Conversions of olive mill wastewater-based media by Saccharomyces cerevisiae ...

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Research ArticleReceived: 17 June 2012 Revised: 24 August 2012 Accepted: 25 August 2012 Published online in Wiley Online Library: 16 October 2012

(wileyonlinelibrary.com) DOI 10.1002/jctb.3931

Conversions of olive mill wastewater-basedmedia by Saccharomyces cerevisiae throughsterile and non-sterile bioprocessesDimitris Sarris,a Marios Giannakis,a Antonios Philippoussis,b

Michael Komaitis,a Apostolis A. Koutinasa and Seraphim Papanikolaoua∗

Abstract

BACKGROUND: Olive mill wastewaters (OMWs) are an important residue and several methods have been proposed for theirtreatment.

RESULTS: Remarkable decolorization (∼63%) and phenol removal (∼34% w/w) from OMW was achieved. In glucose-basedflask sterile cultures, enrichment with OMWs increased ethanol and biomass production compared with cultures withoutOMWs added. Flask sterile and un-sterilized cultures demonstrated similar kinetic results. Batch-bioreactor trials performedshowed higher ethanol and lower biomass quantities compared with the respective shake-flask experiments, while culturesused under un-sterilized conditions revealed equivalent results to the sterile ones. In non-sterile bioreactor cultures, OMWsaddition enhanced biomass production in comparison with culture with no OMWs added, whereas ethanol biosynthesis wasnot affected. The maximum ethanol quantity achieved was 52 g L−1 (conversion yield per sugar consumed of 0.46 g g−1) in abatch bioreactor non-sterilized trial with OMW–glucose enriched medium used as substrate, that presented initial reducingsugars concentration at ∼115 g L−1. Fatty acid analysis of cellular lipids demonstrated that in OMW-based media, cellular lipidscontaining increased concentrations of oleic and linoleic acid were produced in comparison with cultures with no OMWs added.

CONCLUSIONS: S. cerevisiae simultaneously produced bio-ethanol and biomass and detoxified OMWs, under non-sterileconditions.c© 2012 Society of Chemical Industry

Keywords: Saccharomyces cerevisiae; olive-mill wastewaters; bio-ethanol; waste bioremediation

INTRODUCTIONOlive mill wastewaters (OMWs) are the principal waste streamderiving from the olive oil production process. These residues areconsidered to be one of the most difficult-to-treat effluents.1 Theiroverall annual production is estimated to be over 3×107 m3.2,3

In Greece, the production of this residue is estimated to be upto 1.5×106 m3.4 In these wastewaters, BOD and COD values canbe 200–400 times higher than those typically met in municipalsewage, with the organic fraction of these materials beingcomposed of sugars, (poly)-phenolic compounds, organic acidsand residual oil.1,2,5,6 OMW composition is variable and dependson several factors such as the variety of olive fruits, the conditions

and the technology used for the extraction of oil.5–7 In some casesOMWs derived from press extraction systems, besides phenoliccompounds, contain reducing carbohydrates (principally glucose)in very high quantities (i.e. ≥70 g L−1)5 that also pose significantproblems related with their treatment. The OMW dark color andphytotoxic and antimicrobial effects have been attributed to thephenolic compounds that are found in various concentrations in

the residue,4,6,8–10 the breakdown of which is considered to bethe limiting step in OMW treatment by biological means.1,5,9,11,12

The development of cost-effective OMW treatment technolo-gies remains a priority, since OMWs are in most cases discharged

directly into the environment without any other treatment.Several physico-chemical processes have been proposed forOMW treatment, but these methods are limited to small-scaleoperations.2,13,14 On the other hand, recent developments indi-cate that OMWs should be considered as a fermentation mediumto valorize rather than a waste to discharge, being a potentialsubstrate for various fermentation processes.5 OMW-based mediahave been used for the cultivation of molds, prokaryotic microor-ganisms, yeast and yeast-like species leading to the reduction ofCOD values and phenol compounds degradation and also to the

production of added-value compounds.1,5,9,10,12,15–20

The worldwide decrease of petroleum feedstock and theconcomitant rise in the price of crude oil have rendered as a

∗ Correspondence to: S. Papanikolaou, Laboratory of Food Microbiology andBiotechnology, Department of Food Science and Technology, AgriculturalUniversity of Athens, Iera Odos 75, – Athens, Greece. Email: [email protected]

a Department of Food Science and Technology, Agricultural University of Athens,75 Iera Odos, 11855 Athens, Greece

b National Agricultural Research Foundation, Institute of Technology ofAgricultural Products, Laboratory of Edible Fungi, 1 Sofokli Venizelou street,14123 Lykovrysi, Greece

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Bioconversions of olive-mill wastewaters by Saccharomyces cerevisiae www.soci.org

very important priority for the scientific community the discoveryof ‘new’ and ‘renewable’ types of energy sources. First generation(derived from fermentation of hydrolyzed starchy materials orsimple sugars) and principally second generation bio-ethanol(derived through valorization – conversion of several wastestreams) is considered to be one of the most promising and

important types of this renewable energy source.21–24 Researchbased on bio-ethanol production is focused mainly on twoaxes. A main research activity is related to the discovery ofnew natural microorganisms (or the ‘construction’ of geneticallyengineered ones) capable of producing ethanol at significant finalproduct concentrations and high volumetric productivities and/orproducing in small quantities antagonistic to ethanol metabolites(i.e. glycerol). The second axis of scientific activities on bio-ethanolproduction deals with bioprocess optimization and modeling, thedevelopment of several types of fermentation configurations andthe potential of the efficient utilization of various by-products andwaste or raw renewable materials used as substrates in this type of

conversion.23–25 On the other hand, an important innovation thathas recently been developed in biotechnological processes refersto the accomplishment of the bio-process under completely non-sterile conditions, an achievement that can significantly reducethe operating costs of the process.26

The aim of the present study was dual; to investigate thepotential of Saccharomyces cerevisiae strain MAK-1 to grow andproduce bio-ethanol and biomass on glucose-enriched OMWs(in both shake-flask and batch bioreactor experiments). OMW-based media having different initial phenolic and glucose contenthave been created and the ability of the strain to produceethanol and biomass and simultaneously to reduce the colorand the phenol content of the residue was investigated in axeniccultures performed on previously sterilized media. In order tofurther evaluate the physiological behavior of the microorganismused, the impact of the initial glucose and phenolic compoundsquantities upon the biogenesis of cellular lipids and the fatty acidcomposition of cellular lipids produced by the strain were alsoinvestigated. Recent studies have demonstrated that the additionof OMWs to the medium can have serious impact upon both thequantity and composition of cellular lipids synthesized inside theyeast cells.12,20 Besides the physiological aspect of the study, asecond aim concerning the technological development of OMWfermentation rarely applied in the past, namely the potentialof fermentation under completely un-sterilized conditions, wasdeveloped. Application of the non-sterilized process in large-scaleoperations can significantly reduce the overall cost, renderingthe whole concept completely eco-friendly and cost-effective.Physiological, kinetic and technological aspects were consideredand are comprehensively discussed.

EXPERIMENTALMicroorganism and growth mediaS. cerevisiae strain MAK-121 was used in this study. Themicroorganism was conserved in PDA (T = 6±1◦C). In orderto maintain the viability of the strain, the microorganism wassub-cultured every 3 months. OMWs used were obtained from athree-phase decanter olive mill in the region Kalentzi (Corinthia,Peloponnisos – Greece) and were immediately frozen at−20±1◦C.To be used in the experiments, OMWs were thawed and thesolids removed by centrifugation (9000 rpm, 15 min, 21±1◦C)in a Hettich Universal 320R centrifuge. OMW phenolic content,expressed as gallic acid equivalent, was 10.0±0.5 g L−1 while

the sugar concentration of the residue was 30.0±3.0 g L−1,expressed as glucose equivalent. Glucose to a quantity of 90%,w/w, was the principal sugar quantified in the OMWs (analysisperformed by HPLC – see below). Fructose was also found, butits concentration was significantly lower than that of glucose(∼10%, w/w), whereas no other sugars were identified. The OMWsused contained negligible quantities of olive oil (0.4±0.1 g L−1 –determination of oil conducted after triple extraction with hexane).Organic acids were also present in small quantities in the residue.The principal organic acids detected were acetic acid (2.0±0.5 gL−1) and gluconic acid (2.0±0.5 g L−1).

OMWs were diluted in several ratios to yield in liquid mediapresenting various initial phenolic compounds concentrations.Glucose was added in various amounts (resulting in the creation ofmedia with different initial reducing sugars – RS0 concentrations)into the effluent so as to support considerable ethanol production.Inexpensive, commercial-type glucose [with 95% w/w purity andimpurities composed of maltose (2% w/w), malto-dextrines (0.5%w/w), water (1.5% w/w) and salts (0.5% w/w)] provided bythe Hellenic Industry of Sugar SA (Thessaloniki, Greece), wasused as supplementary substrate instead of analytical-gradeglucose, in order to further reduce the cost of the bioprocess.In all OMW-diluted media, salts were added and in all trialsthe final concentration was (in g L−1): KH2PO4, 7.0; Na2HPO4,2.5; MgSO4×7H2O, 1.5; FeCl3×6H2O, 0.15; CaCl2×2H2O, 0.15;ZnSO4×7H2O, 0.02; MnSO4×H2O, 0.06. In all trials, the yeast extractand (NH4)2SO4 concentrations were each 0.5 g L−1. Medium pHwas adjusted to ∼3.5 by adding 1 mol L−1 HCl.

Culture conditionsFermentations were carried out in 250 mL Erlenmeyer flasks,containing 50±1 mL of sterile and non-sterile growth medium,inoculated with 1 mL (2% v/v inoculum) of exponential pre-culture(carried out in the synthetic medium with glucose at ∼35.0 gL−1, (NH4)2SO4 0.5 g L−1 and yeast extract 0.5 g L−1). Flaskswere incubated aerobically in an orbital shaker (New BrunswickScientific, USA) at an agitation rate of 180±5 rpm and incubationtemperature T = 28±1◦C. The medium pH was maintained at 3.50by periodically (and aseptically concerning the trials performedunder sterile conditions) adding into the flasks quantities of 1 molL−1 HCl. The exact HCl solution volume needed for pH correctionwas evaluated by measuring the volume of HCl solution requiredfor pH correction in one (at least) flask. Following, the appropriatevolume of HCl solution was (aseptically) added in the remainingflasks and the value of pH reached was verified to be ∼3.50. Blankexperiments (no OMW addition; commercial glucose added as thesole carbon source) were also carried out.

Sterile and non-sterile batch fermentations were also conductedin a laboratory scale bioreactor (MBR Bioreactor, AG Switzerland),with total volume 3.5 L and working volume 3.0 L, fitted withfour probes and two six-bladed turbines. The culture vessel wasinoculated with 60 mL (2% v/v inoculum) of exponential pre-culture (see above). The incubation temperature was controlledautomatically at T = 28±1◦C. Agitation rate was adjusted to 300rpm. The pH was automatically controlled at 3.50±0.02 by adding1 mol L−1 HCl. Blank sterile experiments (no OMW addition) wereperformed by sparging the media with air (passing through abacteriological filter with 0.2 µm pore size) at a constant flowrate of 1.5 vvm. Thereafter, non-sterile blank fermentations (noOMWs added) or fermentations with OMW added into the mediumwere performed under various aeration conditions (e.g. 1.5, 1.0,

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0.5 and finally 0.0 vvm). Finally, in order to investigate whetherpossible reduction of phenolic compounds concentration or/andcolor reduction occurred due to agitation and aeration alone(no presence of S. cerevisiae cells), three shake-flask experimentswere performed. In these, flasks containing glucose at initialconcentration ∼40 g L−1 in which OMWs were added in orderto yield initial phenolic compounds concentrations of 1.20, 2.00and 2.90 g L−1 were sterilized and thereafter were subjectedto agitation (180±5 rpm) for 200 h without the presence ofmicroorganisms.

Statistical analysisAll trials were performed in duplicate (each experimental pointof the kinetics presented is the mean value of two independentdeterminations). In all experiments, a completely randomizeddesign was applied, using two (2) replicates per treatment. Varianceanalysis was performed by Statgraphics Plus version 5.1 statisticalpackage, using the Least Significant Difference (LSD) test at 5%level of probability to compare mean values. Experimental valuesare given as the mean, and the standard deviations are calculated.

Analytical methodsYeast cell mass was harvested by centrifugation at 9000 rpm,for 10 min at 21±1◦C, washed once with distilled waterand centrifuged again. Biomass concentration (X, g L−1) wasdetermined gravimetrically from dry weight at temperatureT = 100◦C until constant weight (usually within ∼24 h). pHmeasurement was conducted using a Jenway 3020 pH-meter.Ethanol (Eth, g L−1) and reducing sugars (RS, g L−1 – thatconstituting the sum of glucose and fructose) were analyzedthrough high performance liquid chromatography (HPLC) in aWaters Association 600E apparatus equipped with a RI detector(Waters 410). The column used for the separation of moleculeswas an Aminex HPX-87H (Bio-Rad, USA), the mobile phase wasH2SO4 0.005 mol L−1, the column temperature was 65◦C and theflow rate was 0.6 mL min−1. Phenolic compounds concentration inthe residue was determined according to the Folin–Ciocalteaumethod measured at 750 nm and expressed as gallic acidequivalent.9 The decolorization assay of the treated residue wasperformed according to Sayadi and Ellouz.8 The samples were30-fold diluted, their pH was adjusted between 6.0 and 6.3 andthe absorbance was measured at 395 nm. In all trials, initialphenolic compounds concentration and initial color content weredetermined after sterilization (when sterilization occurred).

In the cultures performed under sterile conditions, total intra-cellular lipids (L, g L−1) were extracted with a mixture of chloroformand methanol 2:1 (v/v). Solvents were removed at reducedpressure (Buchi Rotavapor R-114) and lipids were determinedgravimetrically. Lipids were converted to their fatty acid methyl-esters (FAMEs) and analyzed in a gas chromatograph (GC-FID)apparatus (Fisons 8000 series) according to Fakas et al.27 FAMEswere identified by comparison with authentic standards.

RESULTSKinetics of S. cerevisiae strain MAK-1 grown in shake-flaskculturesTo investigate the biochemical response of S. cerevisiae strainMAK-1 grown on OMW-based media, fermentations in sterile(axenic) and non-sterile shake-flask cultures were carried out.OMWs and glucose were added, and media presenting various

initial concentrations of phenolic compounds and RS were created(see Table 1). Initial phenolic compounds concentration of 0.0corresponded to the control experiment (without OMW addition),whereas two non-sterilized trials were also performed. MaximumRS0 concentration selected (∼75.0 g L−1) corresponded to RSquantity that can usually be found in OMWs derived from press-extraction systems.5 Equally, the initial concentrations of phenoliccompounds correspond to quantities that can be found in typicalOMWs.5 Biomass (X, g L−1) and ethanol (EtOH, g L−1) produced andassimilated sugars (RScons, g L−1) as well as the yields of ethanolproduced per sugar consumed (YEtOH/RS, g g) and total lipid in dryweight (YL/X, g g−1) were quantified for all trials (Table 1).

Regarding sterile shake-flask trials, although OMWs containedcompounds that could provoke biomass inhibition (e.g. phenoliccompounds), biomass production achieved, surprisingly, clearlyincreased with OMWs addition into the medium; for all of the RS0

concentrations tested, a two-way ANOVA test indicated that Xmax

concentrations increased significantly with the rise of phenoliccompounds quantities, in the range of 2.0–2.9 g L−1 (see Table 2).For the sterile trials, the highest Xmax value achieved was in mediawith initial phenolic compounds concentration 2.9 g L−1 andRS0∼75.0 g L−1, being 18.7 g L−1, while the Xmax value for thecontrol experiment (RS0∼75.0 g L−1 without OMW addition) wasonly 11.3 g L−1 (see Table 1). Moreover, as was expected, forcultures with varying initial phenolic compounds concentration,statistical analysis revealed significant biomass increase in thehigher range of RS0 concentration (55–75 g L−1) (Table 2). In alltrials, glucose was totally and very rapidly consumed (∼20 h afterinoculation), since the cellular metabolism was shifted towards thesynthesis of ethanol despite the remarkable presence of oxygen inthe medium (this is the so-called ‘Crabtree’ effect).28 After glucoseassimilation, S. cerevisiae consumed previously accumulatedethanol, and new biomass formation was observed (‘diauxicgrowth’). In several cases, complete ethanol re-consumption wasobserved; this is evident in Table 1, as when Xmax concentrationwas recorded, the respective ethanol value was 0.0 g L−1 (it isevident that in a potential process scale-up, fermentation shouldstop ∼25–30 h after inoculation, in order not to lose the valuableethanol). A representative kinetics is shown in Fig. 1 (strain grownon OMW-based media with initial phenol content at 2.0 g L−1 andRS0∼40.0 g L−1).

In order to enhance ethanol production so as to valorize OMWsas process water in such fermentations, commercial glucosewas added into the medium in various amounts. The EtOHmax

concentration achieved for sterile flask cultures was 26.1 g L−1

(yield of ethanol produced per glucose consumed – YEtOH/RS at0.36 g g−1), during growth of the microorganism on the trialwith initial phenolic compounds concentration 2.9 g L−1 andRS0∼75.0 g L−1. The maximum YEtOH/RS value achieved was 0.47 gg−1 (EtOHmax = 17.2 g L−1) and was obtained in the fermentationwith initial phenolic compounds concentration 2.0 g L−1 and RS0

at 40.0 g L−1 (see Table 1). Taking into consideration the yieldYEtOH/RS for all RS0 concentrations tested, statistically significantlyhigher ethanol yield occurred in the media with initial phenoliccompounds adjusted at 2.0 g L−1 (Table 2). Further addition ofOMWs into the culture medium, which resulted in the presence ofinitial phenolics at 2.9 g L−1, somehow lowered the maximum valueof yield YEtOH/RS. In any case, it must be stressed that despite thepresence of inhibitors (e.g. phenolic compounds) in the medium,the addition of OMWs up to a specific level seemed to enhancethe production of ethanol (see Tables 1, 2). In all sterile flask trials,as was expected, maximum ethanol production in absolute values

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Table 1. Kinetics of Saccharomyces cerevisiae strain MAK−1 grown on several concentrations of glucose-based media with OMWs added in variousamounts. Representations of total biomass (X, g L−1), ethanol (EtOH, g L−1), total cellular lipid (L, g L−1) and consumed substrate (RScons, g L−1)concentrations at different fermentation points of each trial: (1) when Xmax concentration was achieved; (2) when EtOHmax concentration wasachieved. Fermentation time, conversion yield of ethanol produced per sugar consumed (YEtOH/RS, g g−1) and total lipid in dry biomass (YL/X, g g−1)are presented for all points of the trials (exception for the representation of lipids in the non-sterile trials). Culture conditions: growth on 250 mLsterile and non-sterile shake flasks at 180±5 rpm, RS0 (approximately in g L−1) 40.0, 55.0 and 75.0 g L−1, initial pH = 3.5±0.1, incubation temperatureT = 28◦C. Each point is the mean value of two independent measurements

Initial phenolics (g L−1) RS0(g L−1) Fermentation time (h) X(g L−1) EtOH(g L−1) L(g L−1) RScons(g L−1) YEtOH/RS (g g−1) YL/X(g g−1)

0.0 ∼40.0242 3.5 13.4 0.2 40.0 0.34 0.06

1321 7.2 0.0 0.6 43.2 — 0.09

∼55.0202 3.5 15.7 0.2 52.8 0.30 0.05

1321 10.7 4.1 0.2 55.8 0.07 0.02

∼75.0362 6.3 19.5 0.2 67.4 0.29 0.03

1361 11.2 4.3 0.1 67.4 0.14 0.01

∼40.0282 4.2 12.9 — 43.2 0.30 —

Non-sterile 1321 7.2 0.0 — 43.2 — —

1.2 ∼40.0162 5.3 15.6 0.2 37.4 0.42 0.04

1321 12.0 0.0 0.2 39.6 — 0.02

∼55.0162 6.8 19.3 0.2 52.7 0.37 0.03

1201 15.8 0.0 0.2 55.4 — 0.01

∼75.0162 7.8 21.7 0.2 66.8 0.32 0.03

1321 16.0 1.3 0.2 68.5 0.02 0.01

2.0 ∼40.0162 5.3 17.2 0.2 36.4 0.47 0.04

1081 13.2 0.0 0.2 39.5 — 0.02

∼55.0162 6.3 19.8 0.2 48.4 0.41 0.03

1321 17.3 0.0 0.2 51.3 — 0.01

∼75.0202 7.9 22.8 0.2 69.3 0.33 0.03

1321 17.7 3.9 0.2 69.3 0.06 0.01

2.9 ∼40.0162 6.8 15.2 0.4 38.3 0.40 0.06

1321 15.0 2.8 0.2 43.9 0.06 0.02

∼55.0162 8.1 17.8 0.3 50.1 0.36 0.04

1321 18.2 0.0 0.2 51.9 — 0.01

∼75.0162 8.2 26.1 0.4 73.1 0.36 0.05

1321 18.7 0.0 0.2 76.8 — 0.01

∼75.0162 8.8 25.8 — 73.8 0.35 —

Non-sterile 1321 18.9 3.9 — 76.8 0.06 —

Table 2. Two-way ANOVA test results for the factors initial phenolic compounds concentration (g L−1) (a) and initial RS concentration (g L−1) (b)during the growth of Saccharomyces cerevisiae strain MAK- on various concentrations of glucose-based media (RS0 levels: 40, 55 and 75 g L−1) withOMWs addition (levels: 0, 1.2, 2.0, 2.9 g L−1). Culture conditions: growth on 250 mL sterile and non-sterile shake flasks at 180±5 rpm, RS0 ∼40.0, 55.0and 75.0 g L−1, initial pH = 3.5±0.1, incubation temperature T = 28◦C. Least Significant Difference (LSD) test at 5% level of probability was used tocompare mean values. Mean values of each parameter and factor within the same column not sharing common letters are significantly different(P<0.05)

Xmax(g L−1) YEtOH/RS(g g−1) YL/X(g g−1)

Initial phenolics(g L−1) P = 0.0000* LSD P = 0.0007 LSD P = 0.0000 LSD

0.0 9.68 a 0.31 a 0.071 b

1.2 14.60 b 0.37 b 0.029 a

2.0 16.07 c 0.40 c 0.038 a

2.9 17.30 c 0.37 b 0.038 a

RS0 (g L−1) P = 0.0000 LSD P = 0.0002 LSD P = 0.0006 LSD

40.0 11.85 a 0.41 c 0.053 b

55.0 15.49 b 0.36 b 0.047 b

75.0 15.90 b 0.33 a 0.030 a

*P-values less than 0.05 indicate a statistically significant effect on tested parameters at the 95.0% confidence level.


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Figure 1. Biomass (X, g L−1), ethanol (EtOH, g L−1) and reducing sugars (RS,g L−1) evolution during growth of Saccharomyces cerevisiae strain MAK-1on OMW-based media (initial phenolic compounds concentration 2.0 gL−1) enriched with commercial glucose. Culture conditions: growth on 250mL sterile flasks at 180±5 rpm, RS0 at 40.0 g L−1, initial pH = 3.5±0.1,incubation temperature T = 28◦C. Each point is the mean value of twoindependent measurements.

(EtOHmax = 13.4–26.1 g L−1) increased with the addition of glucoseinto the synthetic medium. However, two-way ANOVA testsrevealed that maximum yield YEtOH/RS values (ranging between0.29–0.47 g g−1, at the point when EtOHmax was noted) werestatistically significantly reduced when cultures were performedat increasing RS0 concentrations, regardless of the addition ofOMW into the medium (Table 2), and this was probably due tothe fact that when increased RS0 concentrations were employed,media were not equally supplemented with nitrogen and cultureswere performed at high C/N values in the medium (nitrogen-limited experiments), which were potentially not adequate for theelaboration of alcoholic fermentation. Finally, the addition of OMWin the medium reduced the time at which EtOHmax values werenoted, compared with the control experiment. The impact of thedifferent initial RS and initial phenolic compounds concentrationsupon the maximum values of biomass concentration (Xmax, g L−1)and ethanol yield per RS consumed (YEtOH/RS, g g−1) for all trialsare summarized in Fig. 2.

In order to demonstrate the feasibility and the potential of thenon-sterile fermentation process, non-sterile trials (RS0∼40.0 g L−1

when no OMWs were added into the medium and RS0∼75.0 g L−1

when OMWs were added to yield an initial phenolic compoundsconcentration in the medium of 2.9 g L−1) were performed and

Figure 2. Impact of the initial concentration of reducing sugars (RS) andphenolic compounds upon the maximum biomass concentration (Xmax, gL−1) and maximum ethanol yield per reducing sugars consumed (YEtOH/RS,g g−1) for all shake-flask cultures realized, on media composed of differentmixtures of olive-mill wastewaters and glucose. Culture conditions asdetailed in Table 1.

compared with the respective sterile experiments in which axeniccultures were used (see Table 1). Indeed, between sterile and non-sterile cultures no significant statistical differences were observedfor both biomass (by means of Xmax) and ethanol production(by means of YEtOH/RS) (statistical analysis not shown). During non-sterile fermentations, samples were checked under the microscopeafter Gram coloration had been done in order to ensure thepurity of the culture. These microscopic observations revealedthat only cells of the microorganism S. cerevisiae were found inthe fermentation medium. In fact, a slight presence of bacteriawas found during the initial stages of the culture (0–10 or 15 hafter inoculation), but these microorganisms rapidly disappeareddue to the secretion of ethanol and lack of nutrients. However,this slight fermentation diversity observed in comparison with thesterile (axenic) culture, was potentially caused by this (very) littlepresence of bacteria found in the medium during the early growthsteps due to the unsterilized conditions.

Kinetics of S. cerevisiae strain MAK-1 grown in batchbioreactor culturesFermentations were carried out in a lab-scale bioreactor understerile and non-sterile conditions. Initially, no phenolic compoundswere added into the growth medium (control experimentwithout OMW addition) and the influence of sterilization or non-sterilization on the bioprocess was quantified (Table 3). Thereafter,the impact of the aeration rate on the fermentation efficiency wasstudied, and finally OMWs were added to the medium to yield aninitial phenolic compounds concentration of 2.8 g L−1 in orderto study the impact of OMWs addition on the conversion (seeTable 3). Glucose was added into the media giving an initial RSconcentration of ∼75.0 g L−1. In bioreactor trials with no OMWaddition, ANOVA tests demonstrated no significant differencesin biomass and ethanol production (by means of maximumconcentration and yield of product synthesized per RS consumed)when comparing sterile and non-sterile fermentations (statisticalanalysis not shown). Likewise, comparing the various aerationregimes (aeration rate imposed at 1.5, 1.0, 0.5 and 0.0 vvm) inthe non-sterile experiments, no remarkable differences in themaximum concentrations of ethanol and biomass achieved werefound (data not presented). Thus, it may be assumed that glucose-based bioreactor fermentations performed by S. cerevisiae MAK-1under non-sterile conditions in which no aeration was imposedwere ideal concerning bio-ethanol production by this strain. (Allthese findings are advantageous for a potential scale-up of theprocess). In non-sterile bioreactor batch cultures, the addition ofOMWs to the synthetic medium seemed, according to the ANOVAtest, to significantly enhance the production biomass (X = 8.3 gL−1) compared with the equivalent experiment in which no OMWaddition was performed (X = 4.8 g L−1, see Table 3; statisticalanalysis not shown). On the contrary, in non-sterile bioreactorbatch cultures, the addition of OMW into the synthetic medium didnot seem to have a statistically significant impact on the conversionof RS into ethanol compared with the equivalent experiment inwhich no OMW addition was made (Table 3). As in the shake-flaskexperiments, in the non-sterilized fermentations, samples werechecked under the microscope after Gram coloration and it wasrevealed that only the microorganism S. cerevisiae was found inthe fermentation medium. The kinetics of S. cerevisiae MAK-1 onbioreactor experiments with either OMW addition or not (‘control’experiment) under non-sterile conditions and no aeration imposed(0 vvm), is presented in Fig. 3(a), (b) and (c). Clearly higher dry cellmass values were obtained for the experiment with OMW added


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Table 3. Kinetics of Saccharomyces cerevisiae strain MAK−1 grown on glucose-based media (blank experiments in various aeration conditions)and glucose enriched OMW-based media under sterile and non-sterile batch bioreactor fermentations. Representations of biomass concentration(X, g L−1) when maximum ethanol (EtOHmax, g L−1) concentrations were achieved at the different trials; conversion yield of ethanol produced persugars consumed (YEtOH/RS, g g−1) was also presented when EtOHmax concentration was achieved. Culture conditions: growth in batch bioreactorexperiments, agitation rate 300 rpm, initial pH = 3.50±0.02 and incubation temperature T = 28◦C. Each point is the mean value of two independentmeasurements

Initial phenolics (g L−1) RS0 (g L−1) Aeration (vvm) X(g L−1) EtOHmax (g L−1) YEtOH/RS (g g−1)

Sterile 0.0 ∼75.0 1.5 4.9 33.6 0.44

Non-sterile

0.0

∼75.0

1.5 5.2 33.8 0.45

0.0 4.8 33.9 0.45

2.8 ∼75.0 0.0 8.3 33.1 0.45

(a)

(b)

(c)

Figure 3. (a) Biomass (X, g L−1), (b) reducing sugars (RS, g L−1) and (c)ethanol (EtOH, g L−1) evolution during growth of Saccharomyces cerevisiaestrain MAK-1 on blank (no OMW addition; RS0 at ∼75.0 g L−1) and OMW-based (initial phenolic compounds concentration 2.8 g L−1; RS0 at ∼75.0g L−1) non-sterile batch bioreactor cultures enriched with commercialglucose. Culture conditions: agitation at 300 rpm, initial pH = 3.50±0.02,incubation temperature T = 28◦C, no aeration imposed. Each point is themean value of two independent measurements.

to the medium (Fig. 3(a)), while the kinetics of RS assimilation andethanol biosynthesis presented similar trends (Fig. 3(b) and (c)).

In order to perform a process scale-up for several microbialconversions, an important aspect that is taken into considerationand studied is the comparison and the potential differences

in the physiological and kinetic features between experimentsperformed in shake-flask and batch bioreactor cultures, giventhat agitation and aeration conditions may be different in thesefermentation configurations.2,5,9 Comparison was performedbetween the (non sterile) batch bioreactor and shake-flask fermen-tations of OMW-based media that presented almost equal initialRS concentrations (∼75 g L−1) and phenolic compounds (∼2.8g L−1) (Fig. 4(a), (b) and (c)). Cell dry mass production was indeedlower in the bioreactor trial compared with the shake-flasks, whilethe strain reached its kinetics plateau earlier in bioreactor trial(∼18–20 h) than in the shake-flask culture (Fig. 4(a)). In both trials,no RS remained unconsumed at the end of the fermentation, whilethe assimilation rate of glucose was similar in both the shake-flaskand the bioreactor experiment (Fig. 4(b)). Ethanol production wasclearly increased in the bioreactor cultures, while after total deple-tion of glucose in the culture media, the microorganism used thepreviously produced ethanol as carbon source for further biomassproliferation, leading to non-negligible ethanol concentrationreduction (specifically for the shake-flask experiment – Fig. 4(c)).

In order to further increase the maximum ethanol levelachieved, a supplementary batch bioreactor non-sterilized trialwas performed, in which OMWs were added in a (relativelyconcentrated) glucose-based medium, and the initial RS andphenolic compounds concentrations of the fermentation mediumwere ∼115 and ∼2.9 g L−1, respectively. The kinetics of biomassand ethanol production and RS consumption is illustrated inFig. 5. As in all previous fermentations, RS were rapidly consumed(within ∼48 h), although the fermentation was accomplished latercompared with the previous trials (Fig. 3) potentially due to thehigher initial RS quantity. In any case, an EtOHmax concentration of52.0 g L−1 was reported 38 h after inoculation (concomitant YEtOH/RS

value = 0.46 g g−1), while ethanol concentration presented a slightdecrease after sugar consumption from the growth medium. Asin the previous bioreactor experiments, ethanol concentrationdecrease was not followed by biomass concentration rise, thevalue of which remained almost constant (X∼8.5 g L−1).

Decolorization – removal of phenolic compoundsIn order to identify whether reduction of phenolic compoundsconcentration and/or color reduction from the culture mediumoccurred due to agitation (and, thus, aeration) alone, experimentswere carried out in which OMWs and glucose were added to thegrowth medium, as previously, giving initial phenolic compoundsconcentration (in g L−1) 1.2, 2.0 and 2.9 and RS0 = 40.0 g L−1 butwithout inoculating the medium (therefore there was no presenceof S. cerevisiae cells). It was found that the phenolic content of the


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(a)

(b)

(c)

Figure 4. Comparison of Saccharomyces cerevisiae strain MAK-1 kineticsbetween non-sterile shake-flask and batch-bioreactor cultures regarding(a) biomass (X, g L−1), (b) reducing sugars (RS, g L−1) and (c) ethanol(EtOH, g L−1) evolution on OMW-based media enriched with commercialglucose. Culture conditions: non-sterile shake-flask 250 mL culturesagitated at 180±5 rpm, initial phenolic compounds concentration 2.9g L−1, RS0 at ∼75.0 g L−1, initial pH = 3.5±0.1, incubation temperatureT = 28◦C; non-sterile batch bioreactor cultures agitated at 300 rpm, initialphenolic compounds concentration 2.8 g L−1, RS0 at ∼75.0 g L−1, initialpH = 3.50±0.02, incubation temperature T = 28◦C, no air sparging. Eachpoint is the mean value of two independent measurements.

media showed no reduction at all, whereas the color intensityincreased to approximately 5–7%, potentially due to auto-oxidation of phenolic compounds (data not presented). Thereforeneither phenolic compounds nor color were removed from themedium due to the agitation performed (in contrast, as stated, thecoloration of the residue slightly increased due to the agitation).

Remarkable color removal was performed in both sterile andunsterilized shake-flask fermentations (Table 4). It appears thatfor a given RS0 concentration, decolorization rate seemed toincrease with increased initial phenolic content media, while nocorrelation can be established between the removal of phenoliccompounds from the medium and the initial concentrationof phenolics or glucose in the medium. In non-sterile batchbioreactor fermentations, the maximum decolorization andreduction of phenolic compounds concentration achieved was59.6% and 27.3% (w/w) respectively. Comparing unsterile shake-flask and unsterile bioreactor cultures that presented similar initial

Figure 5. Biomass (X, g L−1), ethanol (EtOH, g L−1) and reducing sugars(RS, g L−1) evolution during growth of Saccharomyces cerevisiae strainMAK-1 on OMW-based media (initial phenolic compounds concentration2.0 g L−1) enriched with commercial glucose. Culture conditions: non-sterile batch bioreactor cultures at 300 rpm, initial phenolic compoundsconcentration 2.9 g L−1, RS0 at ∼115.0 g L−1, initial pH = 3.50±0.02,incubation temperature T = 28◦C, no air sparging. Each point is the meanvalue of two independent measurements.

concentrations of RS (∼75 g L−1) and phenolic compounds (∼2.8g L−1), one can conclude that decolorization and reduction ofphenolic compounds values were similar. Moreover, comparisonof bioreactor experiments presenting different RS0 concentrations(75 and 115 g L−1) and almost equal initial phenolic compoundsquantities, demonstrated similar values of decolorization andremoval of phenolic compounds. The kinetics of color and phenoliccompounds removal from the culture medium (comparison of flaskand bioreactor trials) is shown in Fig. 6(a) and (b). From the trendsof the kinetics obtained that were similar to all other shake-flask orbioreactor experiments, it can be readily assumed that rapid colorand phenolic compounds removal occurred (maximum phenoliccompounds removal and decolorization within the first 24–30 h)which coincided with the maximum values of ethanol achieved.

Biogenesis of cellular lipids and fatty acid (FA) compositionanalysis in sterile shake-flask systemsTotal cellular lipids were quantified in all growth phases, andquantities ≤10% (w/w) in dry matter were found (Table 1),indicating, in full accordance with the literature,29 that no lipidaccumulation occurs in S. cerevisiae strains, in spite of the factthat in several cases of the present investigation, the increase ofRS0 concentration rendered the media nitrogen-limited (nitrogenlimitation is prerequisite in order for lipid accumulation to occur29).Two-way ANOVA tests showed that significantly lower quantitiesof lipids per g of dry weight (YL/X) were observed in the mediawith RS0∼75 g L−1, compared with the trials at RS0∼55 g L−1 andRS0∼40 g L−1 (Table 2). Likewise, monitoring of lipid produced bythe strain suggested that the presence of OMWs in the mediumseemed to have effects on the biogenesis of lipids; two-wayANOVA tests revealed that maximum lipid in dry weight YL/X

values were significantly higher in the control experiment (initialphenolic compounds at 0.0 g L−1) compared with the trials inwhich OMW quantities were added to the medium. On the otherhand, maximum YL/X values (ranging between 0.029 and 0.038g g−1) were not statistically significantly different, irrespective ofthe (low or high) initial quantity of phenolic compounds added tothe medium (Table 2). The impact of different initial RS and initialphenolic compounds concentrations on the maximum values oflipid synthesized per g of dry yeast mass (YL/X, in g g−1) for all


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Table 4. Data of Saccharomyces cerevisiae strain MAK−1 concerning removal of phenol compounds and color, obtained from kinetics in media(in sterile and non-sterile shake-flask cultures and non-sterile batch bioreactor culture) containing commercial glucose and various initial OMWconcentrations. Representation of initial and final phenol compounds concentration in the culture medium, phenol compounds removal (%, w/w)and color removal (%) from the medium. Each point is the mean value of two independent measurements

RS0 (g L−1) Initial phenolics (g L−1) Final phenolics (g L−1) Phenol removal (%, w/w) Color removal (%)

∼40.0, shake-flask culture 1.11 0.86 22.5 49.7









∼75.0, non-sterile shake-flask culture 2.95 2.30 22.1 58.5

∼75.0, non-sterile bioreactor culture 2.82 2.10 25.5 59.6

∼115.0, non-sterile bioreactor culture 2.89 2.10 27.3 59.5

(a)

(b)

Figure 6. (a) Phenolic compounds removal (% w/w) and (b) color removal(%) during growth of Saccharomyces cerevisiae strain MAK-1 on OMW-based media enriched with commercial glucose, in 250 mL non-sterileshake-flask (initial phenolic compounds concentration 2.9 g L−1; RS0 at∼75.0 g L−1) and non-sterile batch bioreactor (initial phenolic compoundsconcentration 2.8 g L−1; RS0 at ∼75.0 g L−1) cultures. Culture conditions asdetailed in Fig. 4.

trials is summarized in Fig. 7. Indeed, the presence of phenoliccompounds in the medium, even in small concentrations, as wellas the increasing RS0 quantities negatively affected the quantityof lipids produced by the microorganism. FA composition of intra-cellular lipids was analyzed in all trials at various growth phases(Table 5). The principal FAs detected belonged to the C16 and C18

aliphatic chains. The FA composition changed with fermentationtime and the addition of OMWs into the medium. Specifically,in the absence of OMWs in the medium, the concentration of

Figure 7. Impact of the initial concentration of reducing sugars (RS) andphenolic compounds upon the maximum quantity of lipids produced perunit of dry yeast mass (YL/X, g g−1) for all shake-flask cultures realized,on media composed of different mixtures of olive-mill wastewaters andglucose. Culture conditions as in Table 1.

stearic acid (C18:0) and oleic acid (�9C18:1) clearly decreased withtime since the concentration of palmitoleic (�9C16:1) increased(Table 5). Moreover, in the cultures that were not supplementedwith OMWs, the addition of glucose to the medium also had astrong impact on the total FA composition of the lipids since theconcentration of �9C18:1 and �9C16:1 decreased with RS0 rise inthe medium, while the respective concentration of FA �9,12C18:2increased. The addition of OMW to the culture medium evenin quantities that resulted in minimal elevated initial phenoliccompounds concentration (e.g. 1.20 g L−1) resulted in differencesin the FA composition of cellular lipids produced. In almost all cases,the addition of phenolic compounds resulted in a remarkable rise inthe concentration of the cellular FA �9C18:1 (and to lesser extentof the FA �9,12C18:2) whereas the respective quantities of theFAs C18:0 and �9C16:1 drastically decreased (Table 5). Finally, theaddition of OMWs did not result in drastic FA composition changesas a function of the fermentation time, in contrast with the controlexperiments in which no OMW addition was performed (Table 5).

DISCUSSIONS. cerevisiae strain MAK-1 presented notable biomass and ethanolproduction in sterile and non-sterile shake-flask and batchbioreactor fermentations, when various amounts of OMWs andglucose were added. In several cases, ethanol was produced in


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Table 5. Fatty acid composition (% w/w) of cellular lipids by Saccharomyces cerevisiae MAK−1

Initial phenolics (g L−1) RS0 (g L−1) Time (h) C16:0 �9C16:1 C18:0 �9C18:1 �9,12C18:2

0.00 ∼40.0 12 14.3 14.1 7.2 60.3 4.1

24 14.7 32.0 7.1 40.7 3.8

60 14.1 35.3 6.4 38.8 4.0

132 16.9 29.1 3.9 42.7 7.3

∼55.0 12 16.7 9.9 16.6 54.2 2.6

24 17.5 10.9 15.5 41.3 14.7

60 16.9 24.4 7.4 37.4 13.8

132 17.6 22.5 3.2 40.3 16.4

∼75.0 12 23.6 12.1 5.5 47.6 3.5

24 23.2 15.4 5.5 45.9 4.0

60 20.3 22.1 T. 44.6 12.7

132 17.8 24.0 T. 41.1 16.5

1.20 ∼40.0 12 14.5 T. 8.0 58.0 18.9

24 17.6 T. 6.0 57.4 14.0

60 16.6 7.1 T. 59.7 15.7

132 17.6 8.0 T. 57.1 14.8

∼55.0 12 21.0 T. T. 69.9 7.7

24 15.2 T. T. 65.4 12.4

60 15.4 T. T. 65.2 17.7

132 13.4 7.9 T. 67.6 11.0

∼75.0 12 15.7 10.5 T. 59.6 14.2

24 22.7 T. T. 57.3 12.8

60 15.1 T. T. 68.0 12.9

132 15.5 T. T. 68.5 15.5

2.00 ∼40.0 12 13.1 8.5 T. 61.4 16.3

24 13.8 T. T. 68.4 15.6

60 15.3 T. T. 70.1 13.6

132 16.4 2.0 T. 63.5 15.1

∼55.0 12 14.5 9.9 T. 61.0 14.6

24 16.5 T. T. 68.4 15.1

60 16.7 T. T. 65.3 12.8

132 17.2 T. T. 67.7 11.5

∼75.0 12 16.2 T. T. 70.2 13.5

24 17.5 T. T. 68.8 11.6

60 15.4 3.7 T. 67.8 7.7

132 15.3 1.5 T. 68.9 12.8

2.90 ∼40.0 12 14.8 T. T. 69.4 15.7

24 17.0 T. T. 66.8 16.2

60 14.5 T. T. 68.3 14.1

132 15.9 T. T. 69.0 14.3

∼55.0 12 16.9 2.7 T. 65.0 13.5

24 15.8 T. T. 67.3 16.5

60 14.3 1.3 T. 66.9 14.6

132 19.2 T. T. 68.8 12.0

∼75.00 12 17.3 T. T. 67.8 14.8

24 17.1 T. T. 65.4 16.8

60 17.3 T. T. 66.3 16.0

132 14.3 T. T. 68.0 13.5

T.<0.5%, w/w


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significant quantities in the medium despite the fact that growthwas performed under aerated conditions. This is the ‘Crabtree’(or ‘glucose’) effect, observed in several yeast genera (the so-called ‘Crabtree-positive’ yeasts) in which enzymes involved inthe oxidative part of the metabolism (namely the Krebs cycle andthe oxidative phosphorylation chain) are subjected to cataboliterepression, with the metabolic network being directed towardsthe synthesis of ethanol via fermentative conversion, despite thesignificant presence of oxygen in the medium.28 On the otherhand, S. cerevisiae cells need oxygen (at least during the first hoursafter inoculation). Oxygen-dependent reactions led by the yeastcells result in the biosynthesis of sterols, unsaturated fatty acidsand phospholipids needed in the formation of cell membranesand thus the continuation of growing.28 Additionally, rapidlyafter RS exhaustion from the medium, ethanol was re-consumed,and new cell material was created (principally in the shake-flaskexperiments, since no important biosynthesis of new cell materialwas observed in the bioreactor trials – Figs 4 and 5). It is evidentthat in a potential scale-up of the bioprocess, the fermentationshould stop within the first 24–36 h, in order not to lose thevaluable ethanol (at that time the maximum removal of color andphenolic compounds was also achieved – Fig. 6).

The addition of OMW to the media increased the productionof biomass and ethanol compared with the control experiment(no OMWs added), which is an interesting result consideringthe presence of several inhibitors in the medium. Similar typesof observations have been also observed in other types ofyeasts (e.g. strains of the non-conventional species Yarrowialipolytica) in which the addition of OMWs to the medium seem to‘stimulate’ the production of biomass and other metabolites, suchas citric acid.12,20 Potentially, besides the existence of ‘inhibiting’compounds (such as phenols) found in the waste, ‘useful’ nutrients(e.g. vitamins, growth-stimulating factors, micro- and macro-elements) may also potentially exist, and this could be thereason for the enhancement of biomass and ethanol productionin the presence of waste in the medium. Moreover, although lowquantities of cellular lipids were produced by S. cerevisiae strainMAK-1 throughout all trials, the presence of OMWs in the mediumseemed to decrease (by means of monitoring the quantity of lipidsproduced in dry weight YL/X) lipid biogenesis compared with thecontrol experiment (no OMW added). However, in contrast withthe present investigation, in the case of Y. lipolytica strains, whengrowth was performed in nitrogen-limited media (favoring theaccumulation of storage lipids29), higher quantities of lipids (interms of principally YL/X) were produced in media enriched withOMWs than in the control experiments.20

The main cellular FA of S. cerevisiae produced during all trialswas �9C18:1, but the presence of waste in the growth mediumincreased the amounts of this FA, while the respective quantities of�9C16:1 and C18:0 drastically decreased, in accordance with resultsrecorded for Y. lipolytica in similar trials.12,20 The almost completeabsence of the FA C18:0 in the cellular lipids of the microorganismin OMW-supplemented media, indicated potential activation of thecellular �9 desaturase (catalyzing the reaction C18:0 −→�9C18:1)by the presence of microbial inhibitors (e.g. gallic acid, caffeic acid,polyphenols, etc.). This is an interesting result since the addition of‘inhibitory’ compounds (such as essential oils and cyclopropenicacids) is considered to decrease the activity of cellular desaturasesyielding the synthesis of more saturated cellular lipid in these casesin comparison with the control experiments.30,31

Concerning the ability of yeast strains to decrease the contentof phenols in OMW-based media, some confusion exists in the

literature indicating that phenol removal is a strain-dependentprocess; specifically, three Y. lipolytica strains capable of producinglipid and citric acid12,32 were cultivated in OMW-based media withvarious initial phenolic compounds concentrations imposed.20

Remarkable decolorization (∼63%) and removal of phenol content(∼34% w/w) occurred in most cases. Y. lipolytica ACA-DC 50109when cultivated on OMW-based media reduced the phenoliccontent of the residue by up to ∼15% w/w.12 On the other hand,Scioli and Vollaro17 cultivated Y. lipolytica ATCC 20255 on lowphenolic content OMWs and despite the noticeable biomass andlipase production, phenolic content of the treated OMWs was notreduced at all. Lanciotti et al.1 used low phenolic content (0.7 g L−1)undiluted OMWs for the growth of many Y. lipolytica strains. Somestrains lowered the phenolic compounds concentration to ∼18%w/w, whereas others did not. Bambalov et al.7 cultivated variousSaccharomyces, Torulopsis, Kloeckera and Schizosaccharomycesstrains on olive oil extraction effluents containing ∼8 g L−1 ofphenolic substances. None of the strains showed any growth insuch high initial phenolic compounds media, whereas five strainsamong them did grow only in 3-fold diluted medium. Moreover,Ben Sassi et al.33 highlighted the potential of indigenous yeastsin detoxification of OMWs, and a group of yeast isolates reducedsignificantly the concentration of total phenols (to ∼44% w/w)and produced non-negligible biomass quantities (up to ∼6 g L−1).Ettayebi et al.34 cultivated Candida tropicalis YMEC14 on OMWsand 55% and 69% reduction of polyphenols and monophenols,respectively, was seen. C. cylindracea NRRL Y-17506 flask-culturedon diluted OMWs enriched with olive oil, presented a phenoliccompounds reduction to ∼36% w/w.10 A Trichosporon cutaneumstrain was capable of completely utilizing phenolic compoundswhen used as the sole carbon and energy source (at phenolic com-pounds of 0.8–2.0 g L−1) and removed 85% w/w of total phenoliccontent (initial concentration 6.0 g L−1) using OMWs ethyl-acetateextracts as the only carbon source.35 Literature also reports a

number of molds3-5,8,9,36-38 and bacteria39–41 capable of efficientlyreducing phenolic compounds concentration in OMW-basedmedia. Finally, besides the use of microorganisms there are reportsin the literature of OMW bioremediation by enzyme treatment.42

The ability of higher fungi to break down phenolic compoundsis based on the secretion of extra-cellular oxidases (ligninolyticenzymes) laccase, lignin peroxidase and manganese dependent(or independent) peroxidase.5,9,36,38 The secretion of theseenzymes is strain-dependent and influenced by various culture

conditions.3–5,8,37,43 Non-genetically modified yeast strains (likeS. cerevisiae), in general, do not contain in their genetic arsenalthe potentiality of producing such types of enzymes,5 and, thus,the removal of phenol compounds and the decolorization ofOMWs that are subjected to fermentation by these yeast speciesthrough the use of the above-mentioned enzymes should beexcluded. On the other hand, Rizzo et al.44 suggested a potentialexclusively physical mechanism involving the establishment ofweak and reversible interactions, mainly between anthocyaninsand yeast walls, by means of adsorption. Moreover, (potentiallyvery weak) assimilation of several phenolic compounds by theyeast could, also, not be excluded.35 In the present study, nophenolic compounds concentration reduction occurred due toagitation (and aeration) of the culture medium (no presence ofS. cerevisiae cells), while the color intensity slightly increased (toapproximately 5–7%), regardless of the initial concentration ofphenolic compounds in the medium, providing evidence thatremoval of color and phenolic compounds was due to thebiological activity of S. cerevisiae. It could be supposed, thus,


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that the phenolic compounds concentration reduction noted inour study could be based on the adsorption of the aforementionedcompounds in the yeast cell surface or/and on their partialutilization as carbon source by the microorganism.20,35

OMWs diluted and enriched with low-cost glucose could beproposed as a promising substrate for the biotechnologicalproduction of ethanol. In various fermentation processes,including production of ethanol, utilization of raw OMWs derivedfrom press extraction systems that contain relatively high initialtotal sugars concentration (i.e. ≥70 g L−1) could be used withoutsupplementary glucose addition and thus the cost of the processcould be further reduced.5,20 S. cerevisiae strain MAK-1 has beenused previously in a simultaneous remediation–detoxificationand bio-ethanol production bioprocess; in fact, the strainwas cultivated on pasteurized grape must and the fungicidequinoxyfen was added in various concentrations. Significantquantities of biomass were produced (∼10.0 g L−1) regardless ofthe addition of fungicide to the medium. Ethanol was synthesizedin very high quantities (∼106–119 g L−1) and the fungicideconcentration was reduced to ∼79–82% w/w.21 It is evident thatethanol quantities achieved in the above-mentioned study21 were,in absolute values (g L−1), significantly higher than in the presentstudy (see Fig. 5), since remarkably higher initial carbon sourceconcentrations were employed (initial sugars quantities at ∼260g L−1). Also the medium used in the above-mentioned study(enriched grape must) was considered ideal (including all thenutrients needed) for growth of the microorganism. In any case,the maximum values of conversion yield of ethanol producedper sugar consumed were similar ( = 0.44–0.48 g g−1) to thoserecorded in the current study.

There are only a few reports in the literature suggesting the useof S. cerevisiae strains for the treatment of OMWs and the use ofthis waste fermentation process water directly for the productionof ethanol. Bambalov et al.7 used various Saccharomyces strainsgrowing on olive oil extraction effluents containing around 8.0g L−1 of phenolic substances. Three strains did grow only in the3-fold diluted medium producing ethanol (EtOHmax = 10.8–11.7g L−1; YEtOH/RS = 0.38–0.41 g g−1). Zanichelli et al.45 stated thenecessity for removing the phenolic fraction and of the efficientenzymatic pre-treatment of the waste, before inoculating with aS. cerevisiae strain. The initial phenolic compounds concentration(after dilution) was 2.1 g L−1, the glucose supplementation of themedium was up to 200 g L−1, the total fermentation time was 65days and the ethanol production reached 63–95 g L−1. Massadehand Modallal3 also proposed the use of OMWs as valuableeffluents for the production of ethanol, using Pleurotus sajor-cajufor the pretreatment of the waste so as to degrade the phenols inthe waste. Following the pretreatment, 50% diluted and thermallyprocessed OMW medium was inoculated with a S. cerevisiae strainleading to maximum ethanol production of 14.2 g L−1 after 48h of fermentation. Yeasts are the most common microorganismsfor the biotechnological production of ethanol. Among them,numerous S. cerevisiae strains have been widely used and reportedin literature for the production of high ethanol concentrationswhen cultivated in various substrates including wastes or low-costmaterials like crude molasses (contaminated with fungicides)

grape musts, flour hydrolysates, sorghum stalks, etc.21,46–57

CONCLUSIONSS. cerevisiae strain MAK-1 presented efficient growth whencultivated on glucose-enriched OMW-based media in sterile and

non-sterile shake-flask and bioreactor experiments. Satisfactorybiomass and ethanol quantities were synthesized. The presenceof OMWs in the medium seemed to favor their production.A remarkable decolorization and a non-negligible reductionof phenolic compounds in the OMW-based media occurred.Comparing shake-flask with batch bioreactor cultures it couldbe concluded that in non-sterile bioreactor fermentations,biomass production was reduced whereas ethanol production wassignificantly increased. Non-sterile batch bioreactor conditionscould lead to a dramatic reduction in bioprocess cost. The S.cerevisiae strain MAK-1 tested can be regarded as a satisfactorycandidate for simultaneous OMWs bioremediation and theproduction of added-value metabolites.

NOTATION AND UNITSX – dry biomass (g L−1); RS – reducing sugars (g L−1); EtOH –ethanol (g L−1); L – total lipid (g L−1); YEtOH/RS – ethanol yieldon sugar consumed (g formed per g of RS consumed); YL/X –total lipid yield on biomass (g formed per g of biomass formed).Subscripts 0, cons and max indicate the initial, consumed andmaximum quantity, respectively, of the components in the kineticsperformed.

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