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Environmental factors influencing growth of and exopolysaccharide formation by Pediococcus parvulus 2.6
International Journal of Food Microbiology 111 (2006) 252 – 258 www.elsevier.com/locate/ijfoodmicro
Environmental factors influencing growth of and exopolysaccharide formation by Pediococcus parvulus 2.6 S. Velasco a,b , E. Årsköld a , M. Paese a , H. Grage a , A. Irastorza b , P. Rådström a , E.W.J. van Niel a,⁎ a
b
Applied Microbiology, Lund Institute of Technology, Lund University, PO Box 124, SE-221 00 Lund, Sweden Facultad de Químicas, Dpto. Química Aplicada, Paseo Manuel de Lardizabal, no. 320018 San Sebastián, Spain Received 24 November 2005; received in revised form 28 April 2006; accepted 2 June 2006
Abstract Natural exopolysaccharides (EPSs) from food-grade lactic acid bacteria have potential for development and exploitation as food additives and functional food ingredients with both health and economic benefits. In this study, we have examined the physiological capacity of EPS production in Pediococcus parvulus 2.6. EPS formation by P. parvulus 2.6 was found to be linked to biomass yields, provided that glucose was not limiting. Higher biomass yields and EPS productions were obtained when cultures were pH-controlled at pH 5.2. Various compounds have been tested for their influence on growth rate and EPS formation. Of those, only glucose (up to 75 g l− 1), ethanol (up to 4.9%, w/v) and glycerol (up to 6.6%, w/v) had positive effects on EPS production. EPS production was not directly linked to growth, because its production continued in the stationary phase provided that glucose was present. According to an empirical model, the growth of P. parvulus 2.6 was completely inhibited by 58.9 ± 18.1 g l− 1 lactate. Lactate, the sole fermentation product, was suggested to affect growth by chelation of manganese. The organism grew in an apparent linear fashion due to this imposed manganese limitation. This could be overcome by increasing the manganese concentration to at least 2 mg l− 1 in the medium. The excretion of Mn2+ upon depletion of glucose indicated that maintenance of the high Mn2+ gradient over the cell membrane is an energy requiring process. EPS production was increased from 0.12 g l− 1 to 4.10 g l− 1 in an improved medium that is based on the results from this study. © 2006 Elsevier B.V. All rights reserved. Keywords: Pediococcus parvulus; Exopolysaccharides; Growth; Manganese
1. Introduction The extracellular polysaccharides (EPS) produced by lactic acid bacteria (LAB) have the potential to play an important role as natural viscosifiers and texture enhancers in yogurts and other fermented milks, low-fat cheeses, and dairy desserts, and are therefore of interest to the food industry (De Vuyst, 2000). In addition, EPS such as β-D-glucan has been found to possess antithrombotic, antitumoral or immunomodulatory activity (Sutherland, 1998) and oat β-D-glucan reduces serum cholesterol levels (Braaten et al., 1994). Consequently, the influence of β-D-glucan producing strains of Lactobacillus brevis, L. delbrueckii ssp. bulgaricus and Pediococcus parvulus 2.6 has
⁎ Corresponding author. Tel.: +46 46 2220619; fax: +46 46 2224203. E-mail address: [email protected] (E.W.J. van Niel). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.06.008
been investigated on elaboration and intake of functional oatbased products (Mårtensson et al., 2002). However, EPS productivity by these bacteria is too low to make a production process economical feasible. The study of the optimal culture conditions for EPS production is a first attempt to understand the regulation of product formation. In general, the relative concentration of carbon and nitrogen, and the temperature have been found to be the important parameters for EPS production (Sutherland, 1990; Cerning et al., 1992; Kimmel et al., 1998). Previously, a ropy slime producing strain, P. parvulus 2.6, was isolated from spoiled cider from Basque country (Fernandez et al., 1996). This strain was found to produce maximally 0.12 g l− 1 EPS in cultures without pH-control (Dueñas et al., 2003). The aim of the present paper is to explore the influence of environmental factors on growth and EPS production by P. parvulus 2.6. It is tolerant to relatively high ethanol concentrations and such an environment stimulates its EPS formation (Manca de Nadra and Strasser de Saad, 1995). However, little is
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known of the quantitative effect on EPS formation and even less is known about inhibitors. For example, inhibition of growth and EPS production by LAB other than pediococci have been observed, such as through lower water activity (Fajardo-Lira et al., 1997), uncoupling of growth (Amrane and Prigent, 1999) and chelation of metal ions (Lü-Lü et al., 1992). Especially chelation of manganese appeared to have detrimental effects on streptococcal glucan-binding lectins (Lü-Lü et al., 1992). High demands for Mn2+ have been observed for pediococci (Efthymiou and Joseph, 1972), but the phenomenon has been studied in greater detail in Lactobacillus plantarum (Archibald and Duong, 1984). Pediococcus spp. have no superoxide dismutase, but have a high uptake of Mn2+ to protect itself against oxygen radicals (Archibald, 1986). The results in this study of Mn2+ uptake under anaerobic conditions indicate that P. parvulus 2.6 requires high intracellular Mn2+ levels for growth activation. This process can be jeopardized by the main fermentation product, lactate, which acts as a growth inhibitor most probably through chelation of manganese. As lactate is accumulating in the culture it can reach a critical concentration beyond which it might facilitate the excretion of the free intracellular manganese. This will have implications for the design of fermentation processes for EPS production with P. parvulus. Improved EPS formation could be accomplished in the presence of high glucose, manganese and ethanol concentrations. 2. Materials and methods 2.1. Microorganism and culture medium P. parvulus 2.6 was obtained from the UPV culture collection (Universidad del Pais Vasco, San Sebastian, Spain.). P. parvulus 2.6, formerly called P. damnosus 2.6 (Werning et al., 2006), was isolated from a ropy cider on modified Carr agar medium as described by Fernandez et al. (1996). The strain was stored at −80 °C in Man Rogosa Sharpe (MRS) broth containing 20% (v/v) glycerol. The inoculum cultures were incubated in MRS broth for 46 h, at 30 °C without pH control. The inoculum was collected by centrifugation at 5445 ×g for 20 min. The pellet was resuspended in 0.9% (w/v) NaCl solution. All pH-controlled fermentations were performed in a semi-defined medium (MST) (Levander et al., 2001), containing (per liter): 0.06 g L-alanine, 0.2 g L-serine, 0.03 g L-tryptophan, 0.2 g L-asparagine, 0.5 g Lcysteine, 0.01 g reduced glutathione, 1 ml tween 80 (Merck), 0.06 g uracil, 0.03 g adenine, 0.03 g guanine, and vitamins and trace elements according to Van Niel et al. (2002). The solutions of vitamins, trace elements, nucleic acid bases and amino acids
Table 1 Effect of pH on maximum EPS production pH
EPS (g·l− 1)
Glucose consumed (g·l− 1)
Maximum OD
EPS yield (g.g glucose− 1)
μmax (h− 1)
4 5.2 6
0.51 1.08 0.85
11.80 20.26 17.11
3.66 6.30 6.17
0.042 0.054 0.050
0.037 0.056 0.028
Glucose 50 g·l− 1 and casamino acids 5 g·l− 1 and 50–70 rpm stirring rate.
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Fig. 1. Example of a growth profile of P. parvulus 2.6 culture on glucose. Glucose (■), OD (♦), lactate (▴), EPS (○).
were filter sterilized and added to the media aseptically. The standard MST medium contained 25 g glucose and 7.5 g casamino acids (CA) (Technical; Difco) per liter. All chemicals were from Sigma, unless stated otherwise. 2.2. Cultivations Fermentations were carried out in 1-L fermenters with a working volume of 700 ml. The temperature was kept at 30 °C. The agitation was kept at 50–70 rpm to keep the fermentation broth homogeneous, and nitrogen gas (0.2 l·h− 1) was sparged through the headspace continuously to maintain anaerobic conditions. The pH was adjusted with 5 N NaOH. Three fermentations were run at different pH (4, 5.2 and 6) with 50 g glucose·l− 1 and 5 g CA·l− 1. All other batches were run at pH 5.2. To evaluate their influence, several compounds were added to the medium in various concentrations (see Results and discussion section). In an experiment to check whether there is a relation between EPS production and growth 20 mg l− 1 chloramphenicol was added to the culture when it was in the middle of its exponential growth phase to cease growth prematurely. Fermentation conditions were normally carried out in duplicate, but only once in cases where the focus was on seeking trends in the influence of an effecter on growth and EPS formation. Cell growth was determined by measuring the optical density (OD) of the culture fluid at 620 nm and dry weight of cells. Dry weight (DW) was determined in triplicate by collecting 3 ml cells from fermentation on pre-weighted 0.2 μm filters (Gelman Sciences). The filters were washed three times with 3 ml water and subsequently dried in a microwave oven for 8 min at 350 W. A relation between OD and DW was calculated: DW = 0.22·OD620 + 0.101 mg l− 1 (R2 = 0.95). Samples were taken in the exponential and stationary phase for viable count on MRS agar plates, which were incubated in an anaerobic jar at 30 °C, and growth was checked after 5 days. Glucose and fermentation products were determined by HPLC in triplicate. The compounds were separated on an Aminex HPX87H column (BioRad, Hercules, CA, USA) and detected with a refractometer (Shimadzu Rid-6A, Kyoto. Japan). (Palmfeldt et al., 2004). For total manganese concentrations, liquid samples of 1 ml
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were withdrawn from the cultures and centrifuged at 13,800 ×g for 3 min. The supernatants were acidified with 16 μl of 65% HNO3 and stored in pre-treated micro-centrifuge tubes. The manganese concentrations were determined in triplicate with a graphite furnace atomic absorption spectrophotometer (GBC 932 AA, Shimadzu, Kyoto, Japan). External standards were used to enable quantitative determination. 2.3. EPS isolation and quantification To isolate EPS from the culture broth, first the proteins were precipitated by adding 1/3 volume of 40% (v/v) trichloroacetic acid (TCA), and were subsequently removed by centrifugation at 23,000 ×g for 20 min at 4 °C. The supernatant was transferred to a similar volume of ice-cold acetone. After mixing the solution was kept overnight at 4 °C. The precipitated EPS was picked up with a glass rod and stored in a pre-weighed eppendorf tube. The supernatants of these samples were centrifuged at 23,000 ×g for 20 min. The supernatant was removed and the pellet was dissolved in 6 ml phosphate buffer by shaking for 1 h. The suspension was mixed with 6 ml of ice-cold acetone after which the EPS was precipitated for 4 h at 4 °C. The precipitated EPS was picked up with a glass rod and transferred to the same preweighed eppendorf tube for drying in a vacuum centrifuge at 65 °C for 30 min (DNA Speed Vac DNA 110 (Savant). The eppendorf tubes were weighed to estimate the amount of EPS. 2.4. Model development Lactate inhibition. A generally applicable equation of growth inhibition (Han and Levenspiel, 1988) was adapted to the inhibiting effect of lactate: N S L l ¼ lMAX d ð1Þ d 1− KM þ S LCRIT
EPS were similar at pH 5.2 and 6 but the final concentration and growth rate were higher at pH 5.2 (Table 1). Therefore, pH 5.2 was chosen for the rest of the study. Cells growing under pH-controlled conditions in MST medium had a relatively short exponential growth phase after which growth slowly diminished before it entered the stationary phase (Fig. 1). Cells growing in MRS medium had a maximum specific growth rate that was at least twofold higher. This indicated that growth was either inhibited or limited by a compound other than glucose. When glucose was depleted the EPS concentration often started to decline (data not shown). This is observed frequently in fermentations of other EPSproducing LAB and is possibly due to a combination of glucose limitation and activation of glycohydrolases (Pham et al., 2000). No significant differences were observed in growth rate and EPS production when the casamino acids concentration was varied between 2.5 and 7.5 g·l− 1 (data not shown). This is in contrast to studies with other LAB showing that there is a relationship between EPS and N-source (Kimmel et al., 1998; Degeest and De Vuyst, 1999). Possibly the casamino acids concentration was beyond the range where it might have limited the growth performance of P. parvulus 2.6. A higher glucose concentration was seen to have a positive effect on EPS formation by P. damnosus IOEB8801 (Walling et al., 2005). Indeed, the yield of EPS by P. parvulus 2.6 increased also with the glucose concentration (Fig. 2), which could be due to that glucose did not became limiting. EPS production occurred throughout exponential growth phase and continued in the stationary growth phase. Hence, growth and EPS formation were not directly coupled in P. parvulus 2.6, which is in contrast to P. damnosus IOEB8801 (Walling et al., 2005). However, growthassociated and non-growth-associated production of EPS by LAB has been observed before (Manca de Nadra et al., 1985; Kojic et al., 1992; Looijesteijn and Hugenholtz, 1999).
where μMAX is the maximal specific growth rate (h− 1), KM the apparent affinity constant for glucose (g l− 1), S is the glucose concentration (g l− 1), L is the lactate concentration (g l− 1), LCRIT (g l− 1) is the critical lactate concentration at which growth is inhibited completely, and N is a coefficient pertaining to the lactate concentration. The latter parameter did not remain constant, but could be described as a function of the lactate concentration: N ¼ A þ Bd½LC
ð2Þ
where A, B and C are coefficients. Values and standard errors for the parameters μMAX, KS, LCRIT, A, B and C were estimated by fitting the data using the non-linear least-squares regression method with R software (http://www.r-project.org). 3. Results and discussion 3.1. Effect of the medium composition Three different pH (4, 5.2 and 6) were initially tested to find an appropriate pH for EPS formation. The specific yields of
Fig. 2. Plots of the effect of various compounds on the maximum specific growth rate (a) and EPS production (b) as a function of osmolarity. Glucose (⋄), glycerol (▴), acetate (▵), ethanol (♦), NaCl (■), lactate (●) and citrate (○).
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Table 2 Effect of NaCl and ethanol on the total consumption of glucose and corresponding lactate production (ND = not determined)
NaCl (g·l− 1)
Ethanol% (w/v)
14.0 28.0 40.0 1.65 3.62 4.87
Glucose consumed (g·l− 1)
Lactate produced (g·l− 1)
DW (g·l− 1)
23.43 12.79 1.85 26.28 21.31 24.85
19.57 6.59 1.48 22.52 17.56 24.26
2.15 0.53 0.29 2.82 ND 3.50
Increasing glucose concentrations affected the growth rate and biomass yield negatively, which may be due to the osmolarity. This possibility was tested using different concentrations of NaCl. The decline of the growth rate and growth yield with increasing osmolarity of NaCl was similar to that for glucose (Fig. 2a, Table 2). However, in contrast to glucose, EPS formation was not stimulated by NaCl (Fig 2b). With a NaCl concentration of 2.8% (w/v) (0.89 Os kg− 1) the lag phase was prolonged to about 300 h and hardly any EPS was produced, while in 4% (w/v) (1.24 Os·kg− 1) NaCl the culture failed to grow. With Lactococcus lactis subsp. cremoris NIZO B40 similar results were obtained and it was concluded that its EPS formation is not a stress response (Looijesteijn and Hugenholtz, 1999). 3.2. Effect of compounds present in Basque cider It has been observed that ethanol and glycerol, both present in Basque ciders (Del Campo et al., 2003), have a stimulating effect on EPS formation by P. parvulus. Ethanol affected the specific growth rate negatively (Fig. 2a), but the growth yield positively (Table 2). The best growth rate was found in the lowest ethanol concentration used (Fig. 2a), but the best EPS production (2.8 g l− 1) was found in the presence of 4.9% (w/v) (1.24 Os·kg− 1) of ethanol (Fig. 2b). The specific growth rate and yield were not affected significantly by glycerol (Fig. 2a), confirming the observation that P. parvulus 2.6 does not use glycerol as carbon source
Fig. 4. (A) Influence of different minerals on the growth of P. parvulus 2.6. Trace elements (TE) 7 times more than in control (♦), Co2+ and Mn2+ (■), Cu2+ and Mn2+ (□), Co2+ and Cu2+ (▴), Co2+, Cu2+ and Mn2+ (▵). (B) Concentration profile of the sum of chelated and free Mn2+ in the culture fluid. Control (○), Co2+ and Mn2+ (■), Cu2+ and Mn2+ (□), Co2+, Cu2+ and Mn2+(▵).
(Fernandez et al., 1996). The EPS formation improved only slightly (Fig. 2b) with the best production in the presence of 6.6% (w/v) (0.89 Os·kg− 1) glycerol. The results with ethanol and glycerol differ from NaCl indicating that osmolarity is not the common mechanism behind improved EPS formation. The mechanism behind stimulation by ethanol and glycerol remains unclear, but several possibilities have been described. First, ethanol and glycerol are able to cross the membrane unhindered and subsequently affect various physiological processes, including initiating changes in the lipid constitution of the cell membrane (see e.g. Weber and de Bont, 1996; Silveira et al., 2004). Second, ethanol can be a stress signal to activate a ‘ropy plasmid’ in ropy P. damnosus strains (Lonvaud-Funel et al., 1991). 3.3. Effect of lactate
Fig. 3. Growth of P. parvulus 2.6 on glucose in the presence of different lactate concentrations. Initial lactate concentrations (g l− 1): 6.9 (○), 11.9 (▵), 16.8 (⋄), 29.7 (□). The lines represent best fits of Eq. (1) through the data points.
The inhibiting effect of lactate on growth of microorganisms is frequently described because of its uncoupling (Amrane and Prigent, 1999) or its chelating (Lü-Lü et al., 1992) properties. To investigate whether uncoupling agents, such as weak acids, influence growth rate and EPS production at pH 5.2 P. parvulus 2.6 was grown in different concentrations of acetate, lactate and citrate. The specific growth rate and EPS production were strongly affected by each of these compounds (Fig. 2). In the presence of 20.4 g l− 1 of acetate, 16.8 g l− 1 of lactate (Fig. 3), or 19.2 g l− 1 of citrate growth appeared to progress in a linear
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concentrations sevenfold revealed that P. parvulus 2.6 had only requirements for Mn2+ (Fig. 4a). The specific requirement for Mn2+ among LAB, including pediococci, has been observed before (Efthymiou and Joseph, 1972). Addition of sevenfold increase of trace elements solution was detrimental, most likely because of the concomitant addition of more EDTA (0.1 g l− 1) to the medium (Fig. 4a). 3.5. Mn2+ uptake
Fig. 5. Growth profile of Pediococcus parvulus 2.6 with 1 ppm of Mn Glucose (▵), OD (■), lactate (□), EPS (▴), Mn2+ (⋄).
2+
.
fashion. The effect on the growth rate increased in intensity from acetate to lactate to citrate. This is contrary to the uncoupling effect of weak acids, because in that case acetate (pKA value of 4.75) would have been a stronger inhibitor than lactate (pKA value of 3.86). Each of these weak acids act also as chelators for metal ions and this could explain very well the order of strength of inhibition observed in the cultures (Lü-Lü et al., 1992). In addition, the chelating property of lactate could also be the explanation for the observed behaviour of growth (Fig. 1), since it might impose a trace element limitation on the growing culture. Since lactate is the major fermentation product its accumulation in the fermentation broth is unavoidable. Hence, it is vital to estimate its potential to affect growth and EPS formation in pediococcal cultures. Therefore, P. parvulus 2.6 was grown in the presence of various initial lactate concentrations, ranging approximately 7–37 g l− 1 (Fig. 3). Lactate affected both the growth rate and the mode of growth: the length of exponential growth phase shortened and the apparent linear growth phase prolonged concomitantly with the initial lactate concentration (Fig. 3). Using a KM value of 0.36 g l− 1 in Eq. (1) and the data points of all the fermentations, the values of the remaining parameters were calculated. The model was fitted through the experimental data, revealing the following parameter values: μMAX = 0.12 ± 0.02 h− 1, LCRIT = 58.9 ± 18.1 g l− 1, A = 2.42 ± 2.13, B = 34.4 ± 18.3 and C = − 0.87 ± 0.23. Most of these parameters are quite strongly correlated, which will make the inference about these parameters less straightforward. It further remains to be explained in biological terms why the lactate concentration has a highly non-linear relationship with the growth rate. The inhibiting effect of lactate was mimicked by citrate, albeit that lower concentrations were necessary because of its stronger chelating properties (data not shown). Thus, citrate had no positive effect on growth as has been observed for L. plantarum (Archibald and Duong, 1984).
The extracellular Mn2+ concentration was followed throughout the fermentation (Fig. 4b). Mn2+ uptake occurs only during the exponential phase and then ceases. Notably, after glucose became limited in the stationary phase, the extracellular manganese concentration increased until the level came back closely to the initial concentration of the medium (Fig. 5), indicating that all intracellular free Mn2+ must have been excreted. This was not due to any massive cell death or leakage, because samples for viability tests taken before and after the manganese excretion showed no difference in the number of colony forming units (data not shown). Like with L. plantarum (Archibald and Duong, 1984), higher initial Mn2+ concentrations in the medium led to more Mn2+ uptake. Excretion of Mn2+ upon glucose depletion is an indication that energy is required to maintain the gradient. Like in L. plantarum (Archibald and Duong, 1984), a proton motive force driven Mn2+ transporter, possibly of the MntH-type, will be involved. An ORF with 79% identity at gene level with such a proton symporting transporter of L. brevis have been found in P. pentosaceus (http://genome. ornl.gov/microbial/ppen/). In a fermentation to which 14 g l− 1 of lactate and a relatively high manganese concentration was added prior to inoculation, no uptake of Mn2+ was observed (Fig. 6). This resulted in slow growth for about 120 h, after which growth accelerated probably because of induction of an uptake system with a higher affinity for manganese. The latter was not observed in cultures with low manganese concentration (see e.g. Fig. 3). It clearly demonstrated the necessity of high Mn2+ uptake for relative fast growth. For lactobacilli it has been seen that Mn2+
3.4. Effect of trace element concentrations To determine which trace elements could be potentially become limiting through chelation, the growth test focused on Co2+, Cu2+ and Mn2+, because of their low concentration in the medium: 0.07, 0.07, 0.3 mg l− 1, respectively. Increasing their
Fig. 6. Growth profile of a Pediococcus parvulus 2.6 culture in the presence of 27 ppm of Mn 2+ to which an initial lactate concentration of 14 g·l− 1 was added. Glucose (▵), OD (■), lactate (□), EPS (▴), Mn2+ (⋄).
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Fig. 7. Specific EPS yield in P. parvulus 2.6 cultures with improved environmental conditions. Control with 25 g glucose·l− 1 and 5 mg Mn2+ l− 1 (⋄), 50 g glucose·l− 1, 5 mg Mn2+·l− 1 and 4.9% (w/v) ethanol (□), 25 g glucose·l− 1, 5 mg Mn2+·l− 1 and 5% (w/v) ethanol (▵).
may rapidly trigger growth through creation of a high cytoplasmic [Mn2+]/[Mg2+] ratio by which the activity of many catabolic, but also anabolic, enzymes are enhanced (Archibald, 1986; Kehres and Maguire, 2003). This may be true for P. parvulus too, however, its lactate dehydrogenase, pyruvate kinase, phosphofructokinase and glucokinase did not show any preference for Mn2+ or Mg2+(data not shown). Interestingly, Mn2+ is known to inhibit various glycohydrolases that break down EPS (Pham et al., 2000). Hence, chelation of Mn2+ could very well be the cause of activation of these hydrolases at the end of the fermentations. 3.6. Enhancement of EPS formation Fermentations in batch mode indicated that EPS production increased in the stationary phase (Fig. 1). Therefore, an experiment was carried out to stop growth prematurely, using chloramphenicol, to check whether it would enhance EPS formation. However, the specific EPS yield did not differ in the presence of the antibiotic (data not shown). Hence, a better biomass production will improve EPS production, as was the conclusion of several other studies (Mozzi et al., 1995; Looijesteijn and Hugenholtz, 1999). A significantly higher EPS concentration (4.1 g l− 1) could be obtained with P. parvulus 2.6 by combining the best conditions as they were found in this study, being higher concentrations of Mn2+(5 mg l− 1) glucose (50 g l− 1) and ethanol (4.9% (w/v)) (Fig. 7). In those conditions the EPS concentration was raised more than 30-fold compared to previous reports (Dueñas et al., 2003). In the presence of ethanol the lag phase was prolonged, as a result of adaptation, but still high biomass concentrations could be reached favourable enough for EPS production. 4. Conclusions Conditions leading to higher maximum biomass yields resulted in higher levels of EPS formation in P. parvulus 2.6, provided that adequate concentrations of glucose were present.
Higher biomass yields were obtained when cultures were pHcontrolled at pH 5.2. Glucose, ethanol and glycerol had positive effects on EPS formation. Higher glucose concentrations secured continued EPS formation in the stationary phase. Ethanol and glycerol can be stress signals affecting physiological processes, including initiating changes in the lipid constitution of the cell membrane. Because of the low manganese concentrations in the medium, the chelating action of lactate was such that it limited the growth rate of P. parvulus 2.6. The organism grew in an apparent linear fashion due to imposed manganese limitation. This could be overcome by increasing the manganese concentration to at least 2 mg·l− 1 in the medium. Upon substrate depletion the cells excreted their intracellular free manganese, indicating the need of an energy source to maintain the high Mn2+ gradient over the membrane. It remains unknown in how far the lactate concentration contributes to this excretion process or to the activation of glycohydrolases and, therefore, this merits further investigation. Unlike L. plantarum, P. parvulus 2.6 does not use citrate as functional manganese-binding siderophore. Improved EPS formation (4.1 g l− 1) was obtained in a medium containing high concentrations of glucose (50 g l− 1), Mn2+(5 mg l− 1) and ethanol (4.9% (w/v)). Lactate, as the main fermentation product, is a severe growth inhibitor in P. parvulus cultures. Therefore it is important to know under what conditions it will become an inhibiting factor, and how this burden can be avoided. Further studies are underway addressing these issues. Acknowledgments This study was supported by a Marie Curie grant from the European Commission (QLK3-CT-2001-60077), VINNOVA, the Swedish Agency for Innovation Systems, and “Becas de formación de Investigadores del Departamento de Educación, Universidad e Investigación” from the Basque Country Government. We acknowledge the assistance of Dr Raul Muñoz Torre and Dr Roberto Romero in the GFAAS methodology.
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Lonvaud-Funel, A., Joyeux, A., Ledoux, O., 1991. Specific enumeration of lactic acid bacteria in fermenting grape must and wine by colony hybridization with non-isotopic DNA probes. J. Appl. Bacteriol. 71, 501–508. Looijesteijn, P.J., Hugenholtz, J., 1999. Uncoupling of growth and exopolysaccharide production by Lactococcus lactis subsp. cremoris NIZO B40 and optimization of its synthesis. J. Biosci. Bioeng. 88, 178–182. Lü-Lü, Singh, J.S., Galperin, M.Y., Drake, D., Taylor, K.G., Doyle, R.J., 1992. Chelating agents inhibit activity and prevent expression of streptococcal glucan-binding lectins. Infect. Immun. 60, 3807–3813. Manca de Nadra, M.C., Strasser de Saad, A.M., 1995. Polysaccharide production by Pediococcus pentasaceus from wine. Int. J. Food Microbiol. 27, 101–106. Manca de Nadra, M.C., Strasser de Saad, A.M., Pesce de Ruiz Holgado, A.A., Oliver, G., 1985. Extracellular polysaccharide production by Lactobacillus bulgaricus CRL 420. Milchwissenschaft 40, 409–411. Mårtensson, O., Dueñas-Chasco, M., Irastorza, A., Holst, O., Rudling, M., Norin, E., Midvedt, T., Öste, R., 2002. Effects of fermented, ropy, non-dairy, oat-based products on serum lipids and faecal excretion of cholesterol and short chain fatty acids in germfree and conventional rats. Nutr. Res. 22, 1461–1473. Mozzi, F., Savou de Giori, G., Oliver, G., Font de Valdez, G., 1995. Exopolysaccharide production by Lactobacillus casei. I. Influence of salts. Milchwissenschaft 50, 186–188. Palmfeldt, J., Paese, M., Hahn-Hägerdal, B., Van Niel, E.W.J., 2004. The pool of ADP and ATP regulates anaerobic product formation in resting cells of Lactococcus lactis. Appl. Environ. Microbiol. 70, 5477–5484. Pham, P.L., Dupont, I., Roy, D., Lapointe, G., Cerning, J., 2000. Production of exopolysaccharide by Lactobacillus rhamnosus R and analysis of its enzymatic degradation during prolonged fermentation. Appl. Environ. Microbiol. 66, 2302–2310. Silveira, M.G., Baumgartner, M., Rombouts, F.M., Abee, T., 2004. Effect of adaptation to ethanol on cytoplasmic and membrane protein profiles of Oenococcus oeni. Appl. Environ. Microbiol. 70, 2748–2755. Sutherland, I.W., 1990. Biotechnology of Microbial Exopolysacharides. Cambridge University Press, New York, USA, p. 163. Sutherland, I.W., 1998. Novel and established applications of microbial polysaccharide. Trends Biotechnol. 16, 41–46. Van Niel, E.W.J., Hofvendahl, K., Hahn-Hägerdal, B., 2002. Formation and conversion of oxygen metabolites by Lactococcus lactis subsp lactis ATCC 19435 under different growth conditions. Appl. Environ. Microbiol. 68, 4350–4356. Walling, E., Dols-Lafargue, M., Lonvaud-Funel, A., 2005. Glucose fermentation kinetics and exopolysaccharide production by ropy Pediococcus damnosus IOEB8801. Food Microbiol. 22, 71–78. Weber, F.J., de Bont, J.A.M., 1996. Adaptation mechanisms of microorganisms to the toxic effects of organic solvents on membranes. Biochim. Biophys. Acta 1286, 225–245. Werning, M.L., Ibarburu, I., Dueñas, M.T., Irastorza, A., Navas, J., López, P., 2006. Pediococcus parvulus gtf gene encoding the GTF glycosyltransferase and its application for specific PCR detection of β-D-glucan-producing bacteria in foods and beverages. J. Food Prot. 69, 161–169.
Environmental factors influencing growth of and exopolysaccharide formation by Pediococcus parvulus 2.6 S. Velasco a,b , E. Årsköld a , M. Paese a , H. Grage a , A. Irastorza b , P. Rådström a , E.W.J. van Niel a,⁎ a
b
Applied Microbiology, Lund Institute of Technology, Lund University, PO Box 124, SE-221 00 Lund, Sweden Facultad de Químicas, Dpto. Química Aplicada, Paseo Manuel de Lardizabal, no. 320018 San Sebastián, Spain Received 24 November 2005; received in revised form 28 April 2006; accepted 2 June 2006
Abstract Natural exopolysaccharides (EPSs) from food-grade lactic acid bacteria have potential for development and exploitation as food additives and functional food ingredients with both health and economic benefits. In this study, we have examined the physiological capacity of EPS production in Pediococcus parvulus 2.6. EPS formation by P. parvulus 2.6 was found to be linked to biomass yields, provided that glucose was not limiting. Higher biomass yields and EPS productions were obtained when cultures were pH-controlled at pH 5.2. Various compounds have been tested for their influence on growth rate and EPS formation. Of those, only glucose (up to 75 g l− 1), ethanol (up to 4.9%, w/v) and glycerol (up to 6.6%, w/v) had positive effects on EPS production. EPS production was not directly linked to growth, because its production continued in the stationary phase provided that glucose was present. According to an empirical model, the growth of P. parvulus 2.6 was completely inhibited by 58.9 ± 18.1 g l− 1 lactate. Lactate, the sole fermentation product, was suggested to affect growth by chelation of manganese. The organism grew in an apparent linear fashion due to this imposed manganese limitation. This could be overcome by increasing the manganese concentration to at least 2 mg l− 1 in the medium. The excretion of Mn2+ upon depletion of glucose indicated that maintenance of the high Mn2+ gradient over the cell membrane is an energy requiring process. EPS production was increased from 0.12 g l− 1 to 4.10 g l− 1 in an improved medium that is based on the results from this study. © 2006 Elsevier B.V. All rights reserved. Keywords: Pediococcus parvulus; Exopolysaccharides; Growth; Manganese
1. Introduction The extracellular polysaccharides (EPS) produced by lactic acid bacteria (LAB) have the potential to play an important role as natural viscosifiers and texture enhancers in yogurts and other fermented milks, low-fat cheeses, and dairy desserts, and are therefore of interest to the food industry (De Vuyst, 2000). In addition, EPS such as β-D-glucan has been found to possess antithrombotic, antitumoral or immunomodulatory activity (Sutherland, 1998) and oat β-D-glucan reduces serum cholesterol levels (Braaten et al., 1994). Consequently, the influence of β-D-glucan producing strains of Lactobacillus brevis, L. delbrueckii ssp. bulgaricus and Pediococcus parvulus 2.6 has
⁎ Corresponding author. Tel.: +46 46 2220619; fax: +46 46 2224203. E-mail address: [email protected] (E.W.J. van Niel). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.06.008
been investigated on elaboration and intake of functional oatbased products (Mårtensson et al., 2002). However, EPS productivity by these bacteria is too low to make a production process economical feasible. The study of the optimal culture conditions for EPS production is a first attempt to understand the regulation of product formation. In general, the relative concentration of carbon and nitrogen, and the temperature have been found to be the important parameters for EPS production (Sutherland, 1990; Cerning et al., 1992; Kimmel et al., 1998). Previously, a ropy slime producing strain, P. parvulus 2.6, was isolated from spoiled cider from Basque country (Fernandez et al., 1996). This strain was found to produce maximally 0.12 g l− 1 EPS in cultures without pH-control (Dueñas et al., 2003). The aim of the present paper is to explore the influence of environmental factors on growth and EPS production by P. parvulus 2.6. It is tolerant to relatively high ethanol concentrations and such an environment stimulates its EPS formation (Manca de Nadra and Strasser de Saad, 1995). However, little is
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known of the quantitative effect on EPS formation and even less is known about inhibitors. For example, inhibition of growth and EPS production by LAB other than pediococci have been observed, such as through lower water activity (Fajardo-Lira et al., 1997), uncoupling of growth (Amrane and Prigent, 1999) and chelation of metal ions (Lü-Lü et al., 1992). Especially chelation of manganese appeared to have detrimental effects on streptococcal glucan-binding lectins (Lü-Lü et al., 1992). High demands for Mn2+ have been observed for pediococci (Efthymiou and Joseph, 1972), but the phenomenon has been studied in greater detail in Lactobacillus plantarum (Archibald and Duong, 1984). Pediococcus spp. have no superoxide dismutase, but have a high uptake of Mn2+ to protect itself against oxygen radicals (Archibald, 1986). The results in this study of Mn2+ uptake under anaerobic conditions indicate that P. parvulus 2.6 requires high intracellular Mn2+ levels for growth activation. This process can be jeopardized by the main fermentation product, lactate, which acts as a growth inhibitor most probably through chelation of manganese. As lactate is accumulating in the culture it can reach a critical concentration beyond which it might facilitate the excretion of the free intracellular manganese. This will have implications for the design of fermentation processes for EPS production with P. parvulus. Improved EPS formation could be accomplished in the presence of high glucose, manganese and ethanol concentrations. 2. Materials and methods 2.1. Microorganism and culture medium P. parvulus 2.6 was obtained from the UPV culture collection (Universidad del Pais Vasco, San Sebastian, Spain.). P. parvulus 2.6, formerly called P. damnosus 2.6 (Werning et al., 2006), was isolated from a ropy cider on modified Carr agar medium as described by Fernandez et al. (1996). The strain was stored at −80 °C in Man Rogosa Sharpe (MRS) broth containing 20% (v/v) glycerol. The inoculum cultures were incubated in MRS broth for 46 h, at 30 °C without pH control. The inoculum was collected by centrifugation at 5445 ×g for 20 min. The pellet was resuspended in 0.9% (w/v) NaCl solution. All pH-controlled fermentations were performed in a semi-defined medium (MST) (Levander et al., 2001), containing (per liter): 0.06 g L-alanine, 0.2 g L-serine, 0.03 g L-tryptophan, 0.2 g L-asparagine, 0.5 g Lcysteine, 0.01 g reduced glutathione, 1 ml tween 80 (Merck), 0.06 g uracil, 0.03 g adenine, 0.03 g guanine, and vitamins and trace elements according to Van Niel et al. (2002). The solutions of vitamins, trace elements, nucleic acid bases and amino acids
Table 1 Effect of pH on maximum EPS production pH
EPS (g·l− 1)
Glucose consumed (g·l− 1)
Maximum OD
EPS yield (g.g glucose− 1)
μmax (h− 1)
4 5.2 6
0.51 1.08 0.85
11.80 20.26 17.11
3.66 6.30 6.17
0.042 0.054 0.050
0.037 0.056 0.028
Glucose 50 g·l− 1 and casamino acids 5 g·l− 1 and 50–70 rpm stirring rate.
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Fig. 1. Example of a growth profile of P. parvulus 2.6 culture on glucose. Glucose (■), OD (♦), lactate (▴), EPS (○).
were filter sterilized and added to the media aseptically. The standard MST medium contained 25 g glucose and 7.5 g casamino acids (CA) (Technical; Difco) per liter. All chemicals were from Sigma, unless stated otherwise. 2.2. Cultivations Fermentations were carried out in 1-L fermenters with a working volume of 700 ml. The temperature was kept at 30 °C. The agitation was kept at 50–70 rpm to keep the fermentation broth homogeneous, and nitrogen gas (0.2 l·h− 1) was sparged through the headspace continuously to maintain anaerobic conditions. The pH was adjusted with 5 N NaOH. Three fermentations were run at different pH (4, 5.2 and 6) with 50 g glucose·l− 1 and 5 g CA·l− 1. All other batches were run at pH 5.2. To evaluate their influence, several compounds were added to the medium in various concentrations (see Results and discussion section). In an experiment to check whether there is a relation between EPS production and growth 20 mg l− 1 chloramphenicol was added to the culture when it was in the middle of its exponential growth phase to cease growth prematurely. Fermentation conditions were normally carried out in duplicate, but only once in cases where the focus was on seeking trends in the influence of an effecter on growth and EPS formation. Cell growth was determined by measuring the optical density (OD) of the culture fluid at 620 nm and dry weight of cells. Dry weight (DW) was determined in triplicate by collecting 3 ml cells from fermentation on pre-weighted 0.2 μm filters (Gelman Sciences). The filters were washed three times with 3 ml water and subsequently dried in a microwave oven for 8 min at 350 W. A relation between OD and DW was calculated: DW = 0.22·OD620 + 0.101 mg l− 1 (R2 = 0.95). Samples were taken in the exponential and stationary phase for viable count on MRS agar plates, which were incubated in an anaerobic jar at 30 °C, and growth was checked after 5 days. Glucose and fermentation products were determined by HPLC in triplicate. The compounds were separated on an Aminex HPX87H column (BioRad, Hercules, CA, USA) and detected with a refractometer (Shimadzu Rid-6A, Kyoto. Japan). (Palmfeldt et al., 2004). For total manganese concentrations, liquid samples of 1 ml
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were withdrawn from the cultures and centrifuged at 13,800 ×g for 3 min. The supernatants were acidified with 16 μl of 65% HNO3 and stored in pre-treated micro-centrifuge tubes. The manganese concentrations were determined in triplicate with a graphite furnace atomic absorption spectrophotometer (GBC 932 AA, Shimadzu, Kyoto, Japan). External standards were used to enable quantitative determination. 2.3. EPS isolation and quantification To isolate EPS from the culture broth, first the proteins were precipitated by adding 1/3 volume of 40% (v/v) trichloroacetic acid (TCA), and were subsequently removed by centrifugation at 23,000 ×g for 20 min at 4 °C. The supernatant was transferred to a similar volume of ice-cold acetone. After mixing the solution was kept overnight at 4 °C. The precipitated EPS was picked up with a glass rod and stored in a pre-weighed eppendorf tube. The supernatants of these samples were centrifuged at 23,000 ×g for 20 min. The supernatant was removed and the pellet was dissolved in 6 ml phosphate buffer by shaking for 1 h. The suspension was mixed with 6 ml of ice-cold acetone after which the EPS was precipitated for 4 h at 4 °C. The precipitated EPS was picked up with a glass rod and transferred to the same preweighed eppendorf tube for drying in a vacuum centrifuge at 65 °C for 30 min (DNA Speed Vac DNA 110 (Savant). The eppendorf tubes were weighed to estimate the amount of EPS. 2.4. Model development Lactate inhibition. A generally applicable equation of growth inhibition (Han and Levenspiel, 1988) was adapted to the inhibiting effect of lactate: N S L l ¼ lMAX d ð1Þ d 1− KM þ S LCRIT
EPS were similar at pH 5.2 and 6 but the final concentration and growth rate were higher at pH 5.2 (Table 1). Therefore, pH 5.2 was chosen for the rest of the study. Cells growing under pH-controlled conditions in MST medium had a relatively short exponential growth phase after which growth slowly diminished before it entered the stationary phase (Fig. 1). Cells growing in MRS medium had a maximum specific growth rate that was at least twofold higher. This indicated that growth was either inhibited or limited by a compound other than glucose. When glucose was depleted the EPS concentration often started to decline (data not shown). This is observed frequently in fermentations of other EPSproducing LAB and is possibly due to a combination of glucose limitation and activation of glycohydrolases (Pham et al., 2000). No significant differences were observed in growth rate and EPS production when the casamino acids concentration was varied between 2.5 and 7.5 g·l− 1 (data not shown). This is in contrast to studies with other LAB showing that there is a relationship between EPS and N-source (Kimmel et al., 1998; Degeest and De Vuyst, 1999). Possibly the casamino acids concentration was beyond the range where it might have limited the growth performance of P. parvulus 2.6. A higher glucose concentration was seen to have a positive effect on EPS formation by P. damnosus IOEB8801 (Walling et al., 2005). Indeed, the yield of EPS by P. parvulus 2.6 increased also with the glucose concentration (Fig. 2), which could be due to that glucose did not became limiting. EPS production occurred throughout exponential growth phase and continued in the stationary growth phase. Hence, growth and EPS formation were not directly coupled in P. parvulus 2.6, which is in contrast to P. damnosus IOEB8801 (Walling et al., 2005). However, growthassociated and non-growth-associated production of EPS by LAB has been observed before (Manca de Nadra et al., 1985; Kojic et al., 1992; Looijesteijn and Hugenholtz, 1999).
where μMAX is the maximal specific growth rate (h− 1), KM the apparent affinity constant for glucose (g l− 1), S is the glucose concentration (g l− 1), L is the lactate concentration (g l− 1), LCRIT (g l− 1) is the critical lactate concentration at which growth is inhibited completely, and N is a coefficient pertaining to the lactate concentration. The latter parameter did not remain constant, but could be described as a function of the lactate concentration: N ¼ A þ Bd½LC
ð2Þ
where A, B and C are coefficients. Values and standard errors for the parameters μMAX, KS, LCRIT, A, B and C were estimated by fitting the data using the non-linear least-squares regression method with R software (http://www.r-project.org). 3. Results and discussion 3.1. Effect of the medium composition Three different pH (4, 5.2 and 6) were initially tested to find an appropriate pH for EPS formation. The specific yields of
Fig. 2. Plots of the effect of various compounds on the maximum specific growth rate (a) and EPS production (b) as a function of osmolarity. Glucose (⋄), glycerol (▴), acetate (▵), ethanol (♦), NaCl (■), lactate (●) and citrate (○).
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Table 2 Effect of NaCl and ethanol on the total consumption of glucose and corresponding lactate production (ND = not determined)
NaCl (g·l− 1)
Ethanol% (w/v)
14.0 28.0 40.0 1.65 3.62 4.87
Glucose consumed (g·l− 1)
Lactate produced (g·l− 1)
DW (g·l− 1)
23.43 12.79 1.85 26.28 21.31 24.85
19.57 6.59 1.48 22.52 17.56 24.26
2.15 0.53 0.29 2.82 ND 3.50
Increasing glucose concentrations affected the growth rate and biomass yield negatively, which may be due to the osmolarity. This possibility was tested using different concentrations of NaCl. The decline of the growth rate and growth yield with increasing osmolarity of NaCl was similar to that for glucose (Fig. 2a, Table 2). However, in contrast to glucose, EPS formation was not stimulated by NaCl (Fig 2b). With a NaCl concentration of 2.8% (w/v) (0.89 Os kg− 1) the lag phase was prolonged to about 300 h and hardly any EPS was produced, while in 4% (w/v) (1.24 Os·kg− 1) NaCl the culture failed to grow. With Lactococcus lactis subsp. cremoris NIZO B40 similar results were obtained and it was concluded that its EPS formation is not a stress response (Looijesteijn and Hugenholtz, 1999). 3.2. Effect of compounds present in Basque cider It has been observed that ethanol and glycerol, both present in Basque ciders (Del Campo et al., 2003), have a stimulating effect on EPS formation by P. parvulus. Ethanol affected the specific growth rate negatively (Fig. 2a), but the growth yield positively (Table 2). The best growth rate was found in the lowest ethanol concentration used (Fig. 2a), but the best EPS production (2.8 g l− 1) was found in the presence of 4.9% (w/v) (1.24 Os·kg− 1) of ethanol (Fig. 2b). The specific growth rate and yield were not affected significantly by glycerol (Fig. 2a), confirming the observation that P. parvulus 2.6 does not use glycerol as carbon source
Fig. 4. (A) Influence of different minerals on the growth of P. parvulus 2.6. Trace elements (TE) 7 times more than in control (♦), Co2+ and Mn2+ (■), Cu2+ and Mn2+ (□), Co2+ and Cu2+ (▴), Co2+, Cu2+ and Mn2+ (▵). (B) Concentration profile of the sum of chelated and free Mn2+ in the culture fluid. Control (○), Co2+ and Mn2+ (■), Cu2+ and Mn2+ (□), Co2+, Cu2+ and Mn2+(▵).
(Fernandez et al., 1996). The EPS formation improved only slightly (Fig. 2b) with the best production in the presence of 6.6% (w/v) (0.89 Os·kg− 1) glycerol. The results with ethanol and glycerol differ from NaCl indicating that osmolarity is not the common mechanism behind improved EPS formation. The mechanism behind stimulation by ethanol and glycerol remains unclear, but several possibilities have been described. First, ethanol and glycerol are able to cross the membrane unhindered and subsequently affect various physiological processes, including initiating changes in the lipid constitution of the cell membrane (see e.g. Weber and de Bont, 1996; Silveira et al., 2004). Second, ethanol can be a stress signal to activate a ‘ropy plasmid’ in ropy P. damnosus strains (Lonvaud-Funel et al., 1991). 3.3. Effect of lactate
Fig. 3. Growth of P. parvulus 2.6 on glucose in the presence of different lactate concentrations. Initial lactate concentrations (g l− 1): 6.9 (○), 11.9 (▵), 16.8 (⋄), 29.7 (□). The lines represent best fits of Eq. (1) through the data points.
The inhibiting effect of lactate on growth of microorganisms is frequently described because of its uncoupling (Amrane and Prigent, 1999) or its chelating (Lü-Lü et al., 1992) properties. To investigate whether uncoupling agents, such as weak acids, influence growth rate and EPS production at pH 5.2 P. parvulus 2.6 was grown in different concentrations of acetate, lactate and citrate. The specific growth rate and EPS production were strongly affected by each of these compounds (Fig. 2). In the presence of 20.4 g l− 1 of acetate, 16.8 g l− 1 of lactate (Fig. 3), or 19.2 g l− 1 of citrate growth appeared to progress in a linear
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concentrations sevenfold revealed that P. parvulus 2.6 had only requirements for Mn2+ (Fig. 4a). The specific requirement for Mn2+ among LAB, including pediococci, has been observed before (Efthymiou and Joseph, 1972). Addition of sevenfold increase of trace elements solution was detrimental, most likely because of the concomitant addition of more EDTA (0.1 g l− 1) to the medium (Fig. 4a). 3.5. Mn2+ uptake
Fig. 5. Growth profile of Pediococcus parvulus 2.6 with 1 ppm of Mn Glucose (▵), OD (■), lactate (□), EPS (▴), Mn2+ (⋄).
2+
.
fashion. The effect on the growth rate increased in intensity from acetate to lactate to citrate. This is contrary to the uncoupling effect of weak acids, because in that case acetate (pKA value of 4.75) would have been a stronger inhibitor than lactate (pKA value of 3.86). Each of these weak acids act also as chelators for metal ions and this could explain very well the order of strength of inhibition observed in the cultures (Lü-Lü et al., 1992). In addition, the chelating property of lactate could also be the explanation for the observed behaviour of growth (Fig. 1), since it might impose a trace element limitation on the growing culture. Since lactate is the major fermentation product its accumulation in the fermentation broth is unavoidable. Hence, it is vital to estimate its potential to affect growth and EPS formation in pediococcal cultures. Therefore, P. parvulus 2.6 was grown in the presence of various initial lactate concentrations, ranging approximately 7–37 g l− 1 (Fig. 3). Lactate affected both the growth rate and the mode of growth: the length of exponential growth phase shortened and the apparent linear growth phase prolonged concomitantly with the initial lactate concentration (Fig. 3). Using a KM value of 0.36 g l− 1 in Eq. (1) and the data points of all the fermentations, the values of the remaining parameters were calculated. The model was fitted through the experimental data, revealing the following parameter values: μMAX = 0.12 ± 0.02 h− 1, LCRIT = 58.9 ± 18.1 g l− 1, A = 2.42 ± 2.13, B = 34.4 ± 18.3 and C = − 0.87 ± 0.23. Most of these parameters are quite strongly correlated, which will make the inference about these parameters less straightforward. It further remains to be explained in biological terms why the lactate concentration has a highly non-linear relationship with the growth rate. The inhibiting effect of lactate was mimicked by citrate, albeit that lower concentrations were necessary because of its stronger chelating properties (data not shown). Thus, citrate had no positive effect on growth as has been observed for L. plantarum (Archibald and Duong, 1984).
The extracellular Mn2+ concentration was followed throughout the fermentation (Fig. 4b). Mn2+ uptake occurs only during the exponential phase and then ceases. Notably, after glucose became limited in the stationary phase, the extracellular manganese concentration increased until the level came back closely to the initial concentration of the medium (Fig. 5), indicating that all intracellular free Mn2+ must have been excreted. This was not due to any massive cell death or leakage, because samples for viability tests taken before and after the manganese excretion showed no difference in the number of colony forming units (data not shown). Like with L. plantarum (Archibald and Duong, 1984), higher initial Mn2+ concentrations in the medium led to more Mn2+ uptake. Excretion of Mn2+ upon glucose depletion is an indication that energy is required to maintain the gradient. Like in L. plantarum (Archibald and Duong, 1984), a proton motive force driven Mn2+ transporter, possibly of the MntH-type, will be involved. An ORF with 79% identity at gene level with such a proton symporting transporter of L. brevis have been found in P. pentosaceus (http://genome. ornl.gov/microbial/ppen/). In a fermentation to which 14 g l− 1 of lactate and a relatively high manganese concentration was added prior to inoculation, no uptake of Mn2+ was observed (Fig. 6). This resulted in slow growth for about 120 h, after which growth accelerated probably because of induction of an uptake system with a higher affinity for manganese. The latter was not observed in cultures with low manganese concentration (see e.g. Fig. 3). It clearly demonstrated the necessity of high Mn2+ uptake for relative fast growth. For lactobacilli it has been seen that Mn2+
3.4. Effect of trace element concentrations To determine which trace elements could be potentially become limiting through chelation, the growth test focused on Co2+, Cu2+ and Mn2+, because of their low concentration in the medium: 0.07, 0.07, 0.3 mg l− 1, respectively. Increasing their
Fig. 6. Growth profile of a Pediococcus parvulus 2.6 culture in the presence of 27 ppm of Mn 2+ to which an initial lactate concentration of 14 g·l− 1 was added. Glucose (▵), OD (■), lactate (□), EPS (▴), Mn2+ (⋄).
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Fig. 7. Specific EPS yield in P. parvulus 2.6 cultures with improved environmental conditions. Control with 25 g glucose·l− 1 and 5 mg Mn2+ l− 1 (⋄), 50 g glucose·l− 1, 5 mg Mn2+·l− 1 and 4.9% (w/v) ethanol (□), 25 g glucose·l− 1, 5 mg Mn2+·l− 1 and 5% (w/v) ethanol (▵).
may rapidly trigger growth through creation of a high cytoplasmic [Mn2+]/[Mg2+] ratio by which the activity of many catabolic, but also anabolic, enzymes are enhanced (Archibald, 1986; Kehres and Maguire, 2003). This may be true for P. parvulus too, however, its lactate dehydrogenase, pyruvate kinase, phosphofructokinase and glucokinase did not show any preference for Mn2+ or Mg2+(data not shown). Interestingly, Mn2+ is known to inhibit various glycohydrolases that break down EPS (Pham et al., 2000). Hence, chelation of Mn2+ could very well be the cause of activation of these hydrolases at the end of the fermentations. 3.6. Enhancement of EPS formation Fermentations in batch mode indicated that EPS production increased in the stationary phase (Fig. 1). Therefore, an experiment was carried out to stop growth prematurely, using chloramphenicol, to check whether it would enhance EPS formation. However, the specific EPS yield did not differ in the presence of the antibiotic (data not shown). Hence, a better biomass production will improve EPS production, as was the conclusion of several other studies (Mozzi et al., 1995; Looijesteijn and Hugenholtz, 1999). A significantly higher EPS concentration (4.1 g l− 1) could be obtained with P. parvulus 2.6 by combining the best conditions as they were found in this study, being higher concentrations of Mn2+(5 mg l− 1) glucose (50 g l− 1) and ethanol (4.9% (w/v)) (Fig. 7). In those conditions the EPS concentration was raised more than 30-fold compared to previous reports (Dueñas et al., 2003). In the presence of ethanol the lag phase was prolonged, as a result of adaptation, but still high biomass concentrations could be reached favourable enough for EPS production. 4. Conclusions Conditions leading to higher maximum biomass yields resulted in higher levels of EPS formation in P. parvulus 2.6, provided that adequate concentrations of glucose were present.
Higher biomass yields were obtained when cultures were pHcontrolled at pH 5.2. Glucose, ethanol and glycerol had positive effects on EPS formation. Higher glucose concentrations secured continued EPS formation in the stationary phase. Ethanol and glycerol can be stress signals affecting physiological processes, including initiating changes in the lipid constitution of the cell membrane. Because of the low manganese concentrations in the medium, the chelating action of lactate was such that it limited the growth rate of P. parvulus 2.6. The organism grew in an apparent linear fashion due to imposed manganese limitation. This could be overcome by increasing the manganese concentration to at least 2 mg·l− 1 in the medium. Upon substrate depletion the cells excreted their intracellular free manganese, indicating the need of an energy source to maintain the high Mn2+ gradient over the membrane. It remains unknown in how far the lactate concentration contributes to this excretion process or to the activation of glycohydrolases and, therefore, this merits further investigation. Unlike L. plantarum, P. parvulus 2.6 does not use citrate as functional manganese-binding siderophore. Improved EPS formation (4.1 g l− 1) was obtained in a medium containing high concentrations of glucose (50 g l− 1), Mn2+(5 mg l− 1) and ethanol (4.9% (w/v)). Lactate, as the main fermentation product, is a severe growth inhibitor in P. parvulus cultures. Therefore it is important to know under what conditions it will become an inhibiting factor, and how this burden can be avoided. Further studies are underway addressing these issues. Acknowledgments This study was supported by a Marie Curie grant from the European Commission (QLK3-CT-2001-60077), VINNOVA, the Swedish Agency for Innovation Systems, and “Becas de formación de Investigadores del Departamento de Educación, Universidad e Investigación” from the Basque Country Government. We acknowledge the assistance of Dr Raul Muñoz Torre and Dr Roberto Romero in the GFAAS methodology.
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