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Industrial Crops and Products 56 (2014) 83–93

Contents lists available at ScienceDirect

Industrial Crops and Products

jo ur nal home p age: www.elsev ier .com/ locate / indcrop

erated vs non-aerated conversions of molasses and olive millastewaters blends into bioethanol by Saccharomyces cerevisiaender non-aseptic conditions

imitris Sarrisa, Leonidas Matsakasa, George Aggelisb,c, Apostolis A. Koutinasa,eraphim Papanikolaoua,∗

Department of Food Science and Human Nutrition, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, GreeceUnit of Microbiology, Department of Biology, Division of Genetics, Cell and Development Biology, University of Patras, 26500 Patras, GreeceDepartment of Biological Sciences, King Abdulaziz University, 21589 Jeddah, Saudi Arabia

r t i c l e i n f o

rticle history:eceived 4 October 2013eceived in revised form 25 February 2014ccepted 28 February 2014vailable online 22 March 2014

eywords:accharomyces cerevisiaeeet molasseslive-mill wastewatersioethanol

a b s t r a c t

The ability of Saccharomyces cerevisiae MAK-1 to convert blends of molasses and olive mill wastewaters(OMWs) into compounds of higher added-value under aerated and non-aerated conditions was studiedin the current investigation. Noticeable decolorization (up to 60%) and moderate removal of phenoliccompounds (up to 28%, w/w) was observed. Under aerated conditions in non-sterile shake-flask cultures,cultures in molasses-based media in which supplementation with OMWs had been performed did notsignificantly decrease ethanol and biomass production in comparison with control experiments (culturesin which no OMWs had been added). Ethanol of 34.3 g L−1 (with simultaneous yield of ethanol producedper sugar consumed of ∼0.40 g g−1) and biomass of 7.3 g L−1 (with yield of ∼0.08 g g−1) was observed.Under similar aerated bioreactor cultures, biomass production (up to 5.7 g L−1 with yield of biomassproduced per sugar consumed of ∼0.07 g g−1) decreased while, on the other hand, ethanol biosynthesis

−1 −1

aste bioremediation was notably enhanced (up to 41.8 g L with yield of ethanol produced of ∼0.49 g g – value very close tothe maximum theoretical one). Comparing non-sterile aerated with non-aerated bioreactor experiments,biomass production showed some slight increase and ethanol production slightly increased in the lattercase. It is concluded that S. cerevisiae MAK-1 is a microorganism of importance amenable for simultaneousOMWs remediation and production of added-value compounds.

. Introduction

Olive mill wastewaters (OMWs) are the most important residueshat are implicated with the production of olive oil being one of the

ost difficult to treat industrial effluents (Lanciotti et al., 2005).he total annual production of OMWs is higher than 3 × 107 m3

Massadeh and Modallal, 2008). The dark color and the (phyto)-oxic effect of the OMWs are due to the phenolic compounds thatre found in several concentrations in the residue (Aggelis et al.,003D’Annibale et al., 2006; Ergül et al., 2011; Sayadi and Ellouz,

992; Tsioulpas et al., 2002). Biotechnological methods for thereatment of OMWs have been proposed, with the limiting stepn the treatment being considered the breakdown of the phenolic

∗ Corresponding author at: Laboratory of Food Microbiology and Biotechnology,epartment of Food Science and Human Nutrition, Agricultural University of Athens,

era Odos 75, Athens, Greece. Tel.: +30 210 5294700; fax: +30 210 5294700.E-mail address: spapanik@aua.gr (S. Papanikolaou).

ttp://dx.doi.org/10.1016/j.indcrop.2014.02.040926-6690/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

compounds (Aggelis et al., 2003; Crognale et al., 2006; Ergül et al.,2011; Lanciotti et al., 2005). On the other hand, recent devel-opments have indicated that OMWs should be considered as afermentation feedstock to valorize, either as a process water oras a medium rich in nutrients, rather than a waste to discharge,being a microbial substrate for various bioprocesses (Crognale et al.,2006). OMW-based media have been used for the cultivation ofboth prokaryotic and eukaryotic microorganisms, resulting to theremediation of the waste as also to the synthesis of metabolic com-pounds such as microbial mass (Ben Sassi et al., 2008; Crognaleet al., 2006; Lanciotti et al., 2005; Scioli and Vollaro, 1997), exo-polysaccharides (Crognale et al., 2006), enzymes (Aggelis et al.,2003; Crognale et al., 2006 2006; Scioli and Vollaro, 1997; Tsioulpaset al., 2002), citric acid (Papanikolaou et al., 2008; Sarris et al.,2011; Scioli and Vollaro, 1997; Tsioulpas et al., 2002) and finally

bioethanol (Bambalov et al., 1989; Massadeh and Modallal, 2008;Sarris et al., 2013; Zanichelli et al., 2007).

Molasses, the by-product of the sugar-processing facilities havebeen used as starting material for the biotechnological production

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f various (high-) added value products. The waters remainingfter such bioprocesses, called molasses wastewaters, are char-cterized by high BOD and COD values, strong odor and darkrown color (Satyawali and Balakrishnan, 2008). The composi-ion of this effluent and its release into the environment withoutppropriate previous treatment has serious impact to aquatic envi-onment (FitzGibbon et al., 1998). Molasses wastewaters generallyesist to microbial degradation and to conventional biological treat-ent processes. The decolorization of molasses and degradation

f melanoidins have been achieved by the use of chemical andhysicochemical treatment processes. Those treatment methodsre considered to be quite expensive and unstable especially inarge scale (Tondee et al., 2008). Fungi, bacteria and yeasts haveeen cultivated on molasses for melanoidin degradation, the reduc-ion of BOD and COD values and for the production of added-value

etabolites (Chatzifragkou et al., 2010; Metsoviti et al., 2011; Sarrist al., 2013; Zanichelli et al., 2007).

There is a raise in worldwide need for energy generation deriv-ng from various renewable resources, due to both the decreasef fossil fuel and other non-renewable feedstocks and the insta-ility of their price into the market volume (Dagnino et al., 2013;avila-Gomez et al., 2011; Del Campo et al., 2006; Matsakasnd Christakopoulos, 2013a, 2013b). Bioethanol, one of the mostrincipal renewable energy sources applied worldwide, presentserious economic and environmental benefits (Kopsahelis et al.,009; Lin and Tanaka, 2006; Matsakas and Christakopoulos, 2013a,013b; Sarris et al., 2009). In addition, this compound is an eas-

ly biodegradable and a highly water-soluble material (Kopsahelist al., 2007; Lin and Tanaka, 2006; Matsakas and Christakopoulos,013a, 2013b; Sanchez and Demain, 2008).

Goal of the current submission was to investigate the potentialf S. cerevisiae strain MAK-1 to produce bioethanol and biomassnder aerated and non-aerated conditions when blends of molassesnd OMWs were used as substrates. According to our knowledge,here is a scarce number of investigations indicating the use of. cerevisiae strains for the valorization of OMWs or OMW-basededia for the production of ethanol. The originality of our study

s based upon the simultaneous presence of molasses and OMWsnot pre-treated) as substrate and the growth of the strain usednder completely non-aseptic conditions. The rationale of the uti-

ization of these blends is that it was desirable to study the effect ofhe utilization of these mixtures of residues upon the physiologicalnd kinetic behavior of the strain, since in a scale-up of the pro-ess in large-scale operations, OMWs could be used as tap waterubstitute for molasses dilution. To further reduce the bioprocessost, fermentations under completely non-aseptic conditions wereonducted. Media presenting various initial phenolic and sugarontents were formulated and the ability of the strain to producethanol and biomass together with the simultaneous reduction ofhe color and the phenol content of the medium was investigatednd comprehensively discussed.

. Materials and methods

.1. Microorganism and media

Saccharomyces cerevisiae strain MAK-1 (Sarris et al., 2009)sed in this study was conserved on PDA (T = 6 ± 1 ◦C) and wasub-cultured every 3 months. Beet molasses with total sugarsTS) 573 ± 10 g L−1 expressed as glucose equivalent, and density.38–1.42 g mL−1, used in this study were provided by the “Hellenic

ndustry of Sugar SA” (Orestiada, Greece) and kept at T = 4 ◦C. OMWssed were obtained from a three-phase decanter olive mill in theegion Kalentzi (Corinthia, Peloponnisos, Greece). OMW treatmentefore fermentation (e.g. freeze, removal of solid materials, etc.)

d Products 56 (2014) 83–93

has been presented in details in a previous investigation (Sarriset al., 2011). The used OMW presented total phenolic compoundsconcentration (expressed as gallic acid equivalent) of ∼9.5 g L−1

while its sugar concentration was ∼30.0 g L−1 (expressed as glucoseequivalent). Also OMWs used contained a very small quantity ofolive oil (0.4 ± 0.1 g L−1 – determination of oil conducted after tripleextraction with either hexane or chloroform used as extracting sol-vents). Organic acids were also presented in small concentrations(principally acetic acid and gluconic acids at ∼2.0 g L−1 of each).

Culture media contained blends of OMWs and molasses (dilutedin water in several ratios). The sole external addition of nutrientsbesides the utilization of residues was that of yeast extract and(NH4)2SO4 at a concentration of 2.0 g L−1 of each. In some trialsmineral salts were added in the following concentrations (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. As non-sterile conditions were applied, initial pH medium was adjustedat 3.5 (addition of 1 M HCl was performed).

2.2. Culture conditions

Media composed of diluted molasses and OMWs mixtures wereformulated and fermentations in previously sterilized (sterilizationat 120 ◦C for 20 min) and non-aseptic shake-flask and non-asepticbioreactor cultures were carried out. Fermentations performedin 250-mL non-baffled Erlenmeyer flasks, containing 50 ± 1 mLof the culture medium inoculated with 1 mL (2%, v/v inoculum)of exponential pre-culture, as previously described (Sarris et al.,2013). Flasks were incubated aerobically in an orbital shaker(New Brunswick Scientific, USA) (agitation rate 180 ± 5 rpm andT = 28 ± 1 ◦C). The pH of the culture medium was maintained tothe value of ∼3.5 by periodically (and aseptically for the previ-ously sterilized media) adding into the flasks, previously calculatedamounts of 1 M HCl (see the experimental protocol in Papanikolaouet al., 2008). Blank experiments (in which no OMWs had beenadded) were also carried out. Initially, previously sterilized shake-flask cultures (“control” experiments without OMW addition;TS ∼ 100 g L−1) with added salts were compared to cultures withoutsalts addition. In the second part, previously sterilized shake-flaskcontrol cultures without salts added were compared to non-asepticflask cultures (TS ∼ 100 g L−1). In the third experimental part,shake-flask cultures in non-aseptic conditions were performed, inwhich OMWs were added into the diluted molasses in various ratiosas follows (%, v/v): 0 (control experiment, no OMW addition), 10,20, 30, 40 and 50, yielding at initial quantities of phenolic com-pounds into the medium (in g L−1): 2.6 ± 0.2, 3.9 ± 0.3, 4.5 ± 0.4,5.2 ± 0.4, 5.5 ± 0.5 and 6.3 ± 0.5. The initial total sugars (TS0) con-centration was ∼100 g L−1. Moreover, non-aseptic batch bioreactorfermentations were carried out in a bench top bioreactor (MBR,AG Switzerland), with total volume 3.5 L and working volume 3.0 L.The culture vessel was inoculated with 60 mL (2%, v/v inoculum) ofexponential pre-culture (see above). The culture conditions were asfollows: T = 28 ± 1 ◦C; agitation rate 300 rpm; pH = 3.5 ± 0.1 (con-trolled by automatic addition of 1 M HCl); aeration (air passingthrough a bacteriological filter with 0.2 �m pore size) at 1.2 vvmfor the aerated experiments or 0.0 vvm (thus, with no aeration)for the non-aerated ones (Sarris et al., 2013). In the trials car-ried out without air sparging, OMW was added at a quantity of20% (v/v) and molasses were added in various amounts giving ini-tial total sugars concentration (in g L−1): ∼100, ∼135, ∼150 and∼200. A non-aseptic aerobic bioreactor trial with aeration imposed1.2 vvm was also performed (in this trial OMWs were added into

the medium in a ratio of 20% (v/v) and molasses were added intothe medium in order to have a TS0 ∼ 100 g L−1). All values of totalphenolic compounds previously mentioned, include besides thephenolic compounds of the OMWs also the phenolic compounds

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f molasses (principally melanoidins). For all of the fermentationserformed, two lots of independent cultures with different initial

nocula were realized, while each experimental point of the kineticsresented is the mean value of two independent measurements.

.3. Analytical methods

Biomass was harvested by centrifugation at 9000 rpm, for0 min at T = 21 ± 1 ◦C, washed twice with distilled water andentrifuged again. Biomass concentration was determined gravi-etrically and was expressed as dry cell weight (DCW) in g L−1,

fter placement of wet biomass at T ∼ 100 ◦C until constant weight.H measurement was conducted using a Jenway 3020 pH-meter.thanol was quantified through HPLC analysis according to pre-iously published protocol (Sarris et al., 2013). TS of the mediumere determined according to the protocol proposed by Roukas,

994 as follows: initially, for the hydrolysis of sucrose into glucosend fructose, 4.5 mL of HCl 1 M were added in a test tube with 1 mLf sample. The test tube was led to water bath (100 ◦C; 30 min)nd finally 4.5 mL of KOH 1 M added (Roukas, 1994). The reducingugars concentration was determined according to DNS methodMiller, 1959) measured at 540 nm (Hitachi, U-200) and expresseds glucose equivalent. Phenolic compounds concentration wasetermined according to Folin-Ciocalteau method measured at50 nm and expressed as gallic acid equivalent (Aggelis et al., 2003).ecolorization assay was performed according to Sayadi and Ellouz,992. The samples were 30-fold diluted, their pH was adjustedetween 6.0 and 6.3 and the absorbance was measured at 395 nm.

.4. Notation – units

X – biomass (g L−1); TS – total sugars (g L−1); EtOH – ethanolg L−1); YX/TS – biomass yield on total sugars consumed (g formed−1 sugars consumed); YEtOH/TS – ethanol yield on total sugars con-umed (g formed g−1 sugars consumed); YX/TS and YEtOH/TS are theield values of biomass and ethanol (respectively) produced peremaining total sugars. Subscripts 0, cons, f and max indicate thenitial, consumed, final and maximum quantity, respectively, of theermentation compounds in the kinetic experiments realized.

. Results

.1. Preliminary results – Effect of salts addition and mediumterilization on the growth of S. cerevisiae MAK-1 onolasses-based media

Initially the kinetic behavior of S. cerevisiae was evaluated onedia composed of molasses (without OMW addition – “blank”

xperiments; in these cultures TS0 was adjusted to ∼100 g L−1)n which addition of mineral compounds (see Section 2) hadeen done, with growth without supplementary salts addition.hake-flask experiments were performed, and before inocula-ion sterilization of the culture media had been performed. Theddition of salts into the medium affected negatively biomassXmax = 6.2 against 7.2 g L−1, YX/TS = 0.06 against 0.07 g g−1) andthanol (EtOHmax = 33.5 against 35.7 g L−1, YETOH/TS = 0.34 against.36 g g−1) production. Therefore no necessity of salts additionxisted, and, thus, these elements were not added at the trialshat followed. Previously sterilized (without salts addition) mix-ures of molasses and OMWs (10%, v/v; initial total phenolicst 3.9 ± 0.4 g L−1; TS0 ∼ 100 g L−1) were subjected to shake-flaskermentations and were compared with non-aseptic shake-flask

ultures containing the same initial quantities of phenolic com-ounds and TS (Fig. 1a and b). At the trials performed underseptic conditions, ethanol values were slightly higher compared toon-aseptic trials (EtOHmax = 37.1 against 34.3 g L−1, YETOH/TS = 0.44

d Products 56 (2014) 83–93 85

against 0.40 g g−1), while also the substrate was consumed earlierin the former case. This was attributed to the presence of bacteria(rods) grown together with the yeast strain at the early fermenta-tion stages. Bacteria apparently did not perform glucose breakdowntoward ethanol biosynthesis, and, thus, presence of bacteria (at thefirst fermentation steps), seemed to slightly reduce ethanol produc-tion, as compared with the aseptic trial. On the other hand, bacterialpopulation was almost completely eliminated with the subsequentrise in the concentration of ethanol, as the fermentation proceeded.It must be stressed that DCW production (by means of both Xmax

and YX/TS values) was enhanced at the non-previously sterilizedfermentations compared with the aseptic trials (Xmax = 7.3 against5.8 g L−1, YX/TS = 0.08 against 0.07 g g−1). The results with the trialunder non-aseptic conditions were considered as satisfactory, and,therefore, it was decided to proceed with the following trials withno previous sterilization of the medium.

3.2. Biomass and ethanol production by S. cerevisiae MAK-1cultivated non-aseptically in blends of molasses and OMWs

In the shake-flask trials (TS0 ∼ 100 g L−1; OMWs added in variousamounts) performed under non-aseptic conditions, DCW produc-tion (expressed as Xmax and YX/TS) was not significantly reducedby the addition of phenolic compounds (OMW) into the dilutedmolasses-based media (Table 1). The highest Xmax and YX/TS valueswere presented at the control (without OMW addition) exper-iment (Table 1). The values of total sugars concentration thatremained unconsumed (TSf = 5.1–18.3 g L−1; ∼70 h after inocula-tion) rose proportionally to the addition of OMWs indicating thatthe increasing presence of inhibitors of the effluent into the dilutedmolasses-based media negatively affected the metabolism of thestrain. A representative kinetics is shown in Fig. 2a and b. Additionof OMWs at a ratio of 20% (v/v), gave satisfactory results as regardsthe production of both yeast biomass and ethanol in shake-flaskcultures of S. cerevisiae.

In the next step, it was desirable to perform batch bioreactor tri-als and to compare the results with the respective ones performedin shake-flask cultures (comparative trials with TS0 = 100 g L−1 withOMWs added to a ratio of 20%, v/v). In this step, the effect of aerationor no-aeration on the bioreactor experiments upon the microbialmetabolism was also identified. Thus, in the bioreactor experi-ments, either constant aeration (1.2 vvm) or no aeration (0 vvm)throughout the fermentation was imposed. The obtained resultsare depicted in Fig. 3a–c. When non-aerated and aerated bioreac-tor experiments were compared, biomass production showed someslight increase in the latter case. In any case, though, as regards bothbioreactor trials, biomass production was clearly reduced by meansof DCW values achieved when compared with the respective shake-flask experiment (Fig. 3a). Moreover, DCW evolution reached at itsplateau earlier in bioreactor (∼42 h) than in flask cultures (∼52 h).In the shake-flask fermentations (and in less extent in the biore-actor fermentations performed under aeration), after glucose wasexhausted from the medium, some “diauxic growth” of S. cerevisiaewas observed (Matsakas and Christakopoulos, 2013a, 2013b; Piskuret al., 2006; Pyun et al., 1989; Zhang et al., 1994) and the strainconsumed the previously accumulated into the growth mediumethanol toward the formation of new cell material. On the otherhand, in all trials, glucose was consumed within 52 h after inocula-tion with relatively comparable consumption rates (Fig. 3b), sincevirtually the microbial metabolism was directed toward the accu-mulation of ethanol into the culture medium in spite of the fact thatoxygen (in shake-flask and aerated batch-bioreactor trials) existed

into the medium (this is the so-called “Crabtree” or “glucose”effect – see: Ratledge, 1991). Moreover, comparing non-aeratedand aerated bioreactor trials, it was seen that ethanol productionslightly increased by means of EtOHmax values in the trial under no

86 D. Sarris et al. / Industrial Crops and Products 56 (2014) 83–93

0

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Fig. 1. Total sugars (TS, g L−1), ethanol (EtOH, g L−1) (a) and biomass (X, g L−1) (b) evolution during growth of Saccharomyces cerevisiae MAK-1 on blends of diluted molasses andO onditii of tw

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MWs (10%, v/v; initial phenolic compounds concentration 3.9 ± 0.4 g L−1). Culture cnitial pH = 3.5 ± 0.1, incubation temperature T = 28 ◦C. Each point is the mean value

eration imposed (Fig. 3c). Comparing both bioreactor trials

aeration 1.2 vvm and 0.0 vvm) with the respective non-sterilehake-flask experiment, it was seen that ethanol biosynthesis waslearly favored in the reactor trials by means of EtOHmax valuesather than in the shake-flask experiment (Fig. 3c), suggesting a

able 1uantitative data of Saccharomyces cerevisiae MAK-1 grown on blends of molasses and OMthanol (EtOH, g L−1) and consumed substrate (TScons, g L−1) concentrations at different fehen Xmax concentration was achieved. Fermentation time, conversion yield of biomassroduced per total sugars consumed (YEtOH/TS, g g−1) are presented for all points of the tri80 ± 5 rpm, TS0 ∼ 100 g L−1, initial pH = 3.5 ± 0.1, incubation temperature T = 28 ◦C. Each p

OMWs (%, v/v) Initial phenolics (g L−1) Fermentation time (h) X (g L−1)

0 2.6 ± 0.2 58a 6.7 ± 0.5

73b 8.0 ± 0.6

10 3.9 ± 0.3 53a,b 7.3 ± 0.6

20 4.5 ± 0.4 51a 6.4 ± 0.5

55b 6.7 ± 0.6

30 5.2 ± 0.4 55b 6.3 ± 0.5

56a 6.2 ± 0.5

40 5.5 ± 0.5 54b 6.4 ± 0.5

58a 6.0 ± 0.4

50 6.3 ± 0.5 70a,b 6.5 ± 0.5

ons: growth on 250-mL sterile and non-sterile flasks at 180 ± 5 rpm, TS0 ∼ 100 g L−1,o independent measurements.

clear positive effect of the scale-up of the process toward ethanol

production. The EtOHmax value was noted earlier in bioreactor(∼44–46 h) than in flask cultures (∼52 h).

By taking into consideration that in the non-aseptic biore-actor culture with no aeration imposed, slightly higher ethanol

Ws, with OMWs added at various amounts. Representations of biomass (X, g L−1),rmentation points of each trial: (a) when EtOHmax concentration was achieved; (b)

produced per total sugars consumed (YX/TS, g g−1) and conversion yield of ethanolals. Culture conditions: growth on 250-mL not previously sterilized shake flasks atoint is the mean value of two independent measurements.

EtOH (g L−1) TScons (g L−1) YX/TS (g g−1) YEtOH/TS (g g−1)

37.3 ± 4.0 84.9 ± 6.5 0.08 0.4433.7 ± 3.0 88.3 ± 7.0 0.09 0.3834.3 ± 3.0 86.7 ± 7.0 0.08 0.4033.9 ± 3.0 82.5 ± 6.0 0.08 0.4131.4 ± 2.5 84.7 ± 6.5 0.08 0.3730.2 ± 2.5 87.5 ± 7.0 0.07 0.3531.0 ± 2.5 87.6 ± 7.0 0.07 0.3526.8 ± 2.0 76.3 ± 5.0 0.08 0.3528.4 ± 2.5 80.9 ± 5.5 0.07 0.3524.2 ± 2.0 89.8 ± 7.0 0.07 0.27

D. Sarris et al. / Industrial Crops and Products 56 (2014) 83–93 87

Table 2Quantitative data of Saccharomyces cerevisiae MAK-1 grown on blends of molasses and OMWs (20%, v/v), with molasses added in various quantities. Representations ofbiomass (X, g L−1), ethanol (EtOH, g L−1) and consumed substrate (TScons, g L−1) concentrations. Fermentation time, conversion yield of biomass produced per total sugarsconsumed (YX/TS, g g−1) and conversion yield of ethanol produced per total sugars consumed (YEtOH/TS, g g−1) are presented. Representations are given when maximumethanol concentrations (EtOHmax, g L−1) was achieved. Culture conditions: growth on not previously sterilized batch bioreactor, 300 rpm, OMWs added at 20% (v/v), initialpH = 3.50 ± 0.02, incubation temperature T = 28 ◦C, no aeration imposed (except the trial presented in the last line). Each point is the mean value of two independentmeasurements.

TS0 (g L−1) Fermentation time (h) X (g L−1) EtOH (g L−1) TScons (g L−1) YX/TS (g g−1) YEtOH/TS (g g−1)

∼100 46 5.3 ± 0.3 44.4 ± 3.5 91.4 ± 7.0 0.06 0.49∼135 98 4.6 ± 0.3 52.4 ± 4.0 110.3 ± 8.0 0.04 0.48∼150 72 4.5 ± 0.3 50.6 ± 4.0 112.5 ± 8.0 0.04 0.45∼200 70 2.8 ± 0.2 n.d. 7.8 ± 1.5 0.36 0.00

∼100 (1.2 vvm) 44 5.7 ± 0.4 41.8 ± 3

n.d.: non detected.

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Fig. 2. Total sugars (TS, g L−1), ethanol (EtOH, g L−1) (a) and biomass (X, g L−1) (b) evo-lution during growth of Saccharomyces cerevisiae strain MAK-1 on blends of molassesand OMWs (50%, v/v; initial phenolic compounds concentration 6.3 ± 0.5 g L−1). Cul-tio

qouoiambalmT

a

ure conditions: growth on 250-mL non-sterile flasks at 180 ± 5 rpm, TS0 ∼ 100 g L−1,nitial pH = 3.5 ± 0.1, incubation temperature T = 28 ◦C. Each point is the mean valuef two independent measurements.

uantities were achieved, it was further desirable to study the effectn increasing addition of molasses upon the growth of the strainnder no aeration conditions in media with OMWs added to a ratiof 20% (v/v), therefore, molasses were added in increasing amountsn OMW-based media resulting in increasing TS0 concentrationss follows (in g L−1): ∼100, ∼135, ∼150 and ∼200 g L−1. Incre-ent of molasses quantity into the medium negatively affected

iomass production in terms of absolute values (g L−1) and sugarssimilation by the strain (Table 2), suggesting that high pheno-ic (principally melanoidin) content affected negatively the strain

etabolism. S. cerevisiae did not grow sufficiently at the trial withS0 ∼ 200 g L−1.

As far as ethanol biosynthesis was concerned, in non-septic shake-flask cultures (with TS0 ∼ 100 g L−1), EtOHmax and

.5 84.9 ± 6.5 0.07 0.49

YEtOH/TSmax values achieved were 37.3 g L−1 and 0.44 g g−1, dur-ing growth of the microorganism on the control experiment(Table 1). In all shake-flask trials, supplementation of OMWsinto the molasses medium reduced maximum ethanol produc-tion (EtOHmax = 24.2–37.3 g L−1; YEtOH/TS ∼ 0.27–0.44 g g−1) as thesetrials were compared with the control experiment. Moreover,EtOHmax values were noted earlier at the fermentations of themedia containing blends of molasses and OMWs comparing withthe control experiment (excluding trials with initial phenolic com-pounds concentration 5.5 ± 0.5 and 6.3 ± 0.5 g L−1) (Table 1). Innon-aseptic bioreactor batch cultures (in these media OMWs wereadded to a ratio of 20%, v/v) the increase of total sugars concentra-tion raised ethanol production up to the trial with TS0 ∼ 135 g L−1

(EtOHmax = 52.4 g L−1; YEtOH/TS = 0.48 g g−1). The (overall) maximumYEtOH/TS = 0.49 g g−1 (EtOHmax = 44.4 g L−1) was presented at the fer-mentation with TS0 ∼ 100 g L−1 (0.0 vvm). The fermentation withTS0 ∼ 200 g L−1 was accompanied by no ethanol detected into themedium (detection threshold of ethanol in the HPLC analysis of∼0.1 g L−1) suggesting again that the high phenolic and melanoidincontent negatively affected the metabolism of the strain.

3.3. Decolorization – removal of phenolic compounds

In the flask trials the overall maximum decolorization achievedwas 28–60%. The overall maximum removal of phenolic com-pounds from the culture medium was lower as compared withthe respective decolorization, ranging between 12 and 26% (w/w)(Table 3). The overall maximum removal of color and phenolic com-pounds was achieved at the trial with initial phenolic compounds6.3 ± 0.5 g L−1 (Table 3). In the bioreactor experiments, maximumdecolorization and maximum removal of phenolic compoundswere 54.4% (at TS0 ∼ 135 g L−1) and 27.6% w/w (at TS0 ∼ 100 g L−1;0.0 vvm) respectively. No color and phenol reduction was notedat reactor cultures with TS0 ∼ 200 g L−1 (Table 4). Comparing flaskand bioreactor cultures, one can conclude that removal of color andreduction of total phenolics from the culture medium presentedsimilar kinetic profiles. The evolution of color and phenolic com-pounds removal from the culture medium is shown in Fig. 4a and b.

4. Discussion

S. cerevisiae MAK-1, presented noticeable DCW production innon-aseptic shake-flask and batch bioreactor fermentations, whenvarious quantities of molasses and OMWs were mixed. Moderateremoval of phenolic compounds from the medium (up to ∼28%,w/w) and significant decolorization (up to 60%) were observed.In non-aseptic shake-flask trials (TS0 ∼ 100 g L−1), the addition

of OMWs into molasses-based media not significantly decreasedbiomass production when compared with the control experiment(no OMWs added). The values of total sugars concentration thatremained unconsumed (TSf = 5.1–18.3 g L−1) rose proportionally to

88 D. Sarris et al. / Industrial Crops and Products 56 (2014) 83–93

Table 3Quantitative data of Saccharomyces cerevisiae MAK-1 concerning removal of phenol compounds and color performed in media containing blends of molasses and OMWsadded at various initial concentrations, in which initial total sugars were at ∼100 g L−1. Representation of initial and final phenol compounds concentration in the culturemedium, phenol compounds removal (%, w/w) and color removal (%) from the medium. Cultures performed in not previously sterilized shake-flask experiments.

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

0 2.6 ± 0.3 1.7 ± 0.2 35.1 ± 3.0 35.9 ± 3.010 3.9 ± 0.4 3.2 ± 0.3 19.3 ± 1.5 52.7 ± 4.020 4.5 ± 0.4 3.3 ± 0.3 25.7 ± 2.0 49.6 ± 4.0

tcmsstdt

gmttcmvtilrtcsc

ecbsivmaioes2oniot

TQ(c

30 5.2 ± 0.4 4.4 ± 0.3

40 5.5 ± 0.5 4.9 ± 0.4

50 6.3 ± 0.5 4.6 ± 0.4

he added OMWs indicating that the presence of the inhibitingompounds of that effluent (e.g. the phenolic compounds) into theolasses-based media negatively affected the metabolism of the

train. As far as non-aseptic batch bioreactor cultures with con-tant OMW and increasing addition of molasses were concerned,he addition of molasses into the medium reduced biomass pro-uction. S. cerevisiae strain MAK-1 did not grow sufficiently at therial with TS0 ∼ 200 g L−1.

Ethanol was produced in non-negligible amounts even thoughrowth was performed under aerated conditions (concerning fer-entations in shake-flask and in bioreactor under aeration). This is

he “Crabtree” effect, observed in several yeast genera. In the “Crab-ree” effect several enzymes involved in the oxidative part of theellular metabolism are subjected to catabolite repression, with theicrobial metabolism being shifted toward the synthesis of ethanol

ia fermentative conversion, in spite of the significant oxygen quan-ities found into the culture medium (Ratledge, 1991). However,t should be pointed out that S. cerevisiae cells need oxygen (ateast during the first hours after inoculation). Oxygen-dependenteactions indispensable for the yeast metabolism have as resulthe biosynthesis of sterols, unsaturated fatty acids, etc. that areompounds the biosynthesis of which is a sine qua non prerequi-ite for the formation of the plasma membrane and, thus, for theontinuation of cell proliferation for S. cerevisiae (Ratledge, 1991).

After sugar exhaustion from the medium, previously producedthanol was re-consumed, and new cell material was created (prin-ipally in the shake-flask experiments and in less extent in theioreactor trials under aeration–Fig. 3a–c). In this so-called “diauxichift” or “biphasic growth”, the microorganism’s growth is dividednto two phases. At the first growth phase assimilation of glucoseia aerobic fermentation with ethanol and carbon dioxide as theajor products occurs (Pyun et al., 1989). When ethanol is avail-

ble but virtually the concentration of sugars is substantially lownto the culture medium whereas simultaneously the dissolvedxygen concentration is above a critical level, previously producedthanol is now the sole available carbon source and serves as aubstrate for further yeast growth (Matsakas and Christakopoulos,013a, 2013b; Pyun et al., 1989; Zhang et al., 1994). At this point,nset of biosynthesis of the enzymes responsible for the gluco-

eogenesis occurs and this takes some time and causes a lag

n the yeast growth (Zhang et al., 1994). This phenomenon wasbserved in the present study as shown in Fig. 3a. Accordingo Piskur et al., 2006 the metabolic profile in Crabtree-positive

able 4uantitative data of Saccharomyces cerevisiae MAK-1 concerning removal of phenol com

added at ratio of 20%, v/v), with molasses added in various amounts. Representation ofompounds removal (%, w/w) and color removal (%) from the medium. Cultures performe

TS0 (g L−1) Initial phenolics (g L−1) Final phenolics (g L

∼100 4.7 ± 0.4 3.4 ± 0.3

∼135 5.6 ± 0.5 4.7 ± 0.4

∼150 6.1 ± 0.5 5.2 ± 0.4

∼200 7.8 ± 1.0 7.8 ± 1.0

∼100 (1.2 vvm) 4.7 ± 0.4 3.5 ± 0.3

15.0 ± 1.0 28.4 ± 2.512.1 ± 1.0 46.9 ± 3.526.2 ± 2.0 59.9 ± 4.5

yeast strains changes after exhaustion of glucose and accumulationof ethanol, with the requirement of certain transcription factorsand enzymes. The (ethanol) “make-accumulate-consume” strat-egy (Fig. 5) is relied on the evolution of Saccharomyces against itscompetitors as ethanol is toxic to most other microbes. It is con-sidered therefore than in a (non-aseptic) sugar-rich environment,Saccharomyces strains eliminate their competitors by producingethanol, but in a next fermentation step they consume the pre-viously generated ethanol. Alcohol dehydrogenase (Adh) catalyzesthe acetaldehyde-to-ethanol conversion (during aerobic or anaer-obic fermentation) in both directions. Genes ADH1 (expressedconstitutively) and ADH2 (expressed only when the internal sugarconcentration drops) encode cytoplasmic Adh activity.

S. cerevisiae MAK-1 has also been previously used in simul-taneous remediation-detoxification and bioethanol productionprocess; this strain was cultured in shake-flask experiments onsugar-enriched pasteurized grape must in which the fungicidequinoxyfen had been added in various concentrations. Non-negligible amounts of microbial mass had been synthesized(∼10.0 g L−1) regardless the addition of fungicide into the medium.Ethanol was accumulated into the culture medium in very high con-centrations (∼106–119 g L−1) whereas the fungicide was efficientlyremoved from the medium to a rate of ∼79–82% (w/w) (Sarriset al., 2009). Significantly higher initial carbohydrates amountswere employed (initial sugars quantities at ∼260 g L−1) as com-pared with the present study. The medium that was used (grapemust enriched with glucose and fructose) was considered as idealfor growth and bioethanol production of S. cerevisiae MAK-1. Thus,it appears that not high initial total sugars concentration but poten-tially high melanoidin (and, in essence, high phenolic compounds)content negatively affected the strain’s metabolism in the presentstudy. Finally, comparing non-aseptic bioreactor with shake-flaskfermentations, biomass production was reduced in reactor culturesby means of both Xmax and YX/TS values.

There are various reports in literature suggesting the useof bacteria (Kumar and Chandra, 2006), yeasts (Tondee et al.,2008) and fungal strains (Miranda et al., 1996) employed fordecolorization of molasses. For instance, the fungi Cunning-hamella echinulata and Mortierella isabellina were cultivated on

molasses, and growth was accompanied by non-negligible sub-strate decolorization, reaching up to ∼75% for C. echinulata and∼20% for M. isabellina (Chatzifragkou et al., 2010). Moreover,Metsoviti et al., 2011 used waste molasses as growth medium for

pounds and color performed in media containing blends of molasses and OMWs initial and final phenol compounds concentration in the culture medium, phenold in not previously sterilized batch bioreactor experiments.

−1) Phenol removal (%, w/w) Color removal (%)

27.6 ± 2.0 48.7 ± 4.016.4 ± 1.0 54.4 ± 4.015.2 ± 1.0 51.6 ± 4.0

0.0 0.0

26.5 ± 2.0 51.1 ± 4.0

D. Sarris et al. / Industrial Crops and Products 56 (2014) 83–93 89

0

1

2

3

4

5

6

7

7248240

Bio

mass

(X

, g/L

)

Time (h)

X (g/L) Flasks

X (g/L) Bioreactor (0.0 vvm)

X (g/L) Bioreactor (1.2 vvm)

(a)

0

20

40

60

80

100

7248240

Tota

l S

ugars

(T

.S., g

/L)

Time (h)

T.S. (g/L) Flasks

T.S. (g/L) Bioreactor (0.0 vvm)

T.S. (g/L) Bioreactor (1.2 vvm)

(b)

(c)

0

10

20

30

40

50

7248240

Eth

an

ol

(T

.S., g

/L)

Time (h)

EtOH (g/L) Flasks

EtOH (g/L) Bioreactor (0.0 vvm)

EtOH (g/L) Bioreactor (1.2 vvm)

Fig. 3. Biomass (X, g L−1) (a), total sugars (TS, g L−1) (b) and ethanol (EtOH,g L−1) (c) evolution during growth of Saccharomyces cerevisiae MAK-1 undernon-aseptic conditions on blends of molasses and OMWs (20%, v/v). Cultureconditions: 250-mL flasks at 180 ± 5 rpm, initial phenolic compounds concentra-tion 4.5 ± 0.4 g L−1, TS0 ∼ 100 g L−1, initial pH = 3.5 ± 0.1, incubation temperatureT = 28 ◦C; batch bioreactor at 300 rpm, initial phenolic compounds concentration∼4.7 g L−1, TS0 ∼ 100 g L−1, initial pH = 3.50 ± 0.02, incubation temperature T = 28 ◦C,um

Loppercu

0

5

10

15

20

25

30

35

7248240

Ph

enoli

c co

mp

ou

nd

s re

moval

(% w

/w)

Time (h)

Phenolic compounds removal -Flasks (% w/w)

Phenolic compounds removal -Bioreactor (% w/w)

(a)

0

10

20

30

40

50

60

7248240

Colo

r re

moval

(%)

Time (h)

FlasksColor removal (%) -

BioreactorColor removal (%) -

(b)

Fig. 4. Phenolic compounds (% w/w) (a) and color (%) (b) removal during growthof Saccharomyces cerevisiae MAK-1 on blends of molasses and OMWs (20%, v/v)under non-aseptic conditions, in 250-mL shake-flask (initial phenolic compounds

et al., 2007) to break down phenolic compounds. These enzymes

nder aeration (1.2 vvm) and no aeration (0.0 vvm) conditions. Each point is theean value of two independent measurements.

euconostoc mesenteroides so as to produce bacteriocin. Removalf color up to ∼27% of this residue was performed. As far as theotential of yeast strains to reduce the color and to decrease thehenolic content in OMW-based media is concerned, confusionxists in the literature indicating that in several cases, phenol

emoval from yeasts appears to be a bioprocess that could beharacterized as “strain-dependent”. For instance, the same strainsed in the present study, S. cerevisiae MAK-1, was utilized to

concentration 4.5 ± 0.4 g L−1; TS0 ∼ 100 g L−1) and batch bioreactor (initial phe-nolic compounds concentration 4.7 ± 0.4 g L−1; TS0 ∼ 100 g L−1; 0.0 vvm) cultures.Medium and culture conditions as detailed in Fig. 3.

simultaneously convert glucose-enriched OMWs into ethanol andyeast biomass and perform detoxification (removal of color up to∼63% and removal of phenolic compounds up to ∼34%, w/w) of theemployed residue, under aseptic and non-aseptic flask and biore-actor trials (Sarris et al., 2013). Bambalov et al., 1989 used OMWscontaining ∼8 g L−1 of phenolic compounds to cultivate various Sac-charomyces, Torulopsis, Kloeckera and Schizosaccharomyces strains.None of the strains presented microbial growth, apparently dueto the significantly high initial phenolic compounds concentrationinto the culture media, whereas, on the other hand, five amongst thetested strains presented microbial mass production only in 3-folddiluted media (presenting total phenolic compounds concentra-tions much lower than in the current submission). A summary offindings for the detoxification of OMW media by various microor-ganisms, including the current study is given in Table 5.

In the literature, a number of reports exists dealing withthe capability of molds (Aggelis et al., 2003; Crognale et al.,2006; Massadeh and Modallal, 2008; Sayadi and Ellouz, 1992;Tsioulpas et al., 2002) and bacteria (Ammar et al., 2004; Hachichaet al., 2009; Lamia and Moktar, 2003; Tsioulpas et al., 2002;Tziotzios et al., 2007) to efficiently reduce the phenolic com-pounds content in OMW-based media. Higher fungi have thepotential to secrete extra-cellular ligninolytic enzymes (oxidases)like laccase, lignin peroxidase and manganese dependent (orindependent) peroxidase (Aggelis et al., 2003; Crognale et al.,2006; Lamia and Moktar, 2003; Tsioulpas et al., 2002; Tziotzios

cannot be found in wild S. cerevisiae strains. A possible expla-nation for the reduction of the phenolic compounds duringcultivation of S. cerevisiae on OMW/molasses blends could be

90 D. Sarris et al. / Industrial Crops and Products 56 (2014) 83–93

Fig. 5. Glucose and ethanol assimilation by Saccharomyces cerevisiae under aerobic conditions. The conversion between acetaldehyde and ethanol is catalyzed by alcoholdehydrogenase (Adh). Gene ADH1 is expressed constitutively, whereas gene ADH2 is expressed only when the intra-cellular sugar concentration drops.

Adapted by Piskur et al., 2006.

TDp

able 5etoxification (decolorization and reduction of phenolic compounds) of OMW-based mearison with the present study.

Microorganism OMW-media Detoxification

Phanerochaete chrysosporium Diluted OMW(20%)

50% decolorizatioPleurotus ostreatus 50% decolorizatioGeotrichum candidum Fresh diluted OMW 75% decolorizatioTrichosporon cutaneum OMW ethyl-acetate

extracts85% (w/w) pheno

Geotrichum candidum OMW/cheese whey(20:80)

55% decolorizatio55% phenol remo

Lentinula edodes (Le119) Diluted OMW (10% &20%, v/v)

65% decolorizatio75% phenol remo

Zygomycetes strains Diluted OMW (0–50%,v/v)

48–60% phenol r

Lactobacillus plantarum Fresh OMW 58% decolorizatio46% phenol remo

Lactobacillus paracasei OMW/cheese whey(10:90)

47% decolorizatio23% phenol remo

Yarrowia lipolytica(ATCC 20255)

OMW enriched witholive oil

–

Candida tropicalis(YMEC 14)

OMW/hexadecaneblends

55% monophenol69% polyphenols

Yarrowia lipolytica strains Undiluted OMW 18% (w/w) pheno

Yarrowia lipolytica(ACA-DC 50109)

Diluted OMW glucoseenriched

36% decolorizatio15% (w/w) pheno

Indigenous yeasts OMW (no nutritionalsupplement)

44% (w/w) pheno

Candida cylindracea(NRRL Y-17506)

Diluted OMW enrichedwith olive oil and salts

36% (w/w) pheno

Yarrowia lipolytica strains Diluted OMW glucoseenriched

63% decolorizatio34% (w/w) pheno

Saccharomyces cerevisiaeMAK-1

Diluted OMW glucoseenriched

63% decolorizatio34% (w/w) pheno

Saccharomyces cerevisiaeMAK-1

OMW/molasses 60% decolorizatio28% (w/w) pheno

dia and production of compounds (if occurred) by various microorganisms; com-

Products Reference

n (6 days) – Kissi et al. (2001)n (12 days) –n – Assas et al. (2002)l removal Biomass Chtourou et al., 2004

nval

Biomass Aouidi et al., 2010

nval

Biomass Lakhtar et al., 2010

emoval Microbial oil Bellou et al., 2014

nval

Lactic acid Lamia and Moktar,2003

nval

– Aouidi et al., 2009

BiomassLipase

Scioli and Vollaro, 1997De Felice et al. (1997)

s removal

– Ettayebi et al. (2003)

l removal Citric acidLipase

Lanciotti et al., 2005

nl removal

BiomassCitrate

Papanikolaou et al.,2008

l removal Biomass Ben Sassi et al., 2008

l removal BiomassLipase

DAnnibale et al.,2006DAnnibale et al.,2006

nl removal

BiomassCitrateMicrobial oil

Sarris et al., 2011

nl removal

BiomassEthanol

Sarris et al., 2013

nl removal

BiomassEthanol

Present study

D. Sarris et al. / Industrial Crops and Products 56 (2014) 83–93 91

Table 6Strains producing ethanol from various carbon sources and their comparison with the current investigation.

Strain Carbon source Concentration (g L−1) EtOH (g L−1) Reference

Saccharomyces cerevisiae Bakers’ yeast Carob pod 200–350 ∼62 Roukas, 1994Bakers’ yeast Molasses 150–300 53.0 Roukas, 1996aBakers’ yeast Sucrose 220 96.7 Caylak and Vardar Sukan,

199627817 Glucose 50–200 5.1–91.8 Vallet et al., 1996L-041 Molasses & sucrose

blends∼125 25.0–50.0 Pinal et al., 1997

ATCC 24860 Molasses 2–50 5.0–18.4 Ergun and Mutlu, 2000CMI237 Sugar 160 70.0 Navarro et al., 2000S. cerevisiae sp. Molasses – 44.3 Nahvi et al., 2002Fiso Galactose 20–150 4.8–40.0 da Cruz et al., 2003A3 4.8–36.8L52 2.4–32.0GCB-K5 Sucrose 30 27.0 Kiran et al., 2003GCA-II 42.0KR18 22.52.399 Glucose 32 13.7 Yu and Zhang, 200424,860 Glucose 150 48.0 Ghasem et al., 2004NCYC 1119 Molasses 100 40.0 Baptista et al., 2006AXAZ-1 Molasses ∼216 71.3 Kopsahelis et al., 2007ATCC 26602 Flour hydrolysates 150 76 Wang et al., 2007S. cerevisiae sp. & K.marxianus blends

Henequen juice &molasses blends

∼215 41.2 Cáceres-Farfán et al., 2008

MAK-1 Grape must 250 106.4–119.2 Sarris et al., 2009MAK-1 OMW/glucose 115 52.0 Sarris et al., 2013

Zymomonas mobilis MAK-1 OMW/molasses 135 52.4 Present studyZM4 & ZMI2 Glucose 100–200 47.6–78.0 Sreekumar and Basappa,

1991ATCC 29191 Hydrolyzed starch 120 (glucose) 50.0 Weuster-Botz et al., 1993NS-7 Glucose 150 73.2 Tao et al., 2005ZM4 Hydrolyzed starch 80–110 39–54 Davis et al., 2006

awaitln

utaa2cc2Ketsea2bptTDBbsYa

streamNRRL-B-14023 Glucose

ATCC 29191 Sucrose

ttributed to a physical mechanism involving the creation ofeak interactions, between anthocyanins and yeast walls by

dsorption (Rizzo et al., 2006). Thus, phenolic removal observedn the current investigation could be due to simple adsorption ofhese compounds in the yeast cell surface. Moreover, partial uti-ization of phenolic compounds as carbon and energy source couldot be excluded (Chtourou et al., 2004).

In the international literature, several reports indicate these of various microorganisms grown on molasses for the syn-hesis of added value metabolites such as gluconic acid, citriccid, fructo-oligosaccharides, pullulan, succinic acid, single cell oilnd erythromycin (Chatzifragkou et al., 2010; El-Enshasy et al.,008; Liu et al., 2008; Roukas, 1996a; Sharma et al., 2008). Con-erning ethanol production from S. cerevisiae strains, in manyases molasses were used as growth medium (Baptista et al.,006; Cáceres-Farfán et al., 2008; Caylak and Vardar Sukan, 1996;opsahelis et al., 2007; Nahvi et al., 2002; Pinal et al., 1997; Wangt al., 2007). A summary of findings concerning bioethanol produc-ion is given in Table 6. Besides S. cerevisiae strains, the bacterialpecies Zymomonas mobilis is also used (Cazetta et al., 2007; Davist al., 2006; Lin and Tanaka, 2006; Ruanglek et al., 2006; Sancheznd Demain, 2008; Sreekumar and Basappa, 1991; Wang et al.,007Table 6). Z. mobilis uses the Entner-Doudoroff pathway toreak down glucose, resulting in less biomass production, com-ared with the alcoholic fermentation performed by S. cerevisiaehat uses the Embden-Meyerhof-Parnas (EMP) pathway (Lin andanaka, 2006; Ratledge, 1991; Ruanglek et al., 2006; Sanchez andemain, 2008; Sreekumar and Basappa, 1991; Wang et al., 2007).iomass production by Z. mobilis strains is almost twofold less,

ut the ethanol yield on sugar assimilated for both microbialources used is comparable (maximum theoretical ethanol yieldEtOH/TS = 0.51 g g−1 for both microorganisms used; see: Sancheznd Demain, 2008). Therefore, although the conversion performed

100 40–55 Ruanglek et al., 2006200 55.8 Cazetta et al., 2007

by Z. mobilis strains can, in some cases, have as result slightly highervolumetric productivities achieved compared with the fermenta-tion performed by yeasts (Lin and Tanaka, 2006), cultures led by S.cerevisiae attract interest due to the (higher) concentration of theprocess by-product (biomass) which can be utilized as animal feed.

A viable perspective bioethanol production at industrial-leveloperations refers to the potential of carrying out the biopro-cess under non-aseptic conditions. Remarkably lower energyconsumption attributed to the absence of sterilization processprovides an important advantage of this production approach.Roukas, 1996a has proposed ethanol production using non-previously sterilized beet molasses as microbial substrate by bothfree and immobilized S. cerevisiae cells in fed-batch operations(EtOH = 53 g L−1; YEtOH/TS = 0.31 g g−1). Moreover, Roukas, 1994performed experiments using free and immobilized cells of S.cerevisiae on non-sterile carob pod extract, equally in fed-batchbioprocesses (EtOHmax = 62 g L−1; TS0 = 300 g L−1, F = 167 mL h−1).Roukas, 1996b also introduced continuous ethanol productionfrom non-sterile carob pod extract by immobilized S. cerevisiaeon mineral kissiris using a two-reactor system (average ethanolproductivity 10.7 g L−1 h−1, ethanol yield as % of theoretical ∼72,and sugar utilization 48%). Kopsahelis et al., 2012 have proposed acontinuous ethanol production process performed in a multistagefixed bed tower bioreactor, in which trials with immobilized S.cerevisiae strain AXAZ-1 were performed with waste molasses(at TS0 = 115 g L−1) utilized as substrate. Trials were performedunder both aseptic and non-aseptic conditions and ethanol pro-duction was almost completely unaffected by the non-asepticconditions employed (ethanol up to 51.4 g L−1 with conversion

yield YEtOH/TS ∼ 0.47 g g−1 was reported for the non-sterile trial)(Kopsahelis et al., 2007). Additionally, the mutant strain of Z.mobilis NS-7 has been cultivated on non-sterile glucose media.Maximum ethanol concentration of 73 g L−1 was achieved with the

9 ops an

kiweo39p

ataYmqapwcDwtahlciriOcosr

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2 D. Sarris et al. / Industrial Cr

inetic profiles of glucose consumption and ethanol accumulationnto the culture medium being almost equivalent compared

ith the sterile media (Tao et al., 2005). Finally Weuster-Botzt al., 1993 have developed a continuous fluidized bed reactorperation system for ethanol production by Z. mobilis strain ATCC1821 using hydrolyzed starch without sterilization. The unsterile,9% hydrolyzed, starch conversion resulted in 50 g L−1 ethanolroduction.

OMWs can be considered as a promising tap water substitutend substrate for the biotechnological production of ethanol (inhis study EtOHmax = 52.4 g L−1 and YEtOH/TSmax = 0.48 g g−1 in non-septic batch bioreactor experiments; EtOHmax = 37.3 g L−1 andEtOH/TSmax = 0.44 g g−1 in non-sterile shake-flask cultures). The fer-entation performed in the lab-scale bioreactor, resulted in higher

uantities of ethanol produced, in terms of both absolute and rel-tive values (see also Tables 1 and 2). Given that in some of theerformed bioreactor experiments the conversion yield YEtOH/TSas very close to the maximum theoretical one for this biopro-

ess (Lin and Tanaka, 2006; Ruanglek et al., 2006; Sanchez andemain, 2008; Sreekumar and Basappa, 1991; Wang et al., 2007),hile non-aseptic conditions were employed, secures the fact that

he bioreactor’s dimensions and specifically the aspect ratio aredequate for a process scale-up in a full-scale reactor. On the otherand, in various bioprocesses including production of ethanol uti-

ization of raw OMWs originated from press extraction systemsould be used without supplementary addition of molasses or othernexpensive sugars and thus the cost of the process could be furthereduced (Sarris et al., 2011) (for a state-of-the-art review deal-ng with the production of several valuable metabolites throughMW biotechnological valorization, see: Crognale et al., 2006). S.erevisiae MAK-1, as stated above, has been previously cultivatedn glucose-enriched OMWs under sterile and completely non-terile shake-flask and bioreactor trials. The EtOHmax concentrationeported was of 52.0 g L−1 (YEtOH/TS value = 0.46 g g−1).

Bambalov et al., 1989 reported EtOHmax production of0.8–11.7 g L−1 (YEtOH/TS = 0.38–0.41 g g−1) in media presentingignificantly lower total phenolic compounds compared withhe current study. Zanichelli et al., 2007 indicated that it wasompletely necessary to remove a significant portion of thehenolic fraction of the OMWs through an efficient enzymaticre-treatment, before inoculating with a S. cerevisiae strain inrder to produce bioethanol. After pre-treatment and dilution,he initial phenolic compounds concentration was 2.1 g L−1 andlucose supplementation of the medium was realized (TS0 upo 200 g L−1), with total fermentation time being 65 days andthanol production reaching 63–95 g L−1. Massadeh and Modallal,008 proposed pre-treatment of the OMWs used, with Pleurotusajor-caju in order to sufficiently remove the phenolic com-ounds. Following the pretreatment, 50% diluted and sterilizedMWs were inoculated with a S. cerevisiae strain, and a maxi-um ethanol production of 14.2 g L−1 after 48 h of fermentationas reported (Massadeh and Modallal, 2008). There are some

eports in the literature indicating the use of blends of OMWsith other residues in order to enhance metabolite’s production

r so as to improve the decolorization of the residue. Specifi-ally, Aouidi et al., 2009 used a Lactobacillus paracasei strain tomprove the fermentative decolorization of OMW mixed withheese whey in various proportions resulting in a highest coloremoval of 47% and phenol content removal of 23%. Moreover,ouidi et al., 2010 performed investigations related with biomassroduction and OMW decolorization, using Geotrichum candidum inedia supplemented with cheese whey. Mycelial biomass produc-

ion of ∼9.3 g L−1 was reported, while equally the strain presented decolorization efficiency of 54.5% and a reduction of pheno-ic compounds of 55.3%. To the best of our knowledge, literatureresents no studies at all suggesting the use of mixtures of molasses

d Products 56 (2014) 83–93

and OMWs for the production of ethanol through alcoholicfermentation.

Besides the simultaneous presence of molasses and OMWs assubstrate and the growth of the strain used in completely non-sterile conditions, we also note that the sole external addition ofnutrients was that of yeast extract and (NH4)2SO4, while the pheno-lic content of the used OMW was much higher (i.e. ∼10 g L−1) thanthe typical values found in the literature (i.e. ∼2–4 g L−1), indicatingthat OMWs can partly or even completely substitute tap water inbioethanol fermentation in which molasses are usually used as car-bon substrates, without significant negative effect in the performedbioprocess. S. cerevisiae MAK-1, thus, is a satisfactory candidate forbioremediation of molasses and OMWs blends and simultaneousproduction of added-value metabolites.

Acknowledgments

The current investigation has been partly funded by the projectentitled “Microbial conversions of agro-industrial residues into bio-fuels and other metabolites of biotechnological interest” (BilateralS&T collaboration between Greece and Hungary, financed by GSRT-Ministry of Higher Education and Religious Affairs).

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