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Production and molecular weight characteristics of alginate from free and immobilized-cell cultures of Azotobacter vinelandii
Process Biochemistry 37 (2002) 895– 900 www.elsevier.com/locate/procbio
Production and molecular weight characteristics of alginate from free and immobilized-cell cultures of Azotobacter 6inelandii N. Saude, G.-A. Junter * Faculte´ des Sciences de Rouen, UMR 6522 CNRS, 76821 Mont-Saint-Aignan Cedex, France Received 26 June 2001; received in revised form 26 June 2001; accepted 8 October 2001
Abstract Viable Azotobacter 6inelandii cells were immobilized in composite agar gel layer/microporous membrane structures and tested for alginate production from sucrose during batch incubation. Microporous membranes with varying pore sizes (0.22–3.0 mm) were used in the composite structures. Whatever the membrane pore size (MPS), the duration of the production period was short (ca. 100 h), most sucrose remaining unconsumed by immobilized organisms. The amount of alginate recovered (0.5– 0.9 g dm − 3) and volumetric productivity increased slightly with the MPS, but the average production yield was fairly stable with a mean value of 0.24 g alginate produced g − 1 sucrose consumed, i.e. noticeably higher than that of free-cell counterparts (0.09 g g − 1). Furthermore, immobilization was shown to favour the production of higher molecular weight (MW) polysaccharide macromolecules and to stabilize the MW distribution of alginate produced as compared to conventional free-cell cultures grown in shake flasks. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Alginate; Azotobacter 6inelandii; Bioproduction; Exopolysaccharide; Immobilized cells; Macromolecular weight
1. Introduction A large number of microorganisms, including bacteria, microalgae, yeasts and fungi, can produce extracellular polysaccharide polymers. These microbial exopolysaccharides are of potential interest from an economical point of view, depending on their structural properties and rheological behaviour [1 – 3]. Their production at the industrial level is usually performed by batch culture in stirred-tank bioreactors where the fermentation conditions, in particular shear stress, may be difficult to control. As a consequence, the macromolecular properties (e.g. viscosity, molecular weight) of recovered polymers frequently vary from one batch to another. As cell immobilization represents a possible way to overcome theses difficulties, several attempts to produce microbial exopolysaccharides using immobilized-cell systems have been reported over the last fifteen years [4–9]. While involving biopolymers developed at an industrial scale such as xanthan [4] and dextran [7], these attempts have neglected alginate-like * Corresponding author. Fax: + 33-2-3552-8485. E-mail address: [email protected] (G.-A. Junter).
polysaccharides synthesized, in particular, by the opportunistic human pathogen Pseudomonas aeruginosa and the soil bacterium Azotobacter 6inelandii [10]. In a previous work [11], however, we tested the feasibility of exopolysaccharide production by microbial cultures immobilized in composite agar layer/microporous membrane structures. This study confirmed the difficulty in producing high molecular weight (MW) macromolecules such as xanthan or pullulan by polymer-entrapped organisms, but yielded more promising results on alginate production by immobilized A. 6inelandii. In the present work, we have investigated more particularly the role of the microporous membrane filter on the fermentation efficiency of gel-entrapped A. 6inelandii cultures and on the MW characteristics of the polysaccharide produced.
2. Materials and methods
2.1. Microbial strain, culture medium and inoculum preparation A. 6inelandii NCIMB 9068 was maintained at 4 °C
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on Tryptic Soy Agar plates (Difco). The organisms were grown in the culture medium recommended by the NCIMB [12], containing the following ingredients (g dm − 3): sucrose, 20.0; K2HPO4, 1.0; CaCO3, 1.0; NaCl, 0.2; MgSO4·7H2O, 0.2; FeSO4·7H2O, 0.1; Na2MoO4·2H2O, 0.005. The carbon source and the salt solution (pH adjusted to 7.4 before autoclaving) were autoclaved separately. After incubation for 48 h at 30 °C, the organisms were collected by centrifugation (2200×g, 30 min) and resuspended in liquid medium.
(HPLC) with refractive index detection [11]. Alginate concentrations were obtained gravimetrically. The polysaccharide was precipitated with ethanol (2 vol. ethanol/vol. medium). The precipitate was collected by centrifugation (15 min at 7500× g) followed by filtration on a predried and preweighed glass microfibre filter (type GF/B from Whatman, Maidstone, UK). After washing the polysaccharide deposit with ethanol, the filter was dried at 60 °C for 24 h and reweighed. All determinations were performed in triplicate.
2.2. Shake flask fermentations
2.5. Alginate characterization
Free-cell fermentations were performed in 2-dm3 Erlenmeyer flasks containing 500 cm3 of culture medium. The flasks were incubated at 30 °C in a giratory air convection shaker (agitation speed, 200 rpm). During incubation, the pH value of the culture medium was maintained to its initial level (7.4) by addition of 1 M NaOH.
The polysaccharide was recovered by precipitation with ethanol followed by centrifugation as described above. The alginate pellet was dissolved in deionized water that was next supplied with lithium nitrate to yield a LiNO3 concentration of 0.1 M. The solution was then purified by filtration through a Millex GV filter (Millipore; pore size, 0.22 mm) or, when more stringent purification was needed, by ultracentrifugation (540,000× g for 1 h at 4 °C) followed by pellet resuspension in 0.1 M LiNO3. The molecular weight and size distribution of alginate were obtained by coupling size exclusion chromatography (SEC) with multiangle laser light scattering (MALLS). SEC separations were performed using two columns connected in series (Shodex OHpack SB-804 HQ and SB-806 HQ). The polymer sample (volume, 100 ml; concentration, ca. 1 mg ml − 1) was eluted with filtered (0.1 m pore size filter, Millipore) 0.1 M LiNO3 at a flow rate of 0.6 ml min − 1 (Intelligent Pump 301, Shicoh Engineering, Japan). The MALLS photometer (Dawn-F, Wyatt Technology, Santa Barbara, CA), equipped with a concentration sensitive detector (ERC7515A refractive index detector, Erma CR., Tokyo, Japan), was calibrated with standard pullulan samples (48-kDa molecular weight). SEC/MALLS experiments were performed at room temperature. The ASTRA V.4.50 software package (Wyatt Technology) was used to determine the weight-average molecular weight (M( w) and the MW distribution of alginate. Alginate from Macrocystis pyrifera (sodium salt, low viscosity type, Sigma, St. Louis, MO) was used as reference.
2.3. Cell immobilization and immobilized-cell fermentations Organisms were entrapped in composite agar layer/ microporous membrane structures as described previously [13]. The disc-shaped agar layers [10-cm2 surface; 3-mm thickness; 0.5% (w/v) agar] were loaded with a calibrated amount of A. 6inelandii cells (2.25 mg dry wt. cm − 3 gel). They were stored at 4 °C for 90 min and at room temperature for 30 min before use. The microporous membranes were standard mixed ester cellulose filters (Millipore, Freehold, NJ) with average pore size (i.e. diameter) ranging between 0.22 and 3.0 mm (porosities, 75–82%; thickness, 150 mm). The composite structure was fastened in vertical position to the wall of a 300-cm3 Pyrex reactor, at the level of a circular opening in the vessel wall [11]. The bioreactor was then filled with 250 cm3 of culture medium and placed at 30 °C in an air convection incubator. Magnetic stirring ensured oxygen supply by transfer through the residual air/liquid medium interface (kLa = 5.6 ×10 − 2 min − 1). The pH of the culture broth was maintained to 7.4 by addition of 1 M NaOH.
2.4. Sample analysis Broth samples of 2.5-cm3 volume were collected aseptically. The corresponding changes in broth volume did not exceed 10% over a fermentation run. Free-cell concentrations were monitored by optical density (OD) measurements at 600 nm. OD values were converted to dry weights using a calibration curve. Sucrose and alginate were titrated after elimination of free bacteria by centrifugation (2200× g, 30 min). Sucrose was titrated by high-performance liquid chromatography
3. Results and discussion
3.1. Free-cell experiments Alginate production by conventional free-cell cultures of A. 6inelandii grown in shake flasks mainly occurred during the exponential phase of bacterial growth, i.e. during the first 72 h of incubation, but some polysacharide production was noticed after the
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The weight-average molecular weight of the polysaccharide recovered in the free-cell culture broth at the end of the incubation run (i.e. after incubation for about 140 h) was equal to (1689 58) kDa (n= 6). This M( w value for A. 6inelandii alginate is close to the molecular weight indicated by Clementi [19], but much lower than those given by Pen˜ a et al. [18], ranging from 1400 to 2000 kDa after sucrose fermentation for 72 h in shake flasks. According to the quoted authors, the MW values of alginates produced by batch cultures of A. 6inelandii are dependent strongly on the aeration conditions of the fermentation broth [18] and/or the time at which alginate is recovered from the broth [18,19]. The MW distribution curve of alginate varied significantly from one batch to another (Fig. 2), confirming the difficulty to obtain reproducible macromolecular characteristics for alginate produced by free-cell cultures grown in shake flasks.
Fig. 1. Kinetics of sucrose uptake (), microbial growth (), and alginate production ( ) of a free-cell A. 6inelandii culture.
3.2. Immobilized-cell fermentations In the absence of a microporous membrane filter, cell leakage from the agar gel layer occurred at the very beginning of incubation, and a free-cell culture developed (not shown). Placing a microporous membrane at the gel layer/liquid medium interface— therefore, creating the so-called ‘composite structure’ —delayed significantly cell leakage. The membrane efficiency in preventing cell contamination of the culture broth increased logically as the membrane pore size (MPS) decreased (Table 1). For MPSs equal to or lower than 0.45 mm, the culture medium remained quasi sterile over an incubation period of about 200 h. Conversely, significant numbers of bacteria were released from composite structures with MPSs of 1.2 and 3.0 mm. For all tested MPSs, however, the amount of free cells in the broth remained very low compared to that observed without membrane. The kinetics of sucrose consumption and polysaccharide production were similar for all tested composite structures (Fig. 3). The alginate concentration reached a maximum level after incubation for about 100 h. While some sucrose consumption occurred later, the overall amount of sugar consumed remained very limited compared to that observed during free-cell cultures. The maximum alginate concentration increased slightly with the MPS to reach at 3.0-mm MPS a value close to
Fig. 2. Differential molecular weight distribution curves of alginate samples recovered from several independent free-cell production runs in shake flasks. The MW distribution of algal alginate (solid line) is shown as standard. Ordinate units (given by the MALLS) are arbitrary. M( w values (kDa): 1, 158; 2, 194; 3, 155; 4, 351; algal alginate, 270.
organisms had entered the stationary phase (Fig. 1). This confirms other results showing that alginate production by A. 6inelandii is partially associated to growth [14–18]. The amount of polysaccharide recovered ranged between 0.8 and 1.4 g dm − 3, with a mean of (1.090.2) g dm − 3 corresponding to a production yield of (8996) mg alginate g − 1 sucrose (n = 6). These values are low compared to those reported for diverse A. 6inelandii strains, in particular highly mucoid natural isolates [16,17] or mutants [14,15]. Nevertheless, they are in the range of the values obtained for A. 6inelandii NCIMB 9068 [17] or other strains [16] in shake flask cultures using nitrogen-free incubation media.
Table 1 Influence of the membrane pore size on cell retention inside the agar gel layer Membrane pore size (mm) Time at leakage (h) a
Not determined.
0.22 \220
0.45 \180
0.65 n.d.a
0.80 B120
1.20 B60
3.00 B30
No membrane B20
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Fig. 3. Typical kinetics of alginate production by immobilized A. 6inelandii cells. The pore size of the microporous membrane used in the composite structure was (A) 0.45 mm and (B) 1.2 mm. Symbols: alginate ( ), sucrose () and free cell () concentrations.
that obtained in free-cell cultures (Table 2). This cannot be explained by the very limited cell leakage at high MPS. This increase in alginate productivity with the MPS more probably arises from some polymer accumulation, higher at low MPS, between the agar layer and the microporous membrane, as low amounts of slime were systematically observed on the internal face of the microporous filters after the incubation runs. Average production rates ranged between ca. 0.5 and 0.7 mg alginate h − 1 cm − 3 gel, which, based on a cell content of 2.25 mg dry wt. cm − 3 gel, corresponds to specific productivities between 0.2 and 0.3 g alginate h − 1 g − 1 dry wt. Average production yields, calculated after 100 h of incubation, were slightly dispersed around a mean value of 0.24 g alginate produced g − 1 sucrose consumed. Owing to the low amounts of sugar consumed, such yields were noticeably higher than those obtained for free counterparts (ca. 0.09 g g − 1) and are in the range of yields reported by others for free-cell cultures of highly mucoid strains [14– 17]. This significant improvement in alginate production yield of immobilizedcell cultures compared to free-cell counterparts may be due to a decrease in the amount of sucrose consumed for biomass synthesis. Differences in the oxygenation levels of free and immobilized cells are also probably involved in this gain in yield. It has been reported that alginate production is negatively affected by increasing the oxygen supply to free-cell cultures in shake flasks [18] or stirred tanks [20]. Indeed, it is well known that gel-entrapped organisms are subject to oxygen limitation [21]. Another possible explanation lies in the pecu-
Fig. 4. SEC/MALLS analysis of alginate from immobilized-cell culture (membrane pore size, 0.65 mm): light scattering (solid line) and refractive index (dotted line) signals as a function of the elution volume. The molecular weight distribution as a function of the elution volume is also shown (). The high MW fraction collected between 12.2 and 14.2 cm3 of elution volume represented 4.4% of the total mass injected and had a M( w value of 1730 kDa. Ordinates: arbitrary units.
liar physiology of immobilized cells, which frequently display enhanced metabolic activities when compared to free cells [22]. It has been shown, for example, that natural immobilization of bacteria as biofilms favours cell conversion to mucoidy, resulting in exopolysaccharide overproduction [23]. Bacterial cells artificially immobilized by entrapment in agar gel layers constitute a simple in vitro model structure of natural biofilms [24]. In particular, physiological properties characterizing biofilm bacteria are recovered in gel-entrapped organisms [24]. Therefore, the increase in the production yield of alginate by gel-entrapped A. 6inelandii may also arise from physiological modifications linked to the immobilized state.
3.3. Immobilized-cell alginate characterization Whatever the MPS, the average molecular weight of alginate released from composite structures was somewhat higher than that obtained for free-cell alginate. There was no evident relationship between the MPS and the M( w value, however (Table 2). All analysed alginate samples contained small quantities of highmolecular-weight (i.e. several millions Da) polysaccharide, detected first in the SEC effluent (Fig. 4). Owing to the low amounts recovered in the samples (as shown by the concentration profile given by the RI detector: Fig. 4), however, the presence of these macromolecules
Table 2 Fermentation parameters of immobilized-cell cultures and M( w values of alginate produced. All data were the mean of two independent experiments Membrane pore size (mm) Alginate produced (g dm−3) Yield (g g−1) M( w (kDa) a
Not determined.
0.22 0.55 0.26 324
0.45 0.53 0.25 n.d.a
0.65 0.58 0.21 287
0.80 0.60 0.28 n.d.a
1.20 0.68 0.18 211
3.00 0.92 0.24 239
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ing to the short-lived bioproduction period, however, the amount of polysaccharide produced remained low—though it was in the range of that recovered from free-cell cultures. Future work will therefore be aimed at improving the fermentation performance of immobilized-cell cultures in terms of alginate productivity (choice of a more efficient strain, improvement of the oxygenation level of the gel layer, etc.).
Fig. 5. Differential molecular weight distribution curves of alginate samples recovered from replicated immobilized-cell production runs. The figures refer to the pore size of the microporous membranes used. Ordinates: arbitrary units.
exerted only a limited influence on the M( w values. Previous experiments with dextran fractions of varying molecular weight (10–2000 kDa) have shown that diffusion of the polysaccharide macromolecules through standard mixed ester cellulose Millipore filters was only slightly restricted [25]. Conversely, the diffusion of high MW (] 500 kDa) dextran fractions through sterile agar gel membranes was severely restricted [26]. Therefore, the high-sized polymer macromolecules probably corresponded to aggregates forming in the liquid medium, maybe as a consequence of the low agitation level of the medium, rather than high MW biosynthesized molecules such as those obtained previously by others [18]. In opposition with free-cell polysaccharide samples (Fig. 2), the differential molecular mass distribution curves of alginate recovered from immobilized-cell production runs were very similar for the different MPSs tested and looked fairly reproducible considering replicated experiments (Fig. 5). A number of studies have underlined the prominent influence of the oxygenation level and agitation speed on the MW value of free-cellculture alginate [18– 20,27]. More reproducible incubation conditions inside the gel layer than in shake flasks may be responsible for the stabilization of MW values and distribution observed here.
4. Conclusion A. 6inelandii immobilized in composite agar layer/microporous membrane structures showed efficient alginate production in terms of yield and volumetric productivity. The immobilized state seemed to stabilize the macromolecular characteristics of alginate produced, which is of practical interest since the commercial outlets of microbial polysaccharides rely on these characteristics [3]. The pore size of the microporous membrane preventing cell leakage from the gel layer exerted no noticeable influence on the fermentation performance and the molecular weight of alginate. Ow-
Acknowledgements This work was supported by the Membrane Bioreactors programme of the ‘PIR Grand Bassin Parisien’. References [1] Sutherland IW. The properties and potential of microbial exopolysaccharides. ChimOggi 1990;8(9):9 – 14. [2] Roller S, Dea ICM. Biotechnology in the production and modification of biopolymers for foods. Crit Rev Biotechnol 1992;12:261 – 77. [3] Crescenzi V. Microbial polysaccharides of applied interest: ongoing research activities in Europe. Biotechnol Prog 1995;11:251 – 9. [4] Robinson DK, Wang DIC. A novel bioreactor system for biopolymer production. Ann N Y Acad Sci 1988;506:229 –41. [5] Mulchandani A, Luong JHT, LeDuy A. Biosynthesis of pullulan using immobilized Aureobasidium pullulans cells. Biotechnol Bioeng 1989;33:306 – 12. [6] Wang S-D, Wang DIC. Cell adsorption and local accumulation of extracellular polysaccharide in an immobilized Acinetobacter calcoaceticus system. Biotechnol Bioeng 1989;34:1261 – 7. [7] El-Sayed AHMM, Mahmoud WM, Coughlin RW. Comparative study of production of dextransucrase and dextran by cells of Leuconostoc mesenteroides immobilized on celite and in calcium alginate beads. Biotechnol Bioeng 1990;36:83 – 91. [8] Matsunaga T, Sudo H, Takemasa H, Wachi Y, Nakamura N. Sulfated extracellular polysaccharide production by the halophilic cyanobacterium Aphanocapsa halophytia immobilized on light-diffusing optical fibers. Appl Microbiol Biotechnol 1996;45:24 – 7. [9] West TP, Strohfus BR-H. Polysaccharide production by spongeimmobilized cells of the fungus Aerobasidium pullulans. Lett Appl Microbiol 1996;22:162 – 4. [10] Rehm BHA, Valla S. Bacterial alginates: biosynthesis and applications. Appl Microbiol Biotechnol 1997;48:281 – 8. [11] Lebrun L, Junter G-A, Jouenne T, Mignot L. Exopolysaccharide production by free and immobilized microbial cultures. Enzyme Microb Technol 1994;16:1048 – 54. [12] National Collection of Industrial and Marine Bacteria (NCIMB). In: Dando TR, Young JEP, editors. Catalogue of Strains. Aberdeen: NCIMB, 1990, p. 159. [13] Jouenne T, Bonato H, Mignot L, Junter G-A. Cell immobilization in composite agar layer/microporous membrane structures: growth kinetics of gel-entrapped cultures and cell leakage limitation by the microporous membrane. Appl Microbiol Biotechnol 1993;38:478 – 81. [14] Horan NJ, Jarman TR, Dawes EA. Effects of carbon source and inorganic phosphate concentration on the production of alginic acid by a mutant of Azotobacter 6inelandii and on the enzymes involved in its biosynthesis. J Gen Microbiol 1981;127:185 – 91.
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[15] Chen W-P, Chen J-Y, Chang S-C, Su C-L. Bacterial alginate produced by a mutant of Azotobacter 6inelandii. Appl Environ Microbiol 1985;49:543 –6. [16] Brivonese AC, Sutherland IW. Polymer production by a mucoid strain of Azotobacter 6inelandii in batch culture. Appl Microbiol Biotechnol 1989;30:97 –102. [17] Salvagi V, Salvagi V. Alginate production by Azotobacter 6inelandii in batch culture. J Gen Appl Microbiol 1992;38:641 – 5. [18] Pen˜ a C, Campos N, Galindo E. Changes in alginate molecular mass distributions, broth viscosity and morphology of Azotobacter 6inelandii cultured in shake flasks. Appl Microbiol Biotechnol 1997;48:510 – 5. [19] Clementi F. Alginate production by Azotobacter 6inelandii. Crit Rev Biotechnol 1997;17:327 –61. [20] Parente E, Crudele MA, Ricciardi A, Mancini M, Clementi F. Effect of ammonium sulphate concentration and agitation speed on the kinetics of alginate production by Azotobacter 6inelandii DSM576 in batch fermentation. J Ind Microbiol Biotechnol 2000;25:242 – 8. [21] Omar SH. Oxygen diffusion through gels employed for immobi-
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lization. 2. In the presence of microorganisms. Appl Microbiol Biotechnol 1993;40:173 – 81. Norton S, D’Amore T. Physiological effects of yeast cell immobilization: applications for brewing. Enzyme Microb Technol 1994;16:365 – 75. Deretic V, Martin DW, Schurr MJ, Mudd MH, Hibler NS, Curcic R, Boucher JC. Conversion to mucoidy in Pseudomonas aeruginosa. BioTechnology 1993;11:1133 – 6. Jouenne T, Tresse O, Junter G-A. Agar-entrapped bacteria as an in vitro model of biofilms and their susceptibility to antibiotics. FEMS Microbiol Lett 1994;119:237 – 42. Lebrun L, Junter G-A. Diffusion of dextran through microporous membrane filters. J Membr Sci 1994;88:253 – 61. Lebrun L, Junter G-A. Diffusion of sucrose and dextran through agar gel membranes. Enzyme Microb Technol 1993;15:1057 – 62. Sabra W, Zeng A-P, Lunsdorf H, Deckwer W-D. Effect of oxygen on formation and structure of Azotobacter 6inelandii alginate and its role in protecting nitrogenase. Appl Environ Microbiol 2000;66:4037 – 44.
Production and molecular weight characteristics of alginate from free and immobilized-cell cultures of Azotobacter 6inelandii N. Saude, G.-A. Junter * Faculte´ des Sciences de Rouen, UMR 6522 CNRS, 76821 Mont-Saint-Aignan Cedex, France Received 26 June 2001; received in revised form 26 June 2001; accepted 8 October 2001
Abstract Viable Azotobacter 6inelandii cells were immobilized in composite agar gel layer/microporous membrane structures and tested for alginate production from sucrose during batch incubation. Microporous membranes with varying pore sizes (0.22–3.0 mm) were used in the composite structures. Whatever the membrane pore size (MPS), the duration of the production period was short (ca. 100 h), most sucrose remaining unconsumed by immobilized organisms. The amount of alginate recovered (0.5– 0.9 g dm − 3) and volumetric productivity increased slightly with the MPS, but the average production yield was fairly stable with a mean value of 0.24 g alginate produced g − 1 sucrose consumed, i.e. noticeably higher than that of free-cell counterparts (0.09 g g − 1). Furthermore, immobilization was shown to favour the production of higher molecular weight (MW) polysaccharide macromolecules and to stabilize the MW distribution of alginate produced as compared to conventional free-cell cultures grown in shake flasks. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Alginate; Azotobacter 6inelandii; Bioproduction; Exopolysaccharide; Immobilized cells; Macromolecular weight
1. Introduction A large number of microorganisms, including bacteria, microalgae, yeasts and fungi, can produce extracellular polysaccharide polymers. These microbial exopolysaccharides are of potential interest from an economical point of view, depending on their structural properties and rheological behaviour [1 – 3]. Their production at the industrial level is usually performed by batch culture in stirred-tank bioreactors where the fermentation conditions, in particular shear stress, may be difficult to control. As a consequence, the macromolecular properties (e.g. viscosity, molecular weight) of recovered polymers frequently vary from one batch to another. As cell immobilization represents a possible way to overcome theses difficulties, several attempts to produce microbial exopolysaccharides using immobilized-cell systems have been reported over the last fifteen years [4–9]. While involving biopolymers developed at an industrial scale such as xanthan [4] and dextran [7], these attempts have neglected alginate-like * Corresponding author. Fax: + 33-2-3552-8485. E-mail address: [email protected] (G.-A. Junter).
polysaccharides synthesized, in particular, by the opportunistic human pathogen Pseudomonas aeruginosa and the soil bacterium Azotobacter 6inelandii [10]. In a previous work [11], however, we tested the feasibility of exopolysaccharide production by microbial cultures immobilized in composite agar layer/microporous membrane structures. This study confirmed the difficulty in producing high molecular weight (MW) macromolecules such as xanthan or pullulan by polymer-entrapped organisms, but yielded more promising results on alginate production by immobilized A. 6inelandii. In the present work, we have investigated more particularly the role of the microporous membrane filter on the fermentation efficiency of gel-entrapped A. 6inelandii cultures and on the MW characteristics of the polysaccharide produced.
2. Materials and methods
2.1. Microbial strain, culture medium and inoculum preparation A. 6inelandii NCIMB 9068 was maintained at 4 °C
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on Tryptic Soy Agar plates (Difco). The organisms were grown in the culture medium recommended by the NCIMB [12], containing the following ingredients (g dm − 3): sucrose, 20.0; K2HPO4, 1.0; CaCO3, 1.0; NaCl, 0.2; MgSO4·7H2O, 0.2; FeSO4·7H2O, 0.1; Na2MoO4·2H2O, 0.005. The carbon source and the salt solution (pH adjusted to 7.4 before autoclaving) were autoclaved separately. After incubation for 48 h at 30 °C, the organisms were collected by centrifugation (2200×g, 30 min) and resuspended in liquid medium.
(HPLC) with refractive index detection [11]. Alginate concentrations were obtained gravimetrically. The polysaccharide was precipitated with ethanol (2 vol. ethanol/vol. medium). The precipitate was collected by centrifugation (15 min at 7500× g) followed by filtration on a predried and preweighed glass microfibre filter (type GF/B from Whatman, Maidstone, UK). After washing the polysaccharide deposit with ethanol, the filter was dried at 60 °C for 24 h and reweighed. All determinations were performed in triplicate.
2.2. Shake flask fermentations
2.5. Alginate characterization
Free-cell fermentations were performed in 2-dm3 Erlenmeyer flasks containing 500 cm3 of culture medium. The flasks were incubated at 30 °C in a giratory air convection shaker (agitation speed, 200 rpm). During incubation, the pH value of the culture medium was maintained to its initial level (7.4) by addition of 1 M NaOH.
The polysaccharide was recovered by precipitation with ethanol followed by centrifugation as described above. The alginate pellet was dissolved in deionized water that was next supplied with lithium nitrate to yield a LiNO3 concentration of 0.1 M. The solution was then purified by filtration through a Millex GV filter (Millipore; pore size, 0.22 mm) or, when more stringent purification was needed, by ultracentrifugation (540,000× g for 1 h at 4 °C) followed by pellet resuspension in 0.1 M LiNO3. The molecular weight and size distribution of alginate were obtained by coupling size exclusion chromatography (SEC) with multiangle laser light scattering (MALLS). SEC separations were performed using two columns connected in series (Shodex OHpack SB-804 HQ and SB-806 HQ). The polymer sample (volume, 100 ml; concentration, ca. 1 mg ml − 1) was eluted with filtered (0.1 m pore size filter, Millipore) 0.1 M LiNO3 at a flow rate of 0.6 ml min − 1 (Intelligent Pump 301, Shicoh Engineering, Japan). The MALLS photometer (Dawn-F, Wyatt Technology, Santa Barbara, CA), equipped with a concentration sensitive detector (ERC7515A refractive index detector, Erma CR., Tokyo, Japan), was calibrated with standard pullulan samples (48-kDa molecular weight). SEC/MALLS experiments were performed at room temperature. The ASTRA V.4.50 software package (Wyatt Technology) was used to determine the weight-average molecular weight (M( w) and the MW distribution of alginate. Alginate from Macrocystis pyrifera (sodium salt, low viscosity type, Sigma, St. Louis, MO) was used as reference.
2.3. Cell immobilization and immobilized-cell fermentations Organisms were entrapped in composite agar layer/ microporous membrane structures as described previously [13]. The disc-shaped agar layers [10-cm2 surface; 3-mm thickness; 0.5% (w/v) agar] were loaded with a calibrated amount of A. 6inelandii cells (2.25 mg dry wt. cm − 3 gel). They were stored at 4 °C for 90 min and at room temperature for 30 min before use. The microporous membranes were standard mixed ester cellulose filters (Millipore, Freehold, NJ) with average pore size (i.e. diameter) ranging between 0.22 and 3.0 mm (porosities, 75–82%; thickness, 150 mm). The composite structure was fastened in vertical position to the wall of a 300-cm3 Pyrex reactor, at the level of a circular opening in the vessel wall [11]. The bioreactor was then filled with 250 cm3 of culture medium and placed at 30 °C in an air convection incubator. Magnetic stirring ensured oxygen supply by transfer through the residual air/liquid medium interface (kLa = 5.6 ×10 − 2 min − 1). The pH of the culture broth was maintained to 7.4 by addition of 1 M NaOH.
2.4. Sample analysis Broth samples of 2.5-cm3 volume were collected aseptically. The corresponding changes in broth volume did not exceed 10% over a fermentation run. Free-cell concentrations were monitored by optical density (OD) measurements at 600 nm. OD values were converted to dry weights using a calibration curve. Sucrose and alginate were titrated after elimination of free bacteria by centrifugation (2200× g, 30 min). Sucrose was titrated by high-performance liquid chromatography
3. Results and discussion
3.1. Free-cell experiments Alginate production by conventional free-cell cultures of A. 6inelandii grown in shake flasks mainly occurred during the exponential phase of bacterial growth, i.e. during the first 72 h of incubation, but some polysacharide production was noticed after the
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The weight-average molecular weight of the polysaccharide recovered in the free-cell culture broth at the end of the incubation run (i.e. after incubation for about 140 h) was equal to (1689 58) kDa (n= 6). This M( w value for A. 6inelandii alginate is close to the molecular weight indicated by Clementi [19], but much lower than those given by Pen˜ a et al. [18], ranging from 1400 to 2000 kDa after sucrose fermentation for 72 h in shake flasks. According to the quoted authors, the MW values of alginates produced by batch cultures of A. 6inelandii are dependent strongly on the aeration conditions of the fermentation broth [18] and/or the time at which alginate is recovered from the broth [18,19]. The MW distribution curve of alginate varied significantly from one batch to another (Fig. 2), confirming the difficulty to obtain reproducible macromolecular characteristics for alginate produced by free-cell cultures grown in shake flasks.
Fig. 1. Kinetics of sucrose uptake (), microbial growth (), and alginate production ( ) of a free-cell A. 6inelandii culture.
3.2. Immobilized-cell fermentations In the absence of a microporous membrane filter, cell leakage from the agar gel layer occurred at the very beginning of incubation, and a free-cell culture developed (not shown). Placing a microporous membrane at the gel layer/liquid medium interface— therefore, creating the so-called ‘composite structure’ —delayed significantly cell leakage. The membrane efficiency in preventing cell contamination of the culture broth increased logically as the membrane pore size (MPS) decreased (Table 1). For MPSs equal to or lower than 0.45 mm, the culture medium remained quasi sterile over an incubation period of about 200 h. Conversely, significant numbers of bacteria were released from composite structures with MPSs of 1.2 and 3.0 mm. For all tested MPSs, however, the amount of free cells in the broth remained very low compared to that observed without membrane. The kinetics of sucrose consumption and polysaccharide production were similar for all tested composite structures (Fig. 3). The alginate concentration reached a maximum level after incubation for about 100 h. While some sucrose consumption occurred later, the overall amount of sugar consumed remained very limited compared to that observed during free-cell cultures. The maximum alginate concentration increased slightly with the MPS to reach at 3.0-mm MPS a value close to
Fig. 2. Differential molecular weight distribution curves of alginate samples recovered from several independent free-cell production runs in shake flasks. The MW distribution of algal alginate (solid line) is shown as standard. Ordinate units (given by the MALLS) are arbitrary. M( w values (kDa): 1, 158; 2, 194; 3, 155; 4, 351; algal alginate, 270.
organisms had entered the stationary phase (Fig. 1). This confirms other results showing that alginate production by A. 6inelandii is partially associated to growth [14–18]. The amount of polysaccharide recovered ranged between 0.8 and 1.4 g dm − 3, with a mean of (1.090.2) g dm − 3 corresponding to a production yield of (8996) mg alginate g − 1 sucrose (n = 6). These values are low compared to those reported for diverse A. 6inelandii strains, in particular highly mucoid natural isolates [16,17] or mutants [14,15]. Nevertheless, they are in the range of the values obtained for A. 6inelandii NCIMB 9068 [17] or other strains [16] in shake flask cultures using nitrogen-free incubation media.
Table 1 Influence of the membrane pore size on cell retention inside the agar gel layer Membrane pore size (mm) Time at leakage (h) a
Not determined.
0.22 \220
0.45 \180
0.65 n.d.a
0.80 B120
1.20 B60
3.00 B30
No membrane B20
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Fig. 3. Typical kinetics of alginate production by immobilized A. 6inelandii cells. The pore size of the microporous membrane used in the composite structure was (A) 0.45 mm and (B) 1.2 mm. Symbols: alginate ( ), sucrose () and free cell () concentrations.
that obtained in free-cell cultures (Table 2). This cannot be explained by the very limited cell leakage at high MPS. This increase in alginate productivity with the MPS more probably arises from some polymer accumulation, higher at low MPS, between the agar layer and the microporous membrane, as low amounts of slime were systematically observed on the internal face of the microporous filters after the incubation runs. Average production rates ranged between ca. 0.5 and 0.7 mg alginate h − 1 cm − 3 gel, which, based on a cell content of 2.25 mg dry wt. cm − 3 gel, corresponds to specific productivities between 0.2 and 0.3 g alginate h − 1 g − 1 dry wt. Average production yields, calculated after 100 h of incubation, were slightly dispersed around a mean value of 0.24 g alginate produced g − 1 sucrose consumed. Owing to the low amounts of sugar consumed, such yields were noticeably higher than those obtained for free counterparts (ca. 0.09 g g − 1) and are in the range of yields reported by others for free-cell cultures of highly mucoid strains [14– 17]. This significant improvement in alginate production yield of immobilizedcell cultures compared to free-cell counterparts may be due to a decrease in the amount of sucrose consumed for biomass synthesis. Differences in the oxygenation levels of free and immobilized cells are also probably involved in this gain in yield. It has been reported that alginate production is negatively affected by increasing the oxygen supply to free-cell cultures in shake flasks [18] or stirred tanks [20]. Indeed, it is well known that gel-entrapped organisms are subject to oxygen limitation [21]. Another possible explanation lies in the pecu-
Fig. 4. SEC/MALLS analysis of alginate from immobilized-cell culture (membrane pore size, 0.65 mm): light scattering (solid line) and refractive index (dotted line) signals as a function of the elution volume. The molecular weight distribution as a function of the elution volume is also shown (). The high MW fraction collected between 12.2 and 14.2 cm3 of elution volume represented 4.4% of the total mass injected and had a M( w value of 1730 kDa. Ordinates: arbitrary units.
liar physiology of immobilized cells, which frequently display enhanced metabolic activities when compared to free cells [22]. It has been shown, for example, that natural immobilization of bacteria as biofilms favours cell conversion to mucoidy, resulting in exopolysaccharide overproduction [23]. Bacterial cells artificially immobilized by entrapment in agar gel layers constitute a simple in vitro model structure of natural biofilms [24]. In particular, physiological properties characterizing biofilm bacteria are recovered in gel-entrapped organisms [24]. Therefore, the increase in the production yield of alginate by gel-entrapped A. 6inelandii may also arise from physiological modifications linked to the immobilized state.
3.3. Immobilized-cell alginate characterization Whatever the MPS, the average molecular weight of alginate released from composite structures was somewhat higher than that obtained for free-cell alginate. There was no evident relationship between the MPS and the M( w value, however (Table 2). All analysed alginate samples contained small quantities of highmolecular-weight (i.e. several millions Da) polysaccharide, detected first in the SEC effluent (Fig. 4). Owing to the low amounts recovered in the samples (as shown by the concentration profile given by the RI detector: Fig. 4), however, the presence of these macromolecules
Table 2 Fermentation parameters of immobilized-cell cultures and M( w values of alginate produced. All data were the mean of two independent experiments Membrane pore size (mm) Alginate produced (g dm−3) Yield (g g−1) M( w (kDa) a
Not determined.
0.22 0.55 0.26 324
0.45 0.53 0.25 n.d.a
0.65 0.58 0.21 287
0.80 0.60 0.28 n.d.a
1.20 0.68 0.18 211
3.00 0.92 0.24 239
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ing to the short-lived bioproduction period, however, the amount of polysaccharide produced remained low—though it was in the range of that recovered from free-cell cultures. Future work will therefore be aimed at improving the fermentation performance of immobilized-cell cultures in terms of alginate productivity (choice of a more efficient strain, improvement of the oxygenation level of the gel layer, etc.).
Fig. 5. Differential molecular weight distribution curves of alginate samples recovered from replicated immobilized-cell production runs. The figures refer to the pore size of the microporous membranes used. Ordinates: arbitrary units.
exerted only a limited influence on the M( w values. Previous experiments with dextran fractions of varying molecular weight (10–2000 kDa) have shown that diffusion of the polysaccharide macromolecules through standard mixed ester cellulose Millipore filters was only slightly restricted [25]. Conversely, the diffusion of high MW (] 500 kDa) dextran fractions through sterile agar gel membranes was severely restricted [26]. Therefore, the high-sized polymer macromolecules probably corresponded to aggregates forming in the liquid medium, maybe as a consequence of the low agitation level of the medium, rather than high MW biosynthesized molecules such as those obtained previously by others [18]. In opposition with free-cell polysaccharide samples (Fig. 2), the differential molecular mass distribution curves of alginate recovered from immobilized-cell production runs were very similar for the different MPSs tested and looked fairly reproducible considering replicated experiments (Fig. 5). A number of studies have underlined the prominent influence of the oxygenation level and agitation speed on the MW value of free-cellculture alginate [18– 20,27]. More reproducible incubation conditions inside the gel layer than in shake flasks may be responsible for the stabilization of MW values and distribution observed here.
4. Conclusion A. 6inelandii immobilized in composite agar layer/microporous membrane structures showed efficient alginate production in terms of yield and volumetric productivity. The immobilized state seemed to stabilize the macromolecular characteristics of alginate produced, which is of practical interest since the commercial outlets of microbial polysaccharides rely on these characteristics [3]. The pore size of the microporous membrane preventing cell leakage from the gel layer exerted no noticeable influence on the fermentation performance and the molecular weight of alginate. Ow-
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