32.16

n = 6, ± S.D.

238 G. Markou et al. / Bioresource Technology 112 (2012) 234–241

in R5/1/5. The overall trend for this fraction was to decrease as nutri-ents were limited. As mentioned above, under phosphorus or nitro-gen limitation the micro-algae accumulate mainly carbohydratesor/and lipids against the formation of the other compounds.

3.4. Pollutants removal

The removal of certain organic and inorganic compounds fromthe tOMWW10 after the cultivation of A. platensis is presented inFig. 3. The carbohydrates removal varied from 54.30% to 91.15%.In general the carbohydrates removal was higher in the runs withadded nutrients, in which A. platensis could grow. This higher re-moval of carbohydrates can be explained from the fact that carbo-hydrates, such as sugars, can be used as carbon and/or energy

sources for heterotrophic or mixotrophic algal growth (Andradeand Costa, 2007). The COD removal varied from 28.77% to 66.88%and followed almost the same trends as the carbohydrates removaldid. COD removal of 37% was reported by Travieso Córdoba et al.(2008) in cultures of Chlorella zofingiensis in anaerobic digestedOMWW.

Phenols were removed in a degree of 41.90% up to 100% (Fig. 3).The complete removal of phenols was obtained in all runs with 5%tOMWW10. In general, as the tOMWW10 concentration in the med-ium increased, the phenol removal decreased. In runs in which al-gal growth occurred, the phenols removal was higher than in runswithout algal growth. Thus, phenols removal is a function of theirconcentration and algal density. Pinto et al. (2003, 2002), whostudied the biodegradation of selected phenols from OMWW by

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Fig. 3. Removal of carbohydrates, phenols, COD and phosphorus from the tOMWW10 used as a Arthrospira platensis cultivation medium in various concentrations. tOMWW10

without nutritions added (j), tOMWW10 with 1 g/l NaNO3 (h), tOMWW10 with 5 g/l NaHCO3 (N) and tOMWW10 with 1 g/l NaNO3 + 5 g/l NaHCO3 (D). n = 6, ± S.D.

G. Markou et al. / Bioresource Technology 112 (2012) 234–241 239

green and blue-green algae, report that the removal of phenols wasgreater than 70% within 5 d of cultivation. However, it seems thatphenols are not completely removed, but are bio-transformed intoother aromatic compounds (Pinto et al., 2003).

Carbohydrates, phenols and COD were removed also in runs, inwhich very low or no algal biomass was produced. In these runsthe microscopic examination showed that several microorganismshad grown, i.e. mainly bacteria and fewer fungi and cocci. Thus, it

is assumed that carbohydrates and phenols biodegraded by thebacteria. The biodegradation was enhanced by the continuouslyprovided air. The air enriched the medium with oxygen, whichwas used by the bacteria to perform an enzymatic attack and tobiodegrade (Lika and Papadakis, 2009) the carbohydrates, phenolsor any organic compound (expressed as COD decrease). In runs, inwhich A. platensis could grow, the presence of bacteria or othermicroorganisms was negligible. However, carbohydrates, phenols

Table 4Total removal of certain pollutants from the olive-oil mill wastewater after thetreatment with sodium hypochlorite and the cultivation of A. platensis.

Run COD (%) Carbohydrates (%) Phenols (%) Phosphorus (%)

R5/0/0 42.33 ± 1.35 56.66 ± 5.47 100 32.19 ± 0.21R10/0/0 44.85 ± 2.24 62.74 ± 3.89 88.82 ± 0.33 30.10 ± 4.72R25/0/0 48.60 ± 3.22 67.89 ± 4.68 86.40 ± 1.08 27.79 ± 2.51R5/1/0 51.75 ± 0.27 63.66 ± 0.38 100 100R10/1/0 49.26 ± 0.90 83.01 ± 0.17 98.31 ± 0.10 100R25/1/0 70.43 ± 1.30 91.12 ± 0.30 95.35 ± 0.11 32.29 ± 4.66R5/0/5 43.31 ± 4.74 54.30 ± 1.26 99.78 ± 0.01 81.14 ± 3.05R10/0/5 42.52 ± 1.00 60.36 ± 1.09 88.62 ± 0.11 54.37 ± 0.04R25/0/5 53.38 ± 1.30 68.56 ± 0.32 83.97 ± 0.10 34.75 ± 2.25R5/1/5 59.88 ± 1.25 80.92 ± 0.95 100 100R10/1/5 65.53 ± 2.76 88.41 ± 1.64 100 100R25/1/5 73.18 ± 0.26 91.19 ± 0.33 95.95 ± 0.04 100

n = 6, ± S.D.

240 G. Markou et al. / Bioresource Technology 112 (2012) 234–241

and COD removal was not strictly related to the produced algal bio-mass, but was more related to their concentration in the medium.Thus, the organic compounds removal occurred not just biologi-cally (algal utilization and/or biodegradation) but also physico-chemically (oxidation).

Phosphorus was removed at a degree of 14.69% up to 100%(Fig. 3). In runs with nitrogen and carbon addition, in which algalbiomass could be produced, the removal was complete. In runswith nitrogen addition the removal was complete except R10/1/0,in which the algal growth was relatively low resulting to a low up-take of phosphorus. In the other runs the phosphorus removal de-creased as the tOMWW10 concentration in the media increased. Inthe runs with NaHCO3 addition the phosphorus removal was high-er than in the runs without any nutrient addition. This indicatesthat sodium bicarbonate enhanced the precipitation ofphosphorus.

Nitrates were completely removed in the runs in which nonitrogen was added. In the other runs the nitrates removal washigh and varied from 87.38% up to 95.61% except R25/1/0 with ni-trates removal of 53.77% (Fig. 3). The latter low removal was dueto the low biomass production in this run resulting to a lowernitrogen uptake. Nevertheless, the nitrates removal was consider-ably higher than of the nitrogen taken up (calculated based on thebiomass protein content; data not shown). This was due to the re-moval of nitrates not by algal uptake but by loss. It is supposed thatmicroalgae cultivated in media with an excess of organic com-pounds, use the energy derived by the organic compounds forthe reduction of nitrates to gaseous molar nitrogen (Chojnackaand Zielinska, 2011).

The above discussed results are related to the removal of pollu-tants after the cultivation of A. platensis in the already treatedOMWW10. However, even before the cultivation of A. platensis, apercentage of pollutants were removed by the NaOCl treatmentof the OMWW10. Thus, Table 4 lists the total removal of pollutantsfrom the OMWW10 after the NaOCl treatment and the cultivationof A. platensis in the OMWW10.

3.5. Environmental issues from NaOCl treatment

Chlorination is a widely used method for the disinfection ofwater and for wastewater treatment (Black and Veatch Corpora-tion, 2010). However, the use of NaOCl raises several serious envi-ronmental issues. Hypochlorites in the presence of organic matterform various by-products like chloramines, organochlorinatedcompounds, halogenated volatile organic compounds etc. (Bou-khoubza et al., 2009). The probable presence of these pollutantsformed by the hypochlorites treatment in the cultivation mediumafter the cultivation of microalgae have to be studied in order to se-

cure that the method of hypochlorites treatment of OMWW will besafe. In addition, more research should be made in the direction ofa softer treatment method for phenol and turbidity reduction.Moreover, the addition of NaOCl and nutrient salts resulted in anincreasing of the medium’s electrical conductivity. The highestelectrical conductivity (18.34 ± 0.44) was obtained in R25/1/5. Thisfact rises some additional issues about the disposal of the tOMWWafter it has been used as cultivation medium.

4. Conclusions

OMWW is suitable to be used as medium for the growth of A.platensis after NaOCl treatment, which causes a strong decreasein the OMWW phenol concentration and turbidity. After the NaOCltreatment and the cultivation of A. platensis, a considerable re-moval of certain organic and inorganic pollutants is obtained.However, the use of NaOCl raises some environmental issues andthus more research is needed in order to secure that the methodof hypochlorites treatment of OMWW is safe. Finally, more re-search should take place in the direction of a softer treatmentmethod for phenol and turbidity reduction.

Acknowledgements

We thank Dr Despo Kritsotaki and George Kyriakarakos for theircomments on the manuscript.

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