BIOFILM FORMATION UNDER LAMINAR FLOW CONDITIONS OF YEAST ISOLATED FROM AN APPLE JUICE PROCESSING PLANT

jfpe_336 49..66

L.I. BRUGNONI1, M.A. CUBITTO1 and J.E. LOZANO2,3

1

Department of Biology, Biochemistry and Pharmacy Universidad Nacional del Sur (8000) Baha Blanca Argentina

Pilot Plant of Chemical Engineering (UNS-CO NICET) (8000) Baha Blanca Argentina
Accepted for Publication July 31, 2008

ABSTRACT The attachment of microorganisms to a surface is a critical step of biolm fouling in food processing equipment. The goal of this research was to determine the potential of four yeast strains isolated from an apple juice processing plant, to initiate biolm formation on stainless steel surfaces (SSS) both under static and laminar ow conditions. The isolated yeasts were Kluyveromyces marxianus, Candida krusei, Zygosaccharomyces sp. and Rhodotorula mucilaginosa. Micro-colonies, indicating the starting of biolm formation, were observed after 16 h. Cell numbers per SSS signicantly changed with yeast strain and time. Results seem to indicate that adhesion and colonization of the studied yeast under laminar ow conditions differed from that observed under static conditions. Particularly, when adhesion of K. marxianus, C. krusei and R. mucilaginosa was reduced one log cycle, Zygosaccharomyces sp. increased 0.5 log units as compared with the assays made under static conditions.

PRACTICAL APPLICATIONS The persistence of microorganisms in biolms is a serious hygienic problem in the food industry. Moreover, the microbial attachment greatly reduces the heat transfer and operating efciency of the processing equipment.
3

Corresponding author. TEL: +5420914861600; FAX: +542914861700; EMAIL: jlozano@ plapiqui.edu.ar

Journal of Food Process Engineering 34 (2011) 4966. All Rights Reserved. Copyright the Authors Journal Compilation 2009 Wiley Periodicals, Inc. DOI: 10.1111/j.1745-4530.2008.00336.x

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For example, biolm formation greatly reduces the permeability of ltration membranes. We observed that the yeast strains more frequently isolated from concentrated apple juice processing plants present a rapid capacity of adhesion to stainless steel (the most commonly used surface in food processing plants), after a short time of contact, both under static or laminar ow conditions. The fruit juice industry should be alert to the fast colonization rate of yeast on the surfaces involved in the production processes.

INTRODUCTION The biolm formation is the result of different chemical, physical and biological processes occurring simultaneously, including cell (primary) contact with the surface, cell adhesionattachment and biolm generation through cell division and extracellular matrix production. Biolm detachment is a determining factor for biolm formation as well, because it is a primary process that balances growth (van Loosdrecht et al. 1997). While previous studies of biolm development have been largely devoted to bacterial species, little is known about fungal biolms. The capacity of yeasts to adhere and to form biolms has been basically studied in those species with medical importance (Ramage et al. 2002), or focused on the immobilization of cells in reactors (Yongqiang et al. 2002). The persistence of microorganisms in biolms is a serious hygienic problem in the food industry. Moreover, the microbial attachment greatly reduces the heat transfer and operating efciency of the processing equipment (Bott 1992; Lehmann et al. 1992; Mattila-Sandholm and Wirtanen 1992). For example, biolm formation greatly reduces the permeability of ltration membranes (Flemming et al. 1992). Yeasts are usually contaminants that affect the quality and the shelf life of fruit juices (Zook et al. 1999) and, in many cases, affecting consumers health. Although yeast biolm development may produce adverse effects on processing equipment, causing post-processing contamination and reducing the effectiveness of sanitizing treatments, there is little information about yeast adhesion capacity and biolm formation in fruit juice processing lines. On the other hand, many previous studies on biolm formation used as biolm support translucent abiotic surfaces, such as glass or certain synthetic polymers, because this aids in the visualization of the biolms. These materials are rarely used in the modern food industry. Instead, stainless steel (SS) is widely used in the food industry owing to its excellent corrosion resistance and because it is able to withstand the cleaning and sanitizing regimes usually used in the food industry (Zottola and Sasahara 1994).

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In a previous work (Brugnoni et al. 2007), the adhesion capability on SS surfaces of yeasts Kluyveromyces marxianus, Candida krusei, Zygosaccharomyces and Rhodotorula mucilaginosa isolated from apple juice was investigated. Furthermore, the attempts to characterize the effect of ow velocity on biolm formation are much fewer. Especially in industrial food applications, it is important to consider how the behavior of the liquids owing within the system inuences the processes of cell attachment and detachment on and from the SS surfaces (SSS), controlling in parallel two interlinked parameters: mass transfer and drag. Several microorganism-attachment studies conducted under steady conditions, where there is no ow of liquid relative to the surface, successfully contributed to an understanding of the phenomenon of attachment (Norwood and Gilmour 1999; Beresford et al. 2001). However, in a typical liquid food processing plant, equipment inlet surfaces are either continuously or periodically in contact with owing liquids that contain microorganisms. Fluid ows are characterized by the Reynolds number (Re), dened as rvD/m, where r is the uid density, v its velocity, D a characteristic dimension and m the uid viscosity. In practical terms, this parameter represents the ratio of inertial to viscous ow; values below about 2,000 are termed laminar while those above 4,000 are turbulent (Perry and Green 1998). Although turbulent ow is complex and difcult to predict, and most experimental work by microbiologists involves laminar ow (Brading et al. 1995), the biolm formation under high Reynolds conditions has also been done. Pujo and Bott (1991) operated with Reynolds numbers up to 16,800 and Lewandowski and Stoodley (1995) examined the effect of turbulent ows on the behavior of pseudomonads. The rst authors proposed a type of rule of thumb still widely used in the industry, that velocities of 1 m/s generate enough shear stress to prevent biolm formation. However, some microorganisms increased biolm formation in water piping with turbulence (Perni et al. 2006). Authors considered that turbulent ow resulted in a higher overall mass transfer rate compared with laminar ow, increasing, in that way, oxygen and nutrient availability at the attachment surface, which led to the observed increase in this particular microorganism (Legionella) under turbulent conditions. The case under study in this work contemplates the biolm formation of yeasts on SSS, using apple juice as carrier. Apple juice is basically a sugar solution and the depletion of nutrients under laminar or steady conditions should be irrelevant. The goal of this research was to determine the potential of four yeast strains isolated from an apple juice processing plant to initiate biolm formation on SS under static and laminar ow conditions using a specially designed shear stress ow chamber.

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MATERIALS AND METHODS Yeast Strains and Culture Conditions Apple juice samples were kindly provided by the industry (Cooperativa Agrcola Choele Choel, Ro Negro, Argentina), by sampling from selected processing steps: M1, a 12Brix apple juice from the milling and pressing steps (at room temperature); M2, a 20Brix cloudy apple juice from the aromastripping unit (up to 100C); M3, a 20Brix claried apple juice from the enzymatic clarication tanks (4550C, 24 h); and nally, M4, a 72Brix concentrated apple juice (nal product) from a four-stage falling lm evaporator (<90C). The pH values in all samples were 3.2 0.2. The samples were taken in sterile bottles and kept at 5C until analysis. The different yeast colonies were enumerated and identied by their morphological and biochemical characteristics as described in a previous work (Brugnoni et al. 2007). Stock cultures of each strain were suspended in 20% (v/v) glycerol in yeast extract glucose chloramphenicol (YGC) broth: 0.5% w/v yeast extract (Merck KGaA, Darmstadt, Germany), 2% w/v glucose (Merck KGaA) and 0.01% w/v chloramphenicol (Fluka Chemie AG, Buchs, Switzerland) and stored at -70C. For experiments, a loop of frozen yeast cells was subcultured twice in YGC broth at 25C and 100 rpm on an orbital shaker (Vicking M23, Vicking S.R.L., Bs. As., Argentina) until it reached the stationary phase. Food Soiling System The 12Brix claried apple juice (mean composition: fructose, 70 g/L; glucose, 35 g/L; sucrose, 16 g/L; malic acid, 0.43.4 g/L; citric acid, <1 g/L; ascorbic acid, <40 mg/L; potassium, 1 g/L; calcium, 0.050.4 g/L; phosphorus, 70100 mg/L; sodium, 20 mg/L; free amino acids, 15 g/L), pH, 3.2 0.2; ionic strength, 0.023 mol/L (Lozano 2006) was prepared from 72Brix concentrated juice and sterilized by microltration (pore size 0.45 mm) (Metricel Grid, Gelman Sciences, Ann Arbor, MI). Growth Conditions and Preparation of Yeast Suspensions Each yeast strain cultured as described in the previous section was harvested by centrifugation at 3,000 g for 5 min (Labofuge 200, Kendro, Germany) and subsequently washed and resuspended in sterile 12Brix claried apple juice prepared as explained in the previous section. The optical density (OD) was adjusted at 550 nm to 0.125 (approximately 106 cells/mL) using a visible spectrophotometer (Thermo Spectronic Genesys 20, Thermo Electron Corporation, Waltham, MA).

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Substrate For adhesion experiments, the surface used was AISI 304 SS. In semistatic conditions, it was cut into rectangular chips (15 25 mm), and for assays in ow systems, was cut into 15 75-mm chips. Before the experiments, the chips were soaked for 15 min with 2% w/v of a detergent solution (Extran MA 02 neutral, Merck KGaA) at 50C and rinsed ve times for 5 min each with hot tap water followed by ve rinses with distilled water. Finally, the chips were autoclaved for 15 min at 120C. Adhesion Tests under Static Conditions To simulate the adhesion capacity of the yeast strains to SS under static conditions in the presence of apple juice (typical in a semi-batch processing line), the following experience was performed: A total of nine sterile glass Petri dishes were divided in six sections by glass pieces, to avoid overlapping of the chips during the experiment. One chip was put into each Petri dish section. Six mL of the yeast suspension in apple juice prepared as was indicated in a previous section was poured onto each Petri dish. This volume was enough to cover up the glass divisions. The plates were incubated at 23 1C under low stirring (70 rpm) with an orbital shaker (Vicking M23). After 2.5, 5, 10, 15, 20, 30, 60, 90 and 120 min of incubation, one Petri dish was taken out of the experiment. Each chip was washed twice for 1 min by immersion in sterile distilled water with agitation at 50 rpm to remove the non-adherent cells. Adhesion Test under Laminar Flow Conditions The shear stress ow chamber used in the present study was based on that of Mercier-Bonin et al. (2000) and Chung et al. (2003). A schematic representation of the ow chamber is presented in Fig. 1. The chamber consisted of (1) a bottom plate (130 40 8 mm) with slots for supporting the SS chips (AISI 304 12 75 1 mm; food grade), used as a substrate onto which yeast cells were deposited; and (2) a lid plate of the same dimensions with a channel machined out to allow the uid ow through it and over the coupon. Both plates were made of cast acrylic and held together with three couples of bolts. The uids (yeast suspension in apple juice) entered and left the chamber through 3-mm-diameter tubes connected with exible hoses to a peristaltic pump. The ow channel (90 13 4 mm), in which the biolm formation of yeast cells on chips was studied, followed a divergingconverging channel. This geometry ensures uniform ow both at the entrance and the outlet of the

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130 mm

40 mm

Cast acrylic plates Fluid i n Fluid out

Stainless Steel chip

FIG. 1. SCHEMATIC REPRESENTATION OF THE SHEAR STRESS FLOW CHAMBER

coupon-support rectangular part, where the ow is theoretically a laminar two-dimensional Poiseuille ow. Therefore, the wall shear stress tw can be dened as:

w = 6Q h 2 l

(1)

where Q (mL/min) is the ow rate, m is the uid viscosity (Pas), and l and h are, respectively, the channel width and thickness (m). For the range of ow rates used in this work, the entrance region where shear stress is not uniform was veried to be much smaller than the total length of the rectangular channel. Typical used liquid ow rate, shear stress, Reynolds number (Re = Qr/wh/m) and other required uid properties and ow conditions are listed in Table 1. Moreover, Table 2 includes yeast cell characteristics, as dimensions and sedimentation velocity in claried apple juice (12Brix). Adhesion of cells on the SS chips was performed by recirculating the yeast suspension in apple juice through the ow chamber for 2 h. Assays were made in duplicate at 23 1C from two separately grown cultures. A system in which particles (the nutrient) are carried to the aggregate by a combination of both ow and diffusion was considered. The relative importance of these two effects on nutrient transport can be characterized by the Pclet number (Pe) dened by Pe = ml/D, where m is the mean ow velocity, l a characteristic length and D the diffusion coefcient of the nutrient. Then, the dimensionless number Pclet relates the rate of convection of a ow to its rate of diffusion. A Pclet number > 1, as in this study, indicates the predominance of convection. Actual transport toward the substratum, needed for adhesion to occur, is through diffusion, which makes the PP ow chamber a kinetically slow one.

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TABLE 1. FLUID PROPERTIES AND FLOW CONDITIONS Soluble solids (Brix) Temp (C) m (Pas) Flow rate, Q, (L/h) r (kg/m3) Chamber high, h (m) Chamber width, w (m) Re Yeast mean diameter (m) Shear stress, t (Pa) Shear rate, g (1/s) 12 25 1.24 10-3 3.60 1,052 3.6 10-3 12.8 10-3 66 5 10-6 0.047 37.2

TABLE 2. SIZE AND SEDIMENTATION VELOCITY (Vsed) OF YEAST CELLS IN CLARIFIED APPLE JUICE (12BRIX) Yeast Width (mm) 3.16 0.66 4.00 0.82 1.72 0.29 3.10 0.25 Length (mm) 4.76 0.66 7.60 0.88 10.80 1.15 3.10 0.25 Diameter (mm)* 3.96 0.66 5.80 0.85 6.26 0.72 3.10 0.25 Vsed (m/seg) 0.71 1.53 1.78 0.44

K. marxianus C. krusei Zygosaccharomyces sp. R. mucilaginosa

* Values represented as average SD from >25 cells. Sedimentation velocity (Vsed) was estimated using average cell diameter.

Count of Viable Cells A standard stock solution of 2 mg/mL (0.2% w/v) uorescein diacetate (FDA), (C24H16O7, SigmaAldrich Chemical Co., St. Louis, MO) was prepared in acetone (Dorwil Industria, Grand Bourg, Argentina) and stored at -18C. To evaluate the number of viable yeast cells on SS, the coupons from each time were stained with sterile FDA acetonic solution in 0.1 M phosphate buffer (0.04% w/v), pH 7.5. After 90 min shaking at 23 1C in the darkness, the coupons were rinsed twice with sterile distilled water. The stained surfaces were examined under an Olympus BX 51 epiuorescence microscope (Olympus, Buenos Aires, Argentina) with a suitable lter combination, using a 100 oil-immersion objective. At least 20 elds (area: 0.038 mm2) were examined per coupon. Determination of Yeast Colonization on SS Under static conditions, SS chips submitted to adhesion test for 2 h and washed to remove the non-adherent cells were put into sterile Petri dishes and

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covered with sterilized claried apple juice (12Brix) and left to colonize during 5, 16 and 24 h at 23 1C and washed with sterile water before a colony count and microscopic observation. For scanning electronic microscope (SEM) observations, chips were xed with glutaraldehyde (2.5%) in phosphate buffer (0.1 M, pH 7.2); washed three times with the same buffer and dehydrated in increasing concentrations of acetone (25100%). Samples were vacuum-dried at 40C, gold coated in a Pelco Model 3 Sputter Coater 91,000 metal evaporator (Ted Pella Inc., Tustin, CA) (Sorrivas 1990). Observations were made in an SEM (JEOL Model 35CF, Tokyo, Japan). Under laminar ux conditions, the yeast suspensions were circulated for 2 h through the system in laminar ow conditions (Reynolds = 50) at a ow rate (Q) of 1 mL/seg (ow velocity (v) equal to 1.9 cm/seg) at 23 1C. Then, the coupons were rinsed by circulation of distilled sterile water for 2 min at 23 1C to remove poorly adhering cells. Thereafter, sterile 12Brix claried apple juice was continuously supplied at the same ow rate for 5, 16 and 24 h. Then, the coupons were rinsed by circulation of distilled sterile water for 2 min at 23 1C. The number of viable yeast cells on the SS was evaluated as was indicated previously. Statistical Analysis The resulting data were analyzed using GraphPad Prism version 4.0 computer software (GraphPad Software, Inc., La Jolla, CA). Two-way ANOVA was used to perform multiple analyses of the interactions between all factors. When appropriate, Bonferroni post-test was used for comparison of means. All tests were performed with a condence of 95%.

RESULTS AND DISCUSSION Adhesion and Colonization under Static Conditions Figure 2 shows the number of attached yeast cells of K. marxianus, C. krusei, Zygosaccharomyces sp. and R. mucilaginosa on SSS (SS chips) under static conditions in a 12Brix claried apple juice, respectively (Brugnoni et al. 2007). C. krusei showed the highest and faster adhesion of cells, followed by K. marxianus. Authors observed in this previous work that K. marxianus and R. mucilaginosa presented similar adhesion rate during the rst 20 min of contact with substrate, K. marxianus signicantly increased the number of adhered cells after that time. After 30 min of contact time, the rate of adhesion drastically reduced and the number of adhered cells reached a plateau in all cases.

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FIG. 2. KINETIC ADHESION OF THE YEAST STRAINS SUSPENDED IN CLARIFIED APPLE JUICE TO STAINLESS STEEL (BRUGNONI ET AL. 2007, WITH PERMISSION)

Zygosaccharomyces sp. showed the lowest number of adhered cells and it did not show signicant changes throughout the exposure. Moreover, during the rst 2.5 min, no viable cells were observed on the surface. C. krusei presented the higher initial adhesion rate, a behavior attributable to its elevated hydrophobicity (Brugnoni et al. 2007). The rate of adhesion, as cells/cm2seg, was determined by least-squares tting the data of the rst 20 min of adhesion. The yeasts rate of adhesion in decreasing order was: C. krusei (493 cells/cm2seg) > K. marxianus (125 cells/cm2seg) > R. mucilaginosa (91 cells/cm2seg) > Zygosaccharomyces sp. (4 cells/cm2seg). Figure 3 shows the percentage of SS chips surface covered by the assayed yeasts during adhesion and colonization. While Zygosaccharomyces sp. covered only about 1% of the SS chip during adhesion and colonization, K. marxianus at least tripled the covered surface after 30 min. On the other hand, C. krusei showed a continuous increase from 20 min to 24 h, covering a surface up to 10 times higher than K. marxianus. Results show that except for Zygosaccharomyces sp., the remaining yeast strains suspended in the food matrix (12Brix claried apple juice) adhere and colonize rapidly on SS (Fig. 3). They form a thin monolayer on the SSS, and micro-colonies (the basic structural unit of the biolm) were observed after 16 h (Fig. 4). The shape and alignment of yeasts within the micro-colonies depend on their morphological characteristics and growth, specic for each

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10 0 90 80

% covered surface

70 60 50 40 30 20 10 0 2 ,5 5 10 15

20

30

60

90

18 0

Adhesion time (min)

Zygosaccharomyces sp. R. mucilaginosa K. marxianus

16

24

Colonization time (h)

C. krusei

Time

FIG. 3. PERCENTAGE OF STAINLESS STEEL CHIP COVERED BY ASSAYED YEASTS DURING ADHESION AND COLONIZATION UNDER SEMI-STATIC CONDITIONS

yeast strain. Figure 4 shows the SEM microphotographs of assayed yeasts, with the exception of Zygosaccharomyces sp., because of the low number of attached cells. C. krusei (Table 3) increases the number of cells by 6 106 to 3 107 cells/cm2 at 24 h of colonization, followed by R. mucilaginosa (105 to 106 cells/cm2), Zygosaccharomyces sp. (104 to 105 cells/cm2) and K. marxianus (1 to 2.5 106 cells/cm2). Although in thin biolms, micro-colonies were observed to arrange in a horizontal array, they may form vertical arrays in very thick sessile communities. The consensus is that biolms comprise aggregates of microbial cells within a matrix of extracellular polymeric substances (EPS) and interstitial voids and channels separate the micro-colonies. Several studies indicate that EPS is not necessarily required for the initial attachment of microbial cells to surfaces, but the production of EPS is essential for the development of the architecture of any biolm matrix. The EPS molecules provide the framework into which microbial cells are inserted.

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FIG. 4. (A) CANDIDA KRUSEI; (B) KLUYVEROMYCES MARXIANUS; AND (C) RHODOTORULA MUCILAGINOSA CELLS GROWN ON STAINLESS STEEL AISI 304 FOOD GRADE. COLONIZATION TIME: 16 H IN SEMI-STATIC FLOW CONDITIONS

SEM has provided a high resolution visualization of microbial biolm. In SEM, biolm specimens are prepared by xation, staining, drying and conductively coating prior to imaging under high vacuum. While any pretreatment can alter specimen morphology, drying appears to signicantly alter biolms because of EPS collapsing. Alternatively, environmental SEM, or ESEM,

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TABLE 3. ADHESION AND COLONIZATION UNDER STATIC CONDITIONS AS LOG N CELLS PER cm2 OF SS CHIP Yeast Log (n of cells/cm2*) Time (h) 5 K. marxianus C. krusei Zygosaccharomyces sp. R. mucilaginosa 5.62 0.05 * 5.89 0.09B* 5.12 0.40C* 5.37 0.03D*
A

16 5.90 0.07 6.30 0.05B 5.50 0.06C 5.70 0.07D

A

24 6.13 0.06A 6.43 0.09B 5.70 0.04C 5.89 0.05D

* Values represented as average SD from triplicate determinations. Values with different capital letters in superscripts, within a column, are signicantly different (P < 0.05) determined by Bonferroni post-test. Values with different superscripts letters in each row are signicantly different (P < 0.05) determined by Bonferroni post-test.

FIG. 5. NUMBER OF CELLS OF KLUYVEROMYCES MARXIANUS, CANDIDA KRUSEI, ZYGOSACCHAROMYCES SP. AND RHODOTORULA MUCILAGINOSA (LOG CELL NUMBER/CM2 STANDARD ERROR) ATTACHED ON STAINLESS STEEL AFTER 1, 3, 6, 16 AND 24 H COLONIZATION UNDER LAMINAR FLOW CONDITIONS. ERROR BARS INDICATE STANDARD DEVIATIONS OF TRIPLICATE SAMPLES

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minimizes biolm dehydration and thus preserves native morphologies including surface structures. Confocal scanning laser microscopy (CLSM) has provided some new information on the structural complexity of biolms and has conrmed their heterogeneity (Sutherland 2001). In future studies, colonization times will be increased to observe and characterize a mature biolm by ESEM and CLSM. Adhesion and Colonization under Laminar Flow Conditions Figure 5 shows the number of attached yeast cells of K. marxianus, C. krusei, Zygosaccharomyces sp. and R. mucilaginosa on SSS (SS chips) under a laminar ux of 12Brix claried apple juice. Micro-colonies were detected for all yeast after 16 h of recirculation of the yeast suspension in apple juice (Fig. 6).

FIG. 6. (A) CANDIDA KRUSEI; (B) KLUYVEROMYCES MARXIANUS; (C) RHODOTORULA MUCILAGINOSA; AND (D) ZYGOSACCHAROMYCES SP. CELLS COLONIZED ON STAINLESS STEEL CHIPS AFTER 16 H UNDER LAMINAR FLUX CONDITIONS (MAGNIFICATION 1,000)

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FIG. 7. PERCENTAGE OF STAINLESS STEEL CHIP COVERED BY ASSAYED YEASTS DURING ADHESION AND COLONIZATION UNDER LAMINAR FLOW CONDITIONS

A faster adhesion rate was observed for C. krusei, followed by K. marxianus. The growth of Zygosaccharomyces sp. and R. mucilaginosa were similar during the rst time (P > 0.05), as determined by the Bonferroni multiple comparison test. On the other hand, Fig. 7 shows the percentage of SS chips surface covered by each yeast during colonization under laminar ux conditions. While the percentage of SS chip covered by R. mucilaginosa produced similar results under laminar ux than under static conditions (P > 0.5), K. marxianus reduced 30% and C. krusei signicantly reduced the covered area (P > 0.001). With respect to Zygosaccharomyces sp., it practically triples the covered surface under ux conditions. In this case, both the higher number of cells adhered under ux conditions and the size modication should be considered. Epiuorescense micrographs (Fig. 8) show elongated yeast cells arranged in a net manner, a behavior different from that observed during adhesion under static conditions. Results seem to indicate that adhesion and colonization of the studied yeast under laminar ow conditions differed from that observed under static conditions. Particularly, when adhesion of K. marxianus, C. krusei and R. mucilaginosa was reduced one log cycle, Zygosaccharomyces sp. increased 0.5 log units as compared with the assays made under static conditions. Figure 9 shows that

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FIG. 8. EPIFLUORESCENSE MICROSCOPY CHARACTERISTICS OF ZYGOSACCHAROMYCES SP. COLONIES AFTER 16 H UNDER LAMINAR FLUX CONDITIONS (MAGNIFICATION 1,000)

cell numbers of C. krusei and Zygosaccharomyces sp. increase, for a better comparison between both observed behaviors. Zygosaccharomyces sp. behavior can be attributed to its relatively high sedimentation rate. During colonization and biolm growth, cells are affected by phenotype modications affecting the cell anchor to surfaces (Delissalde and AmabileCuevas 2004). This phenomenon was particularly evident after relatively long cell-substrate contact times when, contrary to the initial period of treatment, the effect of the physicochemical characteristics of cells predominates. It was observed that ow conditions modied the colony growth morphology of Zygosaccharomyces sp. While in the static case, unicellular dispersed pattern were observed, it evolved to a pseudomicelial conguration under laminar ow conditions (Figs. 6 and 8). CONCLUSIONS It was concluded that the yeast strains more frequently isolated from concentrated apple juice in Argentine processing plants present a rapid

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8 7,5 7 6,5 6 5,5 5 4,5 4 3,5 3 0 200 400 600 800 1000 1200 1400 1600 C krusei Zygosaccharomyces sp C krusei LF Zygosaccharomyces sp LF

log cell number per cm2

time (min)

FIG. 9. CELL NUMBER INCREASE OF CANDIDA KRUSEI AND ZYGOSACCHAROMYCES SP. UNDER SEMI-STATIC ( ) AND LAMINAR () FLOW CONDITIONS FOR A BETTER COMPARISON BETWEEN BOTH BEHAVIORS FOUND

capacity of adhesion to SSS, even after the shortest times of assay, both in static and laminar ow conditions. This must alert the juice industry on the fast colonization of the surfaces involved in the production process. This behavior should also warn about the biological stability of processing plants. Furthermore, the parallel ow chamber proved to be a useful tool in yeast biolm studies, imitating the process conditions under laminar ow and allowing the observation of biolm formation in real time. The attachment and biolm formation of yeast strains in turbulent ow conditions will be elucidated in a future work. ACKNOWLEDGEMENTS We thank the Secretara de Ciencia y Tcnica of the Universidad Nacional del Sur, the Consejo Nacional de Investigaciones Cientcas y Tcnicas de la Repblica Argentina (CONICET) and the Comisin de Investigaciones Cientcas de la Provincia de Buenos Aires (CIC) for funding this research.

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