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Process Biochemistry 48 (2013) 1279–1284

Contents lists available at ScienceDirect

Process Biochemistry

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

accharomyces cerevisiae and Oenococcus oeni immobilized in differentayers of a cellulose/starch gel composite for simultaneous alcoholicnd malolactic wine fermentations

oannis Servetasa, Carmen Berbegalb, Nathalia Camachoc, Argyro Bekatoroua,ergi Ferrerb, Poonam Nigamc, Chryssoula Drouzad, Athanasios A. Koutinasa,∗

Food Biotechnology Group, Department of Chemistry, University of Patras, Patras 26500, GreeceENOLAB Laboratory of Enologic Microbiology, Department of Microbiology and Ecology, University of Valencia, Burjassot, Valencia 46100, SpainSchool of Biomedical Sciences, University of Ulster at Coleraine, Coleraine BT52 1SA, Northern Ireland, UKDepartment of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, Lemesos 3603, Cyprus

r t i c l e i n f o

rticle history:eceived 11 March 2013eceived in revised form 11 June 2013ccepted 16 June 2013vailable online 24 June 2013

eywords:

a b s t r a c t

The production of a two-layer composite biocatalyst for immobilization of two different microorganismsfor simultaneous alcoholic and malolactic fermentation (MLF) of wine in the same bioreactor is reported.The biocatalyst consisted of a tubular delignified cellulosic material (DCM) with entrapped Oenococcusoeni cells, covered with starch gel containing the alcohol resistant and cryotolerant strain Saccharomycescerevisiae AXAZ-1. The biocatalyst was found effective for simultaneous low temperature alcoholic fer-mentation resulting to conversion of malic acid to lactic acid in 5 days at 10 ◦C. Improvement of wine

omposite biocatalystmmobilizationenococcus oeniaccharomyces cerevisiae

inealolactic fermentation

quality compared with wine fermented with S. cerevisiae AXAZ-1 immobilized on DCM was attributedto MLF as well as to increased ester formation and lower higher alcohols produced at low fermentationtemperatures (10 ◦C) as shown by GC and headspace SPME GC/MS analysis. Scanning electron microscopyshowed that the preparation of a three-layer composite biocatalyst is also possible. The significance ofsuch composite biocatalysts is the feasibility of two or three bioprocesses in the same bioreactor, thusreducing production cost in the food industry

. Introduction

Co-immobilization of different microorganisms in order toimultaneously conduct different bioprocesses into the same biore-ctor could lead to reduction of both production and investmentosts. However, the biological competition of yeasts and lactic acidacteria (LAB) may cause inhibition problems [1]. Separate entrap-ent of cells in different matrices to form a composite multispecies

iocatalyst could help avoid such problems, and this is investi-ated in the present study regarding simultaneous alcoholic andalolactic fermentations (MLF) of grape must. Many carriers for

mmobilization of yeasts and bacteria have been proposed for wineaking, including inorganic and organic materials, mainly polysac-

harides [2–4], and natural products such as cellulosic materials5,6], cereal grains [7], pieces of fruit [8–11], starch [12], etc. Delig-

ified cellulosic materials (DCM) have been used as immobilizationupports of yeasts [5] and bacteria [13,14] for wine making, demon-trating their promotional effect on alcoholic fermentation and MLF

∗ Corresponding author. Tel.: +30 2610997104; fax: +30 2610997105.E-mail address: a.a.koutinas@upatras.gr (A.A. Koutinas).

359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.procbio.2013.06.020

© 2013 Elsevier Ltd. All rights reserved.

and reduction of the activation energy (Ea) even at very low temper-atures. DCM is a material of food-grade purity, cheap and abundantin nature, and the immobilization method is simple and easy. Fur-thermore, it was recently demonstrated that DCM contains nano-and micro-tubes facilitating extremely low temperature fermen-tation and other bioprocesses [15]. Moreover, other biomaterials,such as starch gel were found interesting for yeast entrapment [12]for use in wine fermentation.

The use of immobilized Oenococcus oeni immobilized on DCMled to improvements in developing MLF in wine making [14]. There-fore, the use of a biocatalyst by co-immobilization of Saccharomycescerevisiae and O. oeni on separate layers of a composite food gradecarrier to provide additional advantages in wine making is inves-tigated. Such improvements could be the increased cell viabilities,better resistance to low pH and ethanol, higher cell densities, simul-taneous alcoholic fermentation and MLF in the same bioreactor,less time for vinification, lower cost, and last but most important,improvement of wine quality. Therefore, the aim of this study was

to develop a composite two-layer biocatalyst consisting of wheatstarch gel and tubular DCM, carrying immobilized S. cerevisiae andO. oeni cells, respectively, and evaluate its suitability for wine mak-ing, to influence both productivity and wine quality.

1 ochemistry 48 (2013) 1279–1284

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280 I. Servetas et al. / Process Bi

. Materials and methods

.1. Microorganisms and media

The strain S. cerevisiae AXAZ-1, isolated from Greek grapes [16], was used in theresent study for the alcoholic fermentation of must, and the strain O. oeni ENOLAB003 isolated from Spanish wine was used for MLF. S. cerevisiae was grown on cultureedium consisting of (g/L): yeast extract 4, (NH4)2SO4 1, KH2PO4 1, MgSO4·7H2O

, and glucose 40. O. oeni was grown on Medium for Leuconostoc oenos (MLO) broth17]. The cells were harvested by centrifugation. All media were sterilized by auto-laving for 15 min. The grape must used contained 160 g/L fermentable sugar, 3 g/L-malic acid and had pH 3.5.

.2. Preparation of the two-layer composite support and cell immobilization

O. oeni was immobilized on DCM, which was prepared from wood sawdust afterreatment with 1% NaOH and boiling for 3 h for lignin removal [5]. For the immobi-ization procedure, 40 g of DCM, 200 mL of MLF medium and 4 g of harvested O. oeniells were placed in a 250 ml conical flask and the mixture was allowed to fermentvernight at room temperature. The liquid was then decanted and the biocatalystDCM with fixed O. oeni cells) was dried at 35 ◦C for 48 h. On the other hand, S. cere-isiae was firstly entrapped in wheat starch gel as follows: 4 g of starch were mixedhoroughly with 50 mL of deionized water, heated to 90 ◦C and then left to cool atoom temperature. Then 5 mL of nutrient medium containing 5 g of harvested S.erevisiae AXAZ-1 were mixed with the prepared gel. Finally, the starch gel/S. cere-isiae biocatalyst was mixed with a specific amount of the DCM/O. oeni biocatalystntil the gel was fully adsorbed on DCM. The composite material was then incubatedt 30 ◦C for 24 h and then dried at 35 ◦C for 48 h. The dried composite biocatalyst washen used for alcoholic fermentation and MLF of grape must.

.3. Scanning electron microscopy

The DCM/O. oeni, starch gel/S. cerevisiae as well as their composite biocatalystere examined by scanning electron microscopy (SEM). They were coated with gold

n a Balzers SCD 004 sputter coater for 2 min and then examined by JSM-6300 SEMicroscope.

.4. Fermentations

An amount of 0.3 g of the composite biocatalyst was added into 50 mL of grapeust and fermentation experiments were carried out at 28 ◦C without agitation.

amples were collected for analysis at various time intervals until the fermentationeased. All experiments were carried out in triplicate (indicated as A, B, C in Table 1)nd 3 fermentation batches were conducted for each separate experiment.

.5. Ethanol and methanol determination

Must and wine samples were analyzed for ethanol and methanol by gas chro-atography (GC) on a Shimadzu Gas Chromatograph GC-8A (Kyoto-Japan) with

C-R6A Chromatopack integrator, a Porapac-S column (2 m) and nitrogen as car-ier gas at a flow rate of 40 mL/min. The injection port and FID detector were sett 210 ◦C and the column was programmed to rise from 140 to 180 ◦C at a rate of0 ◦C/min. Calculations were done by means of standard curves, using standard solu-ions containing 4, 8 and 12%v/v of ethanol and 79, 395 and 948 mg/L of methanol,espectively.

.6. Determination of volatile compounds

Major volatile compounds found in must and wine such as acetaldehyde, ethylcetate, propanol-1, isobutanol and amyl alcohols, were analyzed on a ShimadzuC-8A system, carrying a 80/120 Carbopack B AW/6.6% Carbowax 20 m column, set

o rise from 70 to 100 ◦C at a rate of 5 ◦C/min, and a FID detector. Samples of 4 �Lere injected directly into the column. Quantitative determination was done using

standard solution containing (�L/L): acetaldehyde 10, isobutanol 10, propanol-10, ethyl acetate 10, and 3-methyl-1-butanol 10. The results are shown in Table 1s means plus standard deviations of at least 2 repetitions.

Minor aroma volatile compounds were determined by headspace solid-phaseicro-extraction (SPME) GC/MS. In a 20 mL headspace glass vial 3 g of sodium chlo-

ide (POCH S.A., Polland), 10 mL of sample and 3 �L of 4-methyl-2-pentanol (asnternal standard) were added. The vial was sealed with an aluminum crimp sealined with a rubber septum, and it was placed in a water bath at 60 ◦C. The samples

ere heated for 5 min and then the fiber PDMS–DVB (Supelco U.S.A.) was exposedo the headspace of the vial to absorb volatiles for 45 min. Analysis was performedn a Shimadzu GC-17A system connected to a MS QP5050A mass spectrometer. A

apillary Supelco CO Wax-10 column (60 m, 0.32 mm i.d., 0.25 �m film thickness)as used. Helium was used as carrier gas at a flow rate of 1.8 mL/min. Temperature

f the column oven was set at 35 ◦C for 6 min, then it was raised to 60 ◦C at a rate of◦C/min and was held at this temperature for 5 min, then raised to 200 ◦C at a ratef 5 ◦C/min and finally to 250 ◦C at a rate of 25 ◦C/min where it was maintained for Ta

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ochemistry 48 (2013) 1279–1284 1281

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Fig. 1. SEM micrographs of the DCM/starch gel composite biocatalyst. (a) Tube struc-tures of DCM. (b) O. oeni cells attached in the internal surface DCM tubes. (c) Yeast

I. Servetas et al. / Process Bi

min. The injection port and interface temperatures were 240 ◦C. The mass spec-rometer was operated in a scan range of 2945–4000 m/z. The fiber was exposed tohe injector port for 3 min and the samples were analyzed for a total 59.50 min. Atandard of n-alkanes (C6–C24) was used to determine the Kovats’ Retention Indicesf the volatiles found in the samples. Kovats’ Indices, NIST107, NIST21 and SZTERPibraries were used to identify the compounds (with confidence level above 70%).emi-quantitative analysis was performed by comparison of the area of the inter-al standard and the areas of the peaks of the volatile compounds identified in theamples.

.7. HPLC analysis of residual sugar, lactic and malic acids

Residual sugar, lactic acid and malic acid were determined by HPLC on an Agilenteries 1200 system with an isocratic pump (Agilent G1310A), an Agilent G1322Aegasser, an Agilent G1367B autosampler, and an Aminex HPX-87H precolumn (Bio-ad) coupled with two ion-exclusion columns of 300 mm by 7.8 mm Aminex HPX-7H (Bio-Rad) thermostatically controlled at 65 ◦C. Compounds were detected by aariable-wavelength Agilent G1314B detector set to 210 nm and an Agilent G1362Aefractive index detector in series. The mobile phase consisted of 0.75 mL of an 85%olution of H3PO4 per liter of deionized water, and it was pumped at a flow rate of.7 mL/min. External calibration was performed.

.8. Statistical analysis

The effect of temperature on volatile by-products formation, as well as onarameters such as residual sugar, glycerol, pH, total acidity and volatile acidityas studied. All analyses were performed in triplicate except for volatile acidity

nd lactic acid. Data were analyzed by Statistical software SPSS PASW Statistics 18.0or MAC OS X. Data that complied with homogeneity and normality were assayed byNOVA and significant differences were tested with p < 0.05. Differences betweenairs of groups were tested in Tukey. Data that didn’t comply with these assump-ions were analyzed with the non-parametric Kruskal–Wallis test using InfoStat010, Grupo InfoStat, FCA, Universidad Nacional de Córdova, Argentina.

. Results and discussion

.1. Biocatalyst production

The rationale of this investigation was to produce a compos-te biocatalyst consisting of two different microorganisms, O. oenind S. cerevisiae, entrapped in delignified tubular cellulose andtarch gel, respectively, aiming to avoid species competition andead to improvements in wine making. O. oeni was initially immo-ilized on porous DCM and then starch gel containing entrapped. cerevisiae cells was added until all the gel was adsorbed inhe tubes of cellulose. The composite biocatalyst was thermallyried (for potential lower cost commercial production compared toreeze–drying) and was applied for simultaneous alcoholic fermen-ation and MLF in wine making. Volatiles formation was particularlytudied as they substantially affect the quality of wine. The pro-osed composite biocatalyst consisted of two food grade purityatural polymers, which have previously been found suitable asell immobilization carriers to promote alcoholic fermentation pro-esses even at very low temperatures to improve product quality.specially, low-temperature fermentation in wine making (below5 ◦C) is recognized as a valuable tool to improve flavor, which isttributed to the improved ratios of off-flavor compounds (suchs higher alcohols) to desirable compounds (such as short-chainatty acid esters) on total volatiles produced during fermentation2,5,12–14,18]. Good operational stability of extremely low tem-erature fermentation processes can be facilitated by the use ofsychrophilic or psychrotolerant yeasts combined with cell immo-ilization techniques and suitable bioreactor design [2,19]. Thebility of these species to survive and grow at low temperaturess due to cold-induced properties, such as altering the membraneipid composition and accumulation of carbohydrates and proteinsnvolved in various cellular functions to overcome barriers such

s reduced enzyme activity, decreased membrane fluidity, alteredetabolism, decreased growth rates, protein denaturation, etc.

19]. Alterations in cell growth, physiology and metabolic activ-ty may also be induced by cell immobilization due to various

cells entrapped in starch gel covering the internal surface of a DCM tube.

factors such as mass transfer limitations, surface tension andosmotic pressure effects, reduced water activity, cell-to-cell com-munication, changes in the cell morphology and growth patterns,altered membrane permeability, and media components availabil-ity [2].

Cell immobilization on two different layers of the proposed bio-catalyst was confirmed by SEM showing a series of tubes of DCMwith attached O. oeni cells (Fig. 1a and b). Fig. 1c shows a tube cov-ered with the starch gel with entrapped yeast cells forming thesecond layer. Therefore, alcoholic fermentation and MLF are car-

ried out separately in the same medium and bioreactor, withoutdirect interaction between the two different species.

1282 I. Servetas et al. / Process Biochem

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ig. 2. Lactic acid formation and malic acid consumption during wine fermentationt 10 ◦C using the composite DCM/starch gel biocatalyst.

.2. Fermentations

An effect of fermentation temperature was observed in ethanolnd lactic acid contents, these being inversely related (Table 1). Anmount of 6 g/L of the dry composite biocatalyst corresponded to8 g/L of wet weight biocatalyst and 1.1 g/L wet cells concentration.s shown in Fig. 2, the use of this amount of the biocatalyst for wineaking at 10 ◦C resulted to conversion of malic acid to lactic acid

n 5 days, while glucose and fructose were consumed in twice thatime. Therefore, MLF was completed halfway of the fermentation ofrape must. Likewise, as illustrated in Fig. 3, at 10 ◦C the biocatalystermented a total sugar concentration of 208.4 ± 24.7 g/L in 22 daysielding 10 alcoholic degrees. The ethanol and lactic acid contentst 15 and 25 ◦C had no significant differences (p > 0.05). Therefore,ermentation at low temperatures is ensured even when a relativelyow amount of the composite biocatalyst is used thus affectingroduction cost as previously shown for DCM [5] and starch gel12].

.3. Wine quality

The quality of wine plays a substantial role in the evaluationf a wine making technology, therefore, analysis of volatile by-roducts which define wine flavor, was performed. Specifically,he major volatiles such as acetaldehyde, propanol-1, isobutanolnd amyl alcohols were analyzed by GC-FID, glycerol by HPLC, andinor esters, alcohols, carbonyl compounds and acids in the prod-

ct headspace were determined by SPME GC–MS. Table 1 showshe concentrations of residual sugar, ethanol, glycerol, pH and

ajor volatiles in wines produced using the composite biocata-yst at various temperatures. Regarding wine acidity, an increase

ig. 3. Ethanol and glycerol formation and sugar consumption during wine fermen-ation at 10 ◦C and using the composite DCM/starch gel biocatalyst.

istry 48 (2013) 1279–1284

of pH was observed as expected after malolactic fermentation [13]from 3.5 ± 0.0 in must before fermantation to a maximum valueof 3.8 ± 0.1 in wine fermented at 10 ◦C. Many aspects influencethe synthesis of glycerol among which temperature. As previouslyshowed in the work of Balli et al. [20] the glycerol content reducesas temperature decreases, as also observed in this study. Specifi-cally, glycerol produced by fermentation at 25 ◦C was 50% higherthan that found at 15 ◦C and 10 ◦C. Regarding acetaldehyde, thehighest amounts of acetaldehyde were obtained at 15 ◦C and thelowest at 10 ◦C, which was also observed in previous similar studiesregarding wine making with immobilized cells [12]. Amyl alcoholswere reduced by about 100% at 15 ◦C and 60% at 10 ◦C. Therefore,the use of the composite biocatalyst caused a bigger reduction inthe formation of these compounds compared to the DCM and starchgel biocatalysts separately [12,21]. In a similar manner, isobutanolwas also reduced as the fermentation temperature decreased (e.g.at 25 ◦C it was about 3.5 fold higher than that at 10 ◦C). However,no changes (p > 0.05) were observed in the amounts of propanol-1formed by fermentation at temperatures down to 15 ◦C, while at10 ◦C it was reduced by about 30%. The ethyl acetate formed by fer-mentation at 15 ◦C was also significantly higher (p > 0.05) comparedwith 25 ◦C and slightly increased at 10 ◦C.

Table 2 shows the results of the headspace SPME GC–MS anal-ysis of the produced wines. The total ester concentrations wereincreased as the fermentation temperature was reduced (60%increase at 15 ◦C and 130% at 10 ◦C compared with 25 ◦C). In con-trast to esters, the total concentrations of alcohols were reducedas the fermentation temperature was reduced as observed in thecase of the major alcohol concentrations (Table 1). This reductionwas about 30% at 15 ◦C and 60% for 10 ◦C compared with 25 ◦C.The reduction of total alcohol concentrations in wine making wasalso higher compared with DCM and starch gel biocatalysts sep-arately [12,21]. The improved ratio of alcohols to esters is knownto improve the quality of wine and other beverages fermented atlow temperatures [2]. Esters mainly provide fruity and floral aro-mas, which were specifically observed in wines fermented at 10 ◦Cwhere the highest concentrations of esters were obtained (Table 2).Volatile organic acids, from hexanoic to tetradecanoic, were foundin wine in much higher concentrations compared with grape must,and were reduced significantly to a total 14.7 mg/L at 10 ◦C. Finally,no changes were observed in either the concentrations or numbersof carbonyl compounds identified as the fermentation temperaturedecreased.

3.4. Research and technological perspectives

The production of the DCM/starch gel composite biocatalystmakes the immobilization of two different microorganisms indifferent layers of the material possible, therefore avoiding com-petition problems. The results showed successful simultaneousalcoholic and MLF fermentations in the same bioreactor. The tubu-lar DCM/starch gel composite is of food grade purity and thereforeis suitable for wine making applications. The biocatalyst was driedby a simple thermal drying technique, retaining its viability andfermentation efficiency, which can lead to reduction of commer-cial production cost compared with freeze-drying. This compositebiocatalyst can be therefore manufactured and distributed as driedwine yeast, with advantages such as the (i) lower production costdue to the drying technique used, (ii) lower yeast cell densityrequired, (iii) possibility to co-immobilize yeast and lactic acidbacteria without competition problems, (iv) and ability to conductboth alcoholic and MLF fermentations in the same bioreactor. The

composite biocatalyst could also be used for many other types offood bioprocess development, e.g. for the simultaneous productionof ethanol, lactic acid and citric acid. Due to the fact that the internalsurface of the DCM tubes was covered by the starch gel (forming a

I. Servetas et al. / Process Biochemistry 48 (2013) 1279–1284 1283

Table 2SPME GC–MS analysis (semiquantitative) of minor headspace volatile compounds wines produced using the composite biocatalyst at various temperatures.

KI KI in literature Compound Concentration (mg/L)

Must 25 ◦C 15 ◦C 10 ◦C

Esters934 <800 (31) Ethyl acetate – 6.05 4.58 9.50

1039 1055 (30) Ethyl-2-methylbutanoate 13.81 52.20 52.28 69.121046 925 (20) Ethyl formate – 0.01 0.15 0.011084 1040 (20) Ethyl butanoate 0.90 0.73 0.66 4.531112 1071 (30) Ethyl isovalerate 2.37 7.37 8.22 10.541140 1132 (28) Isoamyl acetate - 3.07 4.22 3.651175 1101 (30) Methyl hexanoate 9.25 2.06 30.45 43.471334 1386 (20) Ethyl lactate – 0.29 – 1.521399 1430 (30) Ethyl octanoate – 0.85 1.87 1.861605 1652 (20) Ethyl decanoate – 0.59 1.42 1.611647 1678 (30) Diethyl succinate – 0.03 – –1661 1691 (30) Ethyl 9-decenoate – 0.23 1.08 0.191794 1811 (30) 2-Phenylethyl acetate – 2.67 19.58 35.071822 1850 (20) Ethyl dodecanoate – 0.89 3.32 1.872038 2049 (30) Ethyl tetradecanoate – 0.61 0.72 0.26

>2200 2255 (30) Ethyl hexadecanoate 0.21 1.27 0.68 0.26Total esters (mg/L) 26.54 78.90 129.22 183.47

Organic acids1244 1260 (31) Lactic acid – 3.29 3.44 3.481465 1452 (28) Acetic acid – 0.06 0.08 0.211812 1885 (30) Hexanoic acid – 0.04 0.80 0.032069 2087 (30) Octanoic acid 0.23 2.14 6.06 1.992171 1213 (20) Nonanoic acid 0.66 2.42 1.43 0.60

>2200 2336 (20) Decanoic acid 0.60 8.25 12.06 5.78>2200 >2600 (30) Tetradecanoic acid 0.14 4.91 7.92 2.63

Total organic acids (mg/L) 1.62 21.12 31.78 14.71

Alcohols1051 1108 (28) 2-Methylpropanol – 13.62 9.58 13.201063 1120 (31) 3-Methyl-1-butanol 0.17 1.56 0.81 1.341114 1174 (30) 1-Penten-3-ol 0.35 0.80 1.46 0.751229 1179 (20) 1-Butanol – 3.90 8.54 7.161239 1210 (30) Isoamyl alcohol – 111.84 62.19 19.001295 1312 (20) 4-Penten-1-ol 1.30 0.09 0.08 0.091313 1350 (20) 3-Methyl-1-pentanol – 0.06 – –1361 1375 (30) 3-Ethoxy-1-propanol – 0.30 0.32 0.111460 1478 (30) 2-Ehtylhexen-1-ol 0.04 0.00 0.68 0.221513 1559 (20) 2,3 Butanediol – 8.13 2.69 1.241548 1590 (20) 1,3 Butanediol – 3.53 1.46 0.491555 1512 (20) 2-Undecanol 0.03 0.61 0.321632 1680 (20) 2-Furanmethanol – 0.51 – 0.361690 1730 (20) 3-(Methylthio)-1-propanol – 1.20 0.56 0.261895 1933 (20) 2-Phenylethanol 0.06 16.89 20.63 16.771944 1998 (20) 1-Dodecanol 0.20 0.33 0.49 0.52

>2200 2296 (28) 2,4-Bis(1,1-dimethylethyl)phenol 0.87 8.14 – 3.38Total alcohols (mg/L) 3.02 1371.15 887.36 509.00Total alcohols (ethanol excluded) (mg/L) 3.02 163.36 109.81 61.50

Carbonyls1115 1091 (31) 3-Methyl butanal – 0.75 0.83 0.131156 1213 (32) 4-Methyl-2-heptanone 2.03 5.92 6.65 9.061161 1213 (32) 4-Methyl-2-heptanone 0.06

1486 (20) Furfural 7.35 0.28 0.021991 2024 (28) Gama-Nonalactone 0.28 0.35 0.49 0.52Total carbonyls (mg/L) 9.66 7.08 8.25 9.74

fibosFlSggbb

lm) leaving available empty spaces, a third biopolymer could alsoe used to fill the gaps, giving the possibility for co-immobilizationf three different microorganisms in different layers of the sameupport, or for co-immobilization of microorganisms and enzymes.or example, a composite tubular DCM/PHB/starch gel biocata-yst could be prepared, carrying different microorganisms such asaccharomyces, Rhizopus, Aspergillus sp. etc., for the production of

luconic acid, citric acid and ethanol etc., or oleaginous microor-anisms, yeast and A. niger to produce simultaneously biodiesel,ioethanol and citric acid in the same bioreactor thus reducingiofuel production and investment costs.

4. Conclusions

Tubular DCM and starch gel produced an effective compositebiocatalyst for co-immobilization of two different microorganisms(S. cerevisiae and O. oeni), for simultaneous alcoholic and MLFfermentations of grape must in the same bioreactor. The microor-ganisms were located in different layers, resulting to simultaneous

performance of the two bioprocesses without any obvious biologi-cal competition among the species. The DCM/starch gel compositebiocatalyst led to improvement of wine quality, regarding the for-mation of volatile by-products, compared with the performance of

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CM and starch gel biocatalysts separately as shown in previoustudies.

cknowledgements

This work was supported by the project CENIT-2008 1002, Vegles scholarship of the University of Valencia and Agrovin S.A.

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