Biotechnological Production of Phenyllactic Acid and Biosurfactants from Trimming Vine Shoot Hydrolyzates by Microbial Coculture Fermentation

Biotechnological Production of PhenyllacticAcid and Biosurfactants from Trimming Vine ShootHydrolyzates by Microbial Coculture Fermentation

Noelia Rodríguez-Pazo & José Manuel Salgado &

Sandra Cortés-Diéguez & José Manuel Domínguez

Received: 13 July 2012 /Accepted: 30 January 2013 /Published online: 16 February 2013# Springer Science+Business Media New York 2013

Abstract Coculture fermentations show advantages for producing food additives fromagroindustrial wastes, considering that different specified microbial strains are combinedto improve the consumption of mixed sugars obtained by hydrolysis. This technologydovetails with both the growing interest of consumers towards the use of natural foodadditives and with stricter legislations and concern in developed countries towards themanagement of wastes. The use of this technology allows valorization of both cellulosicand hemicellulosic fractions of trimming vine shoots for the production of lactic acid (LA),phenyllactic acid (PLA), and biosurfactants (BS). This work compares the study of thepotential of hemicellulosic and cellulosic fractions of trimming vine shoots as cheaper andrenewable carbon sources for PLA and BS production by independent or coculture fermen-tations. The highest LA and PLA concentrations, 43.0 g/L and 1.58 mM, respectively, wereobtained after 144 h during the fermentation of hemicellulosic sugars and simultaneoussaccharification and fermentation (SSF) carried out by cocultures of Lactobacillus planta-rum and Lactobacillus pentosus. Additionally, cell-bond BS decreased the surface tension(ST) in 17.2 U; meanwhile, cell-free supernatants (CFS) showed antimicrobial activityagainst Salmonella enterica and Listeria monocytogenes with inhibition halos of 12.1±0.6 mm and 11.5±0.9 mm, respectively.

Keywords Coculture .Trimmingvinewastes . Phenyllactic acid .Biosurfactants . Lactic acid .

Lactic acid bacteria

Appl Biochem Biotechnol (2013) 169:2175–2188DOI 10.1007/s12010-013-0126-1

N. Rodríguez-Pazo : J. M. Salgado : S. Cortés-Diéguez : J. M. DomínguezAgro-Food Biotechnology Laboratory, CITI-Research, Transfer and Innovation Centre,Parque Tecnológico de Galicia, San Cibrao das Viñas 32900 Ourense, Spain

N. Rodríguez-Pazo : J. M. Salgado : S. Cortés-Diéguez : J. M. Domínguez (*)Chemical Engineering Department, Sciences Faculty, University of Vigo (Ourense Campus),As Lagoas s/n, 32004 Ourense, Spaine-mail: [email protected]

Introduction

Coculture fermentations have been defined by Bader et al. [1] as the combination ofincubation and metabolic activity of different specified microbial strains under asepticconditions. This technology offers advantages for production of food additives, pharma-ceuticals, fuels, antimicrobial substances, enzymes, and bulk and fine chemicals, since itmay result in increased yield and improved control of product quality. Additionally, itmakes possible the use of worthless waste materials, a critical factor in industrialbiotechnology because carbon source is often the greatest contributor to the cost ofmicrobial products [2].

Among these solid wastes, some authors have proposed the use of trimming vineshoots as a source of renewable sugars after a number of thermochemical and biochem-ical processing steps to convert the polymers to monomeric sugars to be furtherconverted by fermentative microorganisms into ethanol, xylitol, lactic acid (LA), andbiosurfactants (BS), among other substances [3]. These studies were carried out evalu-ating independently the cellulosic and hemicellulosic fractions. Thus, in the first step,the hemicellulosic sugar hydrolyzates obtained from trimming vine were assayed toproduce LA and biosurfactants by Lactobacillus pentosus strains [4–6]; meanwhile, thesolid residue obtained after the delignification of trimming vines shoots was employedin a second step to produce LA by simultaneous saccharification and fermentation (SSF)by Lactobacillus rhamnosus [7].

Food and feed spoiling microorganisms cause great economic losses worldwide,being necessary to develop efficient biopreservatives. In this way, LA bacteria (LAB)can be applied at an industrial scale for the production of bioactive molecules such asorganic acids, phenolic compounds, fatty acids, hydrogen peroxide, diacetyl bacteriocins,and BS with antimicrobial activity against organisms and pathogens, thus offering apotential alternative to the use of synthetic preservatives [8]. The coculture of LABallows the valorization of both cellulosic and hemicellulosic fractions of trimming vineshoots for the production of biopreservatives such as LA, with wide use in foodindustry, mainly as acidifying, coagulant, and preservative agent against several patho-genic bacteria, fungi, and yeast species [9]; phenyllactic acid (PLA), a novel antimi-crobial compound produced through the phenylalanine degradation metabolism that isactive against Gram-(+), Gram-(−) bacteria, yeast, and fungi [10–13]; and BS, amphi-philic compounds of microbial origin with a pronounced surface activity and withseveral advantages over chemical surfactants including lower toxicity, higher biodegrad-ability, and effectiveness at extreme temperatures or pH, being potential candidates formany commercial applications in the biomedical, petroleum, and food processing indus-tries [9, 14–16].

The main purpose of this work was to develop a profitable technology for the use ofboth hemicellulosic and cellulosic fractions of trimming wastes by coculture of Lacto-bacillus plantarum and L. pentosus. Different bioactive compounds such as LA, PLA,and BS were analyzed in the exhausted culture media. This is, in our knowledge, thefirst report about the simultaneous use of both fractions, which can simplify theoperational conditions at an industrial scale considering that different sugars are metab-olized simultaneously in one bioreactor, thus making the overall process more compet-itive and controllable. The antimicrobial activity of cell-bond BS and cell-freesupernatants (CFS) was also assayed against the pathogenic bacteria Salmonella entericaand Listeria monocytogenes.

2176 Appl Biochem Biotechnol (2013) 169:2175–2188

Materials and Methods

Reagents

DL-3-PLA (product code P7251) was purchased from Sigma-Aldrich (Milan, Italy), while DL-phenylalanine (product code 78040) was provided by Fluka (Sigma-Aldrich, Buchs, Switzer-land). Ultrapure water was produced using a Millipore Milli-Q System (Millipore, Bedford,MA, USA). Methanol high-performance liquid chromatographic (HPLC) grade and trifluoro-acetic acid (TFA, 99.8 %) were supplied by Panreac Química SAU (Barcelona, Spain). Formicacid 98 %v/v (product code 131030) and acetic acid (product code 122703.1611) from PanreacQuímica SAU CULTIMED (Barcelona, Spain) as well as D(+) xylose (product code 95729),D(+) glucose (product code 49139), D(−) arabinose (product code A3131), 5-hydroxymethyl-2-furaldehyde (product code H40807), and 2-furaldehyde (product code 48070) were bought toSigma-Aldrich, (St. Louis, MO, USA) for HPLC analysis using H2SO4 (product code131058.1211) as mobile phase from Panreac Química SAU. Glycerol for maintenance ofstrains in cryovials and the Man–Rogosa–Sharpe (MRS) broth were obtained from PanreacQuímica SAU.

Raw Material and Analysis

Trimming vine shoots were locally collected during the campaign of 2011. These wasteswere dried, milled to a particle size less than 1 mm, homogenized in a single lot to avoidcompositional differences, and stored until use. Aliquots from the homogenized lot weresubmitted to moisture determination and quantitative hydrolysis in a two-stage acid treat-ment according to Vázquez et al. [17]. The first stage used sulfuric acid (72 % weight) at30 °C during 1 h. The second step started after sulfuric acid dilution to 4 % weightfollowed by treatment at 121 °C during another hour. The resulting solid was consid-ered as Klason lignin; meanwhile, hydrolyzates were analyzed by HPLC as describedbelow. This method allowed determination of glucose, xylose, arabinose, acetic acid,furfural, and hydroxymethylfurfural.

Acid Hydrolysis (Prehydrolysis) and Enzymatic Activity of Enzymes for SimultaneousSaccharification and Fermentation

Hydrolyzates of trimming wastes were obtained according to the conditions reported byBustos et al. [4] in an autoclave at 130 °C with 3 % sulfuric acid solutions during15 min using a liquid/solid ratio of 8 g/g. Hydrolyzates were cooled, filtered through0.45-μm membranes, and analyzed by HPLC, providing hemicellulosic hydrolyzates;meanwhile, the solid residue was treated with 8 % (w/w) NaOH solution at 130 °C for120 min using a liquid/solid ratio of 10 g/g to a delignification treatment to obtain twoother phases: a solid residue with cellulosic sugars and a liquid phase with solublelignin. The cellulosic fraction obtained in this second step was employed for SSFexperiments, employing two commercial enzyme concentrates “Celluclast” and “Novo-zym 188,” with cellulase and β-glucosidase activities, respectively. Both enzymes werekindly provided by Novozymes (Denmark). The cellulase activity of concentrates wasassayed according to previous reports [18] by the filter paper activity (FPA) test andexpressed as filter paper units (FPU) per milliliter; meanwhile, the β-glucosidaseactivity was measured according to Paquot and Thonart [19].

Appl Biochem Biotechnol (2013) 169:2175–2188 2177

Microorganisms and Fermentation Conditions

L. pentosus CECT-4023 and L. plantarum CECT-221 used in this study for coculture and S.enterica subsp. enterica CECT-724 and L. monocytogenes CECT-934 employed to evaluatethe antimicrobial activity of cell-free extracts and BS obtained after fermentation processagainst pathogenic bacteria were obtained from the Spanish Collection of Type Cultures(Valencia, Spain) and maintained in cryovials at −80 °C in MRS medium with 15 %v/vglycerol as cryoprotector. The MRS medium contains (per liter): 10 g peptone, 8 g beefextract, 4 g yeast extract, 20 g D-glucose, 2 g K2HPO4, 2 g diammonium hydrogen citrate,5 g CH3COONa, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·2H2O, and 1 g Tween 80. The samemedium was employed for seed activation.

Inocula were prepared by inoculating one glycerol stock vial into a 250-mL Erlenmeyerflask containing 100 mL of activation medium, followed by growth in an incubator shaker(Optic Ivymen System, Comecta S.A., distributed by Scharlab, Madrid, Spain) at 31.5 °Cand 100 rpm during 12 h. Two generations of activation cultures were required beforefermentation. Finally, cells were recovered and resuspended in the fresh broth medium forinoculation with a 5 % of the final culture volume. After inoculation, fermentations werecarried out in a 2-L Braun-Biostat fermenter (Braun, Melsungen AG, Melsungen, Germany)at 31.5 °C, with pH automatically controlled to 6.2 with 5 N NaOH.

Hemicellulosic sugars supplemented with MRS nutrients (except glucose) and 0.6 g/L Phewere fermented by L. plantarum (Fermentation 1A) or applying coculture of L. plantarum andL. pentosus, adding the second microorganism after 20-h fermentation (Fermentation 1B) at100 rpm with a final working volume medium of 1,800 mL. SSF was conducted using 30 g/gliquid/solid ratio:30 g of distilled water per gram of cellulosic fraction by L. plantarum(Fermentation 2) or 30 g of hemicellulosic sugars (Fermentation 3) per gram of cellulosicfraction by coculture of L. plantarum and L. pentosus, supplemented, in both cases, with MRSnutrients (except glucose) and 0.6 g/L Phe and with a final working volume medium of1,500 mL. The agitation was fixed in 150 rpm to ensure complete mixing. Enzymes wereadded with a cellulose/substrate ratio of 28 FPU/g and cellobiase/cellulose ratio of 13 UI/FPU.

All media were sterilized at 101 °C for 60 min in an autoclave (Trade Raypa SL, Terrassa,Barcelona, Spain) prior to the experiments and inoculated with each producer strain in apercentage of 5 % of the final culture volume. Samples were taken at given fermentation timesand centrifuged at 2,755g, 10 °C for 15 min. Supernatants were filtered (0.22 μm pore-sizemembrane, Millipore) and employed for HPLC analysis; meanwhile, cells were used for biomassdetermination and BS extraction. Culture filtrates were produced in triplicate for each experiment.

Analytical Methods

Glucose, xylose, arabinose, ethanol, and lactic, formic, and acetic acid concentrations duringfermentations were measured by a HPLC system (Agilent, model 1200, Palo Alto, CA,USA) equipped with a refractive index detector and an Aminex HPX-87H ion exclusioncolumn (Bio-Rad 300×7.8 mm, 9 μm particles) with a guard column, eluted with 0.003 Msulfuric acid at a flow rate of 0.6 mL/min at 50 °C. PLA and Phe were measured by an HPLCsystem (Agilent, model 1200, Palo Alto, CA, USA) equipped with an Agilent Zorbax SB-AqC18 column (4.6×150 mm, 5 μm particles) with a guard column and using a UV detector.Linear gradient elution was used with methanol/0.05 % TFA (solvent A) and water/0.05 %TFA (solvent B) at 1 mL/min and A/B ratios of 10:90, 100:0, 100:0, and 10:90, with runtimes of 0, 20, 23, and 25 min, respectively. PLA and Phe were detected at 210 nm accordingto the procedure described by Valerio et al.[20].


Cell concentration in experiments was measured by dry cell weight. Cells of knownvolume of culture media were centrifuged at 2,755g, 10 °C for 15 min and washed twicewith distilled water and centrifuged under the same conditions. The resulting pellets wereoven-dried at 105 °C to constant weight.

Determination of Biosurfactants

The surface activity of BS produced by L. pentosus CECT-4023 and/or L. plantarum CECT-221 was determined by measuring the surface tension (ST) of samples using the Ring method[21] employing a KRÜSS Tensiometer (Hamburg, Germany) equipped with a 1.9-cmDuNoüyplatinum ring at room temperature. To obtain the samples for BS determination, cells wererecovered by centrifugation (10,000g, 15 min, 10 °C) from fermentation media, washed twicein demineralized water, resuspended in phosphate buffer saline (PBS: 10 mMKH2PO4/K2HPO4 and 150 mM NaCl with pH adjusted to 7.0) using a fermentation mediumvolume/PBS volume ratio of 6, incubated for 2 h at room temperature, and centrifuged torecover the PBS extract free of biomass. The BS concentration [BS] was calculated using thecalibration curve reported by Portilla-Rivera et al. [22]: [BS] (mg/L)=(ST (mN/m)−76.984) /−8.64658 that was obtained using surfactin at different concentrations below the critical micelleconcentration (CMC) with known values of ST.

Antimicrobial Activity

The antimicrobial activity of the CFS and BS extracts obtained from all different fermentationswas determined by the well diffusion technique using tryptocase soy broth (TSB) with 20% (w⁄v)agar as culture media, held at 45 °C, and inoculated with 100 μL of active culture of the targetorganism. About 25 mL of the seeded agar was poured into a sterile Petri dish and allowed tosolidify at room temperature. Wells were then cut in the solidified agar with a sterile metalcylinder and filled with 50 μL of each extract (CFS or BS extract). CFS were obtained fromcultures of producer organisms after centrifugation (2,755g, 15 min, 10 °C) and filtration using0.22-μm pore-size filter-sterilized membranes (Millipore); meanwhile, BS extracts were obtainedas it was described before. Agar plates were then kept for 4 h at 4 °C for diffusion of the test CFSand BS into the inoculated agar. Then, plates were incubated aerobically for 24 h at 37 °C using S.enterica or L.monocytogenes as indicator microorganisms. The inhibition zones around the wellswere examined for clearing around the wells, measured, and recorded after plate's incubation.

Results and Discussion

In order to study the feasibility of using the hemicellulosic and cellulosic fractions of vinetrimmings for the production of LA, PLA, and BS, different strategies were explored for thesolubilization of sugars and further fermentation.

The Use of Hemicellulosic Sugars Supplemented with 0.6 g/L of Commercial Phe (F1)

Trimming vine shoots were hydrolyzed as described previously [4] to release the hemicellulosicsugars under mild conditions. The average composition of the hemicellulosic liquors obtainedwas 16.1 g/L xylose, 11.7 g/L glucose, 1.6 g/L arabinose, 0.45 g/L acetic acid, 0.6 g/L furfural,and 0.1 g/L hydroxymethylfurfural. Neutralized hydrolyzates were supplemented with thenutrients of MRS broth, except glucose, and 0.6 g DL-Phe/L as precursor for the PLA


production by L. plantarum in fermenter after sterilization. This microorganismwas selected byRodríguez et al. [23] as the best PLA producer among different LAB. However, althoughglucose and arabinose were rapidly consumed by L. plantarum in the first few hours offermentation, the depletion of these sugars interrupted the consumption of xylose, and only5.2 g/L was consumed after 144-h fermentation to produce 14.5 g/L LA, corresponding to aglobal volumetric productivity of 0.101 g/Lh and a product yield of 0.77 g/g. Additionally,negligible amounts of acetic acid and formic acid were produced. Figure 1a shows the coursewith time for glucose, xylose, and arabinose consumption as well as LA and biomass formation;meanwhile, Table 1 provides additional information. The results indicated that although LAwasthe main metabolite of reaction, Phe was efficiently converted into PLA (see Fig. 1b) producingup to 1.32 mM with a product yield of 0.48 mM/mM.

In spite of the good results obtained in terms of PLA, the poor consumption of xyloseindicated that L. plantarum is a good PLA producer but is not suitable for metabolizingpentoses. In this way, Bustos et al. [4] reported that L. pentosus ferments glucose to LA byhomolactic fermentation (C6H12O6 → 2C3H6O3) but also xylose to LA and acetic acid byheterolactic fermentation (C5H10O5 → C3H6O3 + CH3COOH). Consequently, to increasethe consumption of xylose, L. pentosus was added after 20 h, and fermentation proceeded

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Fig. 1 Course with time for hem-icellulosic sugar consumption,lactic acid and biomass formation(a), and Phe to PLA biosynthesis(b) by L. plantarum. Unfilledcircle glucose, unfilled diamondxylose, unfilled triangle arabi-nose, asterisk biomass, filledcircle lactic acid, filled diamondPhe, filled triangle PLA. Standarddeviations were less than 2.1 %of the mean


until 144 h. During this period, xylose, although slowly, was completely metabolized(Fig. 2a); meanwhile, acetic acid concentration increased only by 5 g/L to achieve a finalconcentration of 18.9 g/L (Fig. 2b). However, conversely to the expected, acetic acid andformic acid were hardly produced. Compared with the fermentation in the absence of L.pentosus, the final LA concentration increased from 14.5 to 18.9 g/L and the globalvolumetric productivity increased from 0.101 to 0.131 g/Lh. The downside was the reduc-tion in the yield achieved, which decreased from 0.77 to 0.68 g/g. Contrarily, the concen-tration of PLA was not influenced, reaching similar values (1.27 mM), although the Phe toPLA yield increased slightly up to 0.82 mM/mM.

Table 1 Summary of fermentative parameters and surface tension at the beginning and the end of 144-h fermentation

F1A F1B F2 F3

A. Hemicellulosic sugars transformation into organic acids

Glucoset = 0 (g/L) 12.1 11.2 – –

Glucoset = 144 h (g/L) 0 0 0 0

Xyloset = 0 (g/L) 16.9 15.4 – –

Xyloset = 144 h (g/L) 11.7 0.4 – 0

Arabinoset = 0 (g/L) 1.59 1.7 – –

Arabinoset = 144 h (g/L) 0 0 – 0

Sugars consumed (g/L) 18.9 27.8 – –

Lactic acid produced (g/L) 14.5 18.9 19.5 36.4

Acetic acid produced (g/L) 0.1 0.23 2.3 5.1

Formic acid produced (g/L) 0.6 0 2.0 1.5

Organic acids produced (g/L) 15.2 19.2 23.8 43.0

QLA (g/Lh) 0.101 0.131 0.140 0.253

QS (g/Lh) 0.131 0.193 – –

YLA/S (g/g) 0.77 0.68 – –

YOA/S (g/g) 0.80 0.69 – –

B. Phe to PLA bioconversion

Phet = 0 (mM) 4.5 5.6 5.2 5.2

Phet = 144 h (mM) 1.8 4.0 4.0 2.7

PLA produced (mM) 1.32 1.27 1.11 1.58

YPLA/Phe (mM/mM) 0.48 0.82 0.95 0.63

C. Biosurfactants

STPBS (mN/m) – 68.5 73.9 72.3

STB (mN/m) – 54.2 61.9 55.1

STreduction (mN/m) – 14.3 12.0 17.2

FCMC – 3.4 2.0 1.3

[BS] (mg/L) – 5.6 2.7 2.6

F1A fermentation of hemicellulosic sugars by L. plantarum, F1B coculture of hemicellulosic sugars by L.plantarum and L. pentosus, F2 simultaneous saccharification and fermentation carried out by L. plantarum,F3 fermentation of hemicellulosic sugars and SSF carried out by cocultures of L. plantarum and L. pentosus,QLA global volumetric productivity of lactic acid, QS sugars (glucose, xylose, and arabinose) consumptionrate, YLA/S sugars (glucose, xylose, and arabinose) to lactic acid yield, YOA/S sugars (glucose, xylose, andarabinose) to organic acid (lactic acid, acetic acid, and formic acid) yield, STPBS surface tension of PBS at thebeginning of extraction, STB surface tension of PBS containing cell-bond biosurfactants, FCMC dilution ratioto achieve the CMC, [BS] total concentration of cell-bond biosurfactants


Simultaneous Saccharification and Fermentation of Cellulosic Fraction Carriedout by L. plantarum Supplemented with 0.6 g/L of Commercial Phe (F2)

Once the feasibility of fermenting hemicellulosic sugars by cocultures of L. plantarum andL. pentosus was demonstrated, experiments were carried out by simultaneous performanceof enzymatic hydrolysis and microbial conversion. Figure 3a shows the LA production andglucose generated and consumed during SSF of cellulosic residues. This technology allowedincreasing the LA concentration up to 19.5 g/L with a global volumetric productivity of0.140 g/Lh. Small amounts of acetic acid (2.3 g/L) and formic acid (2.0 g/L) were alsoquantified. During the process, 1.11 mM PLA was also achieved after 144-h fermentation,with a product yield of 0.95 mM/mM. Figure 3b depicts the course with time for Pheconsumption and PLA production.

Fermentation of Hemicellulosic Sugars and Simultaneous Saccharification and Fermentationof Cellulosic Fraction Carried Out by Cocultures of L. plantarum and L. pentosus (F3)

Once the use of single cultivation of microorganisms in liquors obtained from hemicellulosicand cellulosic fractions of vine shoots was studied, separately, the final step was the study ofa combined mixture of sugars by coculture of Lactobacilli strains. Under these conditions,

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Fig. 2 Course with time forhemicellulosic sugars consump-tion, lactic acid and biomassformation (a), and Phe to PLAbiosynthesis (b) by coculture ofL. plantarum and L. pentosus.Unfilled circle glucose, unfilleddiamond xylose, unfilled trianglearabinose, asterisk biomass, filledcircle lactic acid, filled diamondPhe, filled triangle PLA. Standarddeviations were less than 2.2 %of the mean


the sugars present in the hemicellulosic fraction as well as the glucose released from thecellulosic fraction were entirely consumed after 92 h of fermentation, as shown in Fig. 4a,yielding 36.4 g/L LA (with a global volumetric productivity of 0.253 g/Lh) and lowconcentrations, 1.5 g/L and 5.1 g/L, of formic acid and acetic acid, respectively, thus makinga final concentration of organic acids of 43.0 g/L. This increment was also observed duringthe biosynthesis of PLA, since the concentration increased up to 1.58 mM at the end offermentation (see Fig. 4b), with a product yield of 0.63 mM/mM.

Production of Biosurfactants

During the previous fermentations, the ability of both bacteria to produce extracellularand/or associated to cell membrane (cell-bond) BS was also analyzed by measuring thechanges in ST. A substance is considered BS if the ST of the media where it was added orproduced decreases by at least 8 U [24]. In all the conditions studied, there were nosignificant reductions in ST in culture broths at the end of fermentations, meaning that therewas no extracellular BS. Conversely, ST of PBS (the ST of PBS is approximately 72 mN/m)after cell-bond BS extraction decreased by more than 8 U (see Table 1), ranging between12.0 in Fermentation 2 and 17.2 mN/m in Fermentation 3 that is in concordance with the bestsugars to LA fermentation described upwards due to a synergic effect of both microorganisms.

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Fig. 3 Profiles for lactic acidformation (a) and Phe to PLAbiosynthesis (b) during the si-multaneous saccharification of thesolid residue obtained after pre-hydrolysis and fermentation byL. plantarum. Unfilled circle glu-cose, filled circle lactic acid,unfilled square formic acid, filledsquare acetic acid, filled diamondPhe, filled triangle PLA. Standarddeviations were less than 2.4 %of the mean


The production of BS with these microorganisms grown on various hydrolyzates was scarcelyinvestigated. For instance,Moldes et al. [25] reported similar values by using L. pentosus grownin hemicellulosic hydrolyzates obtained from different origins, reporting reduction valuesranging from 16 to 21 U when the microorganism was grown on barley bran husks or trimmingvine shoot hemicellulosic hydrolyzates, respectively; meanwhile, Portilla-Rivera et al. [22],working with the same microorganism grown in distilled grape marc hydrolyzates, achievedslightly lower values (9.55–16.05 mN/m).

Additionally, the concentration of BS was calculated (see Table 1) using the calibra-tion curve obtained by Portilla-Rivera et al. [22] employing different concentrations ofsurfactin below the CMC with known values of ST. These values depend on thecomposition of fermentation media [26]. In our case, the highest concentration wasconsiderably higher (5.6 mg/L) in Fermentation 1B, where both bacteria were employedsequentially to metabolize all hemicellulosic sugars. A similar pattern was observed withthe dilution ratio (FCMC) necessary to achieve the CMC calculated as indicated in Fig. 5and which can be defined as the dilution ratio necessary to match the lowest concen-tration of BS that allows obtaining the highest ST reduction of the media (the CMC).The results revealed that it is necessary to make higher dilutions in cell-bond BSobtained in Fermentation 1B to achieve the CMC, as can be seen in Fig. 5. Conversely,the simultaneous fermentation of hemicellulosic sugars and SSF of cellulosic fractioncarried out by cocultures of both bacteria (Fermentation 3) decreased drastically the

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Fig. 4 Course with time forhemicellulosic and cellulosicsugars consumption, lactic acidand biomass formation (a), andPhe to PLA biosynthesis (b) bycoculture of L. plantarum and L.pentosus. Unfilled circle glucose,unfilled diamond xylose, unfilledtriangle arabinose, filled circlelactic acid, unfilled square formicacid, filled square acetic acid,filled diamond Phe, filled trianglePLA. Standard deviations wereless than 2.3 % of the mean


final concentration of BS (2.6 mg/L) and FCMC (1.3), obtaining values lower than thoseachieved during the SSF by L. plantarum (Fermentation 2). Nevertheless, all fermenta-tions showed to be effective in producing BS. Considering that the commercial appli-cations of BS are determined by their cost and properties in relation to competingsynthetic compounds, the use of BS in the food and chemical industries is not generallycompetitive because of the relatively high production costs [26], and the source ofalternative renewable resources and the use of cocultures can improve the profitabilityof the technology.

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/m)

1/F

Fig. 5 Calculation of dilutionratio (FCMC) for achieving thecritical micellar concentration(CMC) of cell-bound biosurfac-tants obtained by a coculture of L.plantarum and L. pentosus usinghemicellulosic hydrolyzates, b si-multaneous saccharification andfermentation carried out by L.plantarum, and c fermentation ofhemicellulosic sugars and simul-taneous saccharification and fer-mentation carried out by cocultureof L. plantarum and L. pentosus.Standard deviations were lessthan 3.1 % of the mean


Antimicrobial Activity

Finally, the well diffusion technique was assayed in order to evaluate the antimicrobialactivity of CFS and BS. No antimicrobial inhibition was observed in any case using the CFSobtained from samples or BS extracts at the beginning of fermentations (0 h). By contrast,CFS obtained at the end of fermentation (144 h) showed different patterns. Thus, measuringinhibition halos (see Fig. 6) revealed no inhibition against any pathogenic bacteria whenCFS from Fermentation 1B was assayed (see halo “a” in each pathogenic bacteria in Fig. 6).Conversely, a measurable inhibition of growth was observed against S. enterica and L.monocytogenes when CFS from F2 and F3 were assayed (see halos “b” and “c” in Fig. 6aand b), showing the strongest antimicrobial activity using CFS from Fermentation 3 (12.1±0.6 mm of inhibition halo against S. enterica and 11.5±0.9 mm against L. monocytogenes);meanwhile, using CFS from Fermentation 2, the inhibition halos were reduced to 9.5±0.3 mm against S. enterica and 9.0±0.7 mm against L. monocytogenes.

Conclusions

Various biocompounds (LA, PLA, and BS) can be produced by L. plantarum and L.pentosus in independent cultures or coculture fermentations using hemicellulosic and/orcellulosic fractions of trimming vine shoots as a renewable source of carbon. Although allthe technologies assayed proved to be effective, the coculture of both microorganismsincreased the concentration of LA and PLA and also augmented the reduction of ST.Consequently, it can be concluded that this technology enables the synergistic utilizationof metabolic pathways of the participating microorganisms with the additional advantage ofconducting the process in one step within the same reactor, i.e., significantly simplifying theprocess.

Acknowledgments We are grateful for the financial support of this work from the Spanish Ministry ofScience and Innovation (project CTQ2011-28967) that has partial financial support from the FEDER funds ofthe European Union. Additionally, we wish to thank the University of Vigo for the doctoral fellowship ofNRP.

Fig. 6 Clearing halos formation around wells charged with cell-free supernatant extracts obtained from aFermentation 1B, b Fermentation 2, and c Fermentation 3 or with biosurfactants extracts obtained from dFermentation 1B, e Fermentation 2, and f Fermentation 3 against pathogenic bacteria: a Salmonella entericaand b Listeria monocytogenes


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Biotechnological Production of Phenyllactic Acid and Biosurfactants from Trimming Vine Shoot Hydrolyzates by Microbial Coculture Fermentation