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Applied and Environmental Microbiology, July 2003, p. 3833-3839, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.3833-3839.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Curing Agent Sodium Nitrite, Used in the Production of Fermented Sausages, Is Less Inhibiting to the Bacteriocin-Producing Meat Starter Culture Lactobacillus curvatus LTH 1174 under Anaerobic Conditions

Jurgen Verluyten, Winy Messens, and Luc De Vuyst*

Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing, Department of Applied Biological Sciences, Vrije Universiteit Brussel, B-1050 Brussels, Belgium

Received 25 November 2002/ Accepted 17 April 2003


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ABSTRACT
 
Curvacin A is a listericidal bacteriocin produced by Lactobacillus curvatus LTH 1174, a strain isolated from fermented sausage. The response of this strain to an added curing agent (sodium nitrite) in terms of cell growth and bacteriocin production was investigated in vitro by laboratory fermentations with modified MRS broth. The strain was highly sensitive to nitrite; even a concentration of 10 ppm of curing agent inhibited its growth and both volumetric and specific bacteriocin production. A meat simulation medium containing 5 ppm of sodium nitrite was tested to investigate the influence of the gas phase on the growth and bacteriocin production of L. curvatus LTH 1174. Aerating the culture during growth had no effect on biomass formation, but the oxidative stress caused a higher level of specific bacteriocin production and led to a metabolic shift toward acetic acid production. Anaerobic conditions, on the other hand, led to an increased biomass concentration and less growth inhibition. Also, higher maximum volumetric bacteriocin activities and a higher level of specific bacteriocin production were obtained in the presence of sodium nitrite than in fermentations under aerobic conditions or standard conditions of air supply. These results indicate that the inhibitory effect of the curing agent is at least partially masked under anaerobic conditions.


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INTRODUCTION
 
Lactic acid bacteria (LAB) have traditionally been used in food processing because of their characteristic flavor changes but mainly because of their ability to lower the pH and to produce antimicrobial agents. The latter property results in a more stable and safer fermented end product (30). LAB preserve foods as a result of competitive growth and the production of inhibitory substances, such as lactic and acetic acids, ethanol, diacetyl, hydrogen peroxide, reuterin, and bacteriocins (38). However, some compounds are not formed in sufficient amounts (e.g., reuterin) to be active, and others, such as diacetyl and hydrogen peroxide, interfere with the sensorial properties of the food product (30). In addition, the use of chemical preservatives has fallen into disfavor with consumers, who are requesting fresh, natural food products that are mild and light, with less acid, sugar, salt, or fat (15, 30). These consumer preferences have led to an increased interest in bacteriocins and bacteriocinogenic starter cultures during the last decade (9, 38).

Bacteriocins are antibacterial peptides or proteins active against other gram-positive, mainly closely related bacterial species, including some undesirable spoilage bacteria and food-borne pathogens (11). The in situ production of bacteriocins enhances the growth of the starter organisms toward the fortuitous flora and hence ensures a stable and safe end product (17, 40). Bacteriocinogenic starter cultures and cocultures and their bacteriocins could therefore be useful in preserving meat and meat products (38). However, among the uncertainties are the levels of production and activities of bacteriocins in situ. Therefore, it is important to know both the effects of environmental factors, such as temperature and acidity, and the influence of specific sausage ingredients, such as salt and nitrite, on the growth characteristics and production of bacteriocins by the starter cultures used in European fermented sausage, in particular, Lactobacillus sakei and Lactobacillus curvatus (26, 27, 31).

At the start of a sausage fermentation, the addition of the curing agent nitrite is important, since at that time characteristics such as a low redox potential (Eh), a low water activity (aw), and a low pH have not yet been established. Nitrite is added to the meat batter to inhibit the growth of salmonellae and clostridia, to aid in color development, to prevent lipid rancidity, and to produce a typical cured flavor (7, 25, 34). With respect to Salmonella, Listeria, and Staphylococcus aureus, the first hours and days (high aw and pH) are critical. Therefore, the rapid development of LAB competing with the spoilage bacteria is very important, causing a rapid reduction in the pH to below 5.4 (23). The dominance of desirable LAB is favored by anaerobic conditions, added sugars and curing salt, and a low initial pH of the mixture (30). However, little is known about the influence under aerobic and anaerobic conditions of nitrite as a sausage batter ingredient and antimicrobial substance on the functionality of bacteriocin-producing strains as meat starter cultures used for sausage fermentation.

L. curvatus LTH 1174, a strain isolated from a naturally fermented sausage and a producer of the listericidal bacteriocin curvacin A, has been shown to be a successful starter strain for European sausage fermentations (39, 40). It was shown previously in vitro that this strain displays maximum bacteriocin activity levels under temperature and pH conditions used for such fermentations (31). The aim of the present study was to examine how various oxygen levels and anaerobic conditions affect the growth and bacteriocin production of the meat starter culture L. curvatus LTH 1174 in the presence or absence of sodium nitrite (NaNO2).


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MATERIALS AND METHODS
 
Microorganisms and media.
L. curvatus LTH 1174 was used as the producer of the antilisterial bacteriocin curvacin A (39). Listeria innocua LMG 13568 was used as a curvacin A-sensitive indicator organism to determine bacteriocin activity levels (26). Both strains were maintained and propagated as described previously (31).

For the experiments with nitrite, a modified version of MRS medium (8) was used (mMRS). This modification contained twice the concentrations of the complex nutrient sources, i.e., bacteriological peptone (Oxoid, Basingstoke, United Kingdom), Lab Lemco (Oxoid), and yeast extract (VWR International, Darmstadt, Germany), present in standard MRS medium. This modification prevented the severe growth limitation of L. curvatus LTH 1174 due to nutrient depletion (J. Verluyten, W. Messens, F. Leroy, V. Schrijvers, and L. De Vuyst, unpublished results). In addition, calculations of the amino nitrogen content of mMRS medium (as described in The Oxoid Manual, 8th ed.) showed that it approximates more closely the actual sausage environment (7). Furthermore, maximum curvacin A activity was found when the concentrations of the complex nutrient sources were doubled; the slightly lower level of specific bacteriocin production was counterbalanced by the higher level of biomass production; and the apparent bacteriocin inactivation rate was comparable to that in standard MRS medium (Verluyten et al., unpublished). For the fermentations without added NaNO2, 20 g of glucose per liter was used. For all other fermentations, 15 g of glucose per liter was used to approximate the actual sausage environment.

For examining growth and bacteriocin production under anaerobic and aerobic conditions, a meat simulation medium (MSM) was used. This medium contained, per liter, the following: 20 g of bacteriological peptone, 16 g of Lab Lemco, 8 g of yeast extract, 0.2 g of MgSO4 · 7H2O, 0.038 g of MnSO4 · H2O, 1 ml of Tween 80, 5 g of lactic acid (sterilized separately), 40 g of NaCl, and 0.005 g of NaNO2. A stock solution of NaNO2 (10 g per liter) was sterilized separately by microfiltration (Acrodisc; Pall Gelman Sciences, Ann Arbor, Mich.). The amount of NaNO2 added was representative of residual nitrite levels encountered in fermented sausage, since nitrite is rapidly depleted when added to the sausage batter (1, 14, 32, 37). Additionally, a pH profile was imposed (Fig. 1). This profile was based on the course of the pH during an actual German-type sausage fermentation with L. curvatus LTH 1174 (18).



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  		FIG. 1. pH profile for the simulation of an L. curvatus LTH 1174 fermentation in MSM (for the composition of MSM, see Materials and Methods). The pH profile was chosen from an actual German-type sausage fermentation with L. curvatus LTH 1174 (18).

Fermentation experiments.
To determine the inhibitory effect of added sodium nitrite, a series of small-scale experiments were performed. One-liter flasks containing 750 ml of mMRS medium with various concentrations of sodium nitrite (0, 50, 100, and 200 ppm) were incubated at 30°C without agitation. The optical density at 600 nm and the pH were measured at various times (0, 10, and 25 h). Additionally, laboratory fermentations were carried out in duplicate with mMRS medium containing 10 ppm of NaNO2 and under standard conditions of air supply. Standard conditions of air supply were obtained by continuous blowing of 4 liters of filtered air per min through the headspace of the fermentor, a step which is necessary for online measurement of the gases given off with the exhaust analyzer system used (EGAS 8; B. Braun Biotech International, Melsungen, Germany). The fermentation temperature was controlled at 25°C, and the pH was kept constant at 5.5 through automatic addition of 10 N NaOH.

The influence of anaerobic and aerobic conditions on the growth of and curvacin A production by L. curvatus LTH 1174 was investigated by sparging the medium (MSM) with filtered nitrogen gas (N28; Air Liquide, Paris, France) and air, respectively, at flow rates of 2 and 3 liters per min, respectively. These fermentations were carried out at a controlled temperature of 20°C and with the pH profile shown in Fig. 1, the pH being controlled through automatic addition of 10 N NaOH. Finally, laboratory fermentations were carried out in triplicate to clarify the influence of anaerobic conditions on nitrite inhibition by sparging mMRS medium with 2 liters of nitrogen gas (N28) per min at a controlled temperature of 25°C and a constant pH of 5.5.

All laboratory fermentations were carried out by using a 15-liter laboratory fermentor (BiostatC; B. Braun Biotech International) with a working volume of 10 liters. Online control was performed as described previously (26), except that online analyses of both input gases and gases given off were performed (EGAS 8). The inoculum was prepared as described previously (31).

Assays.
At regular time intervals, samples were withdrawn aseptically from the fermentor to determine cell counts (CFU), biomass (cell dry mass [CDM]), the level of soluble bacteriocin activity in a cell-free culture supernatant, the lactic acid concentration, and the residual glucose concentration as described previously (10, 26). The standard deviations for the CDM, glucose, and lactic acid measurements were 0.11, 0.04, and 0.02 g liter-1, respectively.

Modeling.
Primary modeling of cell growth, glucose consumption, lactic acid production, and bacteriocin production and inactivation was performed both to fit the data and to estimate the biokinetic parameters representative of growth and curvacin A production. The equations listed in Table 1 were used. They are the same as those reported by Messens et al. (31), except that bacteriocin production was made dependent on the minimum biomass concentration required for the onset of bacteriocin production due to induction (12).


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  		TABLE 1. Equations used for primary model development

The equations were integrated with the Euler integration technique in Microsoft Excel 97 (version 8.0a). All parameters needed for the modeling were estimated by manual adjustment until a good visual fit was obtained. This procedure was applied previously and led to reliable results (26, 31). It has the advantage of avoiding unrealistic fitting solutions, such as those due to local minima or convergence problems, which are sometimes generated by computational solving.


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RESULTS
 
Influence of sodium nitrite under standard conditions of air supply.
Preliminary shake flask experiments indicated that the growth of L. curvatus LTH 1174 is inhibited by as little as 10 ppm of NaNO2 but still grows at 50 to 200 ppm, although growth is very much inhibited from 50 ppm on (Fig. 2). During the fermentations carried out in mMRS medium without and with 10 ppm of added NaNO2 under standard conditions of air supply, L. curvatus LTH 1174 grew exponentially for approximately 9 to 10 h, after which growth slowed down (Fig. 3a). After 12 to 13 h of fermentation, growth completely ceased. The addition of NaNO2 caused the bacteria to enter into the stationary phase earlier, thereby markedly reducing biomass production and modifying the maximum specific growth rate only under anaerobic conditions (Fig. 3a). The growth arrest coincided with a slowing down of lactic acid production (Fig. 3b). Hence, the conversion of glucose into lactic acid was retarded but finally reached the same level (20 g liter-1). The cell yield coefficient was unaffected by the addition of NaNO2 (0.22 g of CDM/g of glucose). In other words, L. curvatus LTH 1174 was still able to produce the same amount of cells per unit of substrate but, due to the addition of NaNO2, cell production stopped much earlier. This decreased biomass production resulted in a lower maximum bacteriocin activity, which was less than one-third the activity observed in the fermentation without added NaNO2 (0.8 instead of 2.9 mega-arbitrary units [MAU] liter-1) (Fig. 3c). However, the lower curvacin A activity was the result not only of the decrease in cell growth but also of the decrease in specific bacteriocin production, which was reduced by 40% to a value of 1.2 MAU/g of CDM in the presence of 10 ppm of NaNO2, compared with 2.0 MAU/g of CDM in the absence of NaNO2. Since bacteriocin production started early in the growth phase, no minimum biomass concentration was necessary to initiate curvacin A production.



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  		FIG. 2. Influence of different concentrations of sodium nitrite on the growth of L. curvatus LTH 1174, as measured by the optical density at 600 nm (OD600) (closed symbols) and pH (open symbols) as a function of time. Sodium nitrite was added at 0 ppm (diamonds), 50 ppm (squares), 100 ppm (triangles), and 200 ppm (circles).



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  		FIG. 3. Experimental (symbols) and modeled (lines) values for the production of biomass (X; in grams of CDM per liter) (a), lactic acid formation (in grams of lactic acid per liter) (b), and curvacin A production (B; in MAU per liter) (c) by L. curvatus LTH 1174. Values were determined in mMRS medium without added sodium nitrite under standard conditions of air supply ({blacksquare}) and under anaerobic conditions ({square}) and with 10 ppm of added sodium nitrite under standard conditions of air supply ({blacktriangleup}) and under anaerobic conditions ({triangleup}) at a controlled temperature of 25°C and at a constant pH of 5.5.

Influence of aerobic and anaerobic conditions.
To determine the influence of the gas phase on the growth and bacteriocin production of L. curvatus LTH 1174 in MSM (containing 5 ppm of NaNO2) at a constant temperature of 20°C and the pH profile shown in Fig. 1, fermentations were performed under anaerobic and aerobic conditions.

When the different biokinetic parameters used for the primary model were compared (Table 2), it was clear that bacterial growth was unaffected by the gas phase. No changes in the maximum specific growth rate were observed. However, the maximum attainable biomass concentration was higher under anaerobic conditions than under standard conditions of air supply or aerobic conditions (Fig. 4a). Under anaerobic conditions, the higher biomass concentration also expedited glucose consumption and, remarkably, the retardation of lactic acid production caused by the addition of NaNO2 was not observed (Fig. 4b). In contrast, under standard conditions of air supply or aerobic conditions, this nitrite inhibition phenomenon was obvious (Fig. 4b). Additionally, when aeration was applied, not all glucose was converted into lactic acid, but part of it (approximately 20%) was converted into acetic acid (Fig. 4b).


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  		TABLE 2. Influence of aerobic and anaerobic conditions on various parameters for L. curvatus LTH 1174a



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  		FIG. 4. Influence of oxygen availability on production of biomass (X; in grams of CDM per liter) (a), lactic acid production (in grams per liter) (b), and bacteriocin production (B; in MAU per liter) (c) by L. curvatus LTH 1174. Values were determined in MSM containing 5 ppm of sodium nitrite as a function of time. Fermentation was carried out under standard conditions of air supply ({blacksquare}), under aerobic conditions ({blacktriangleup}), and under anaerobic conditions ({square}). The symbols represent experimental values; lines were drawn according to the model. Experimental values for acetic acid produced during fermentation under aerobic conditions are also shown ({triangleup}).

Maximum bacteriocin activity almost doubled under aerobic conditions compared to standard conditions of air supply and was even slightly higher under anaerobic conditions (Fig. 4c). Aerobic conditions improved curvacin A activity due to an increase in specific bacteriocin production, since the maximum attainable biomass concentration under aerobic conditions was comparable to the maximum attainable biomass concentration under standard conditions of air supply. The higher curvacin A activity obtained under anaerobic conditions was the consequence of both a higher maximum attainable biomass concentration and an increase in specific bacteriocin production. The values obtained for the apparent rate of bacteriocin inactivation were higher under aerobic conditions and comparable for anaerobic and standard conditions of air supply. The minimum biomass concentrations to initiate bacteriocin production were comparable for all three fermentations (Table 2). The results indicated that oxygen availability did not influence the production or the stability of the induction factor.

Influence of sodium nitrite under anaerobic conditions.
When the influence of oxygen availability was studied, a slowing down of lactic acid production was not observed under anaerobic conditions (see above). Therefore, two additional fermentations were carried out under anaerobic conditions, one without added NaNO2 and one in the presence of 10 ppm of NaNO2, in mMRS medium. The fermentations without added NaNO2 were comparable, except for a slightly lower maximum attainable biomass concentration under anaerobic conditions than under standard conditions of air supply (2.00 instead of 2.20 g of CDM liter-1, respectively), which resulted in a slightly lower maximum bacteriocin activity (2.6 instead of 2.9 MAU liter-1, respectively) (Fig. 3c), although the specific bacteriocin production levels were the same. In the presence of NaNO2, anaerobic conditions resulted in a higher biomass concentration than standard conditions of air supply (1.65 instead of 1.00 g of CDM liter-1), which was still lower than that obtained in the absence of NaNO2 (2.00 g of CDM liter-1) (Fig. 3a). Again, the nitrite-dependent slowing down of lactic acid production was not observed (Fig. 3b). The reduction in bacteriocin activity due to the addition of NaNO2 was less pronounced under anaerobic conditions (from 2.6 to 1.9 MAU liter-1) than under standard conditions of air supply (from 2.9 to 0.8 MAU liter-1) (Fig. 3c). This result was due to a higher maximum attainable biomass concentration (1.6 instead of 1.0 g of CDM liter-1) and an increase in specific bacteriocin production (1.7 instead of 1.2 MAU/g of CDM).


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DISCUSSION
 
At present, a large variety of fermented sausages are being produced. Raw sausage technology allows for diversification toward low acid, sugar, salt, or fat content, provided the basic concept of reduction of pH and/or aw is maintained (25). Concomitantly, the rational application of LAB starter cultures in sausage fermentations is important to suppress undesirable microorganisms, to stabilize the raw sausage, and to ensure high product quality. However, the meat-borne pathogen Listeria monocytogenes can survive acidic conditions, low aw, and high sodium chloride and sodium nitrite concentrations during the fermentation and drying of meat (13, 20, 21). Therefore, interest in bacteriocin-producing starter cultures with listericidal capacities has increased (9).

Both L. sakei CTC 494 and L. curvatus LTH 1174 are interesting bacteriocin-producing meat starter cultures (26, 27, 28, 31). Previously, it was shown that sodium chloride drastically influenced the bacteriocin production of L. sakei CTC 494 by decreasing both biomass production and specific bacteriocin production (27). In this study, it was shown that stress imposed on L. curvatus LTH 1174 due to added NaNO2 inhibited biomass formation and decreased both volumetric and specific curvacin A production. Clearly, there are distinct adaptive responses to environmental stress conditions, not all of which have a positive effect on bacteriocin production (10). For Enterococcus faecium CTC 492, the addition of 100 ppm of NaNO2 significantly inhibited enterocin production compared with that seen in the standard MRS control (4). In contrast, Lactococcus lactis DPC 4275 produced significantly larger amounts of lacticin 3147 (1,300 arbitrary units/g) in sausages to which a low concentration of sodium nitrite (20 ppm) had been added (36). L. curvatus LTH 1174 showed a marked decrease in maximum bacteriocin activity even at a sodium nitrite level of 10 ppm in mMRS medium. This decrease in bacteriocin activity was due to a marked inhibition of biomass production as well as to a significant decline in specific bacteriocin production. In contrast, in L. sakei CTC 494, the decline in bacteriocin titer was solely due to the inhibitory effect of sodium nitrite on biomass production; specific bacteriocin production was unaffected (27).

Hence, it appears that L. curvatus LTH 1174 is much more sensitive to nitrite than L. sakei CTC 494. The latter strain still displays growth and bacteriocin production at sodium nitrite levels of 400 ppm and is also more salt tolerant (27). Although L. sakei and L. curvatus are the dominant LAB strains encountered in European sausage starter cultures, both commercial and natural (16), it seems that L. curvatus is less adapted to the sausage environment than L. sakei. The higher sensitivity toward salt (22), especially the very high sensitivity toward a curing agent, may, at least partially, explain why L. curvatus is less frequently isolated from traditionally fermented sausages. Moreover, when investigating the cold-room environments of an artisanal sausage manufacturing plant, Andrighetto et al. (3) isolated only L. sakei, a result which is also indicative of the higher resistance and adaptability of this species to these environments. However, a concentration of about 30 to 50 ppm of nitrite is sufficient to obtain the full curing color in fermented sausages (19, 23). In this situation, low nitrite levels are not a severe problem for L. curvatus, and its higher sensitivity toward salt may be one factor disfavoring L. curvatus compared to L. sakei.

The maximum curvacin A activity and the specific bacteriocin production of L. curvatus LTH 1174 were dependent on the atmospheric conditions applied, whereas the onset of bacteriocin production characterized by its minimum biomass concentration was not. The minimum biomass concentration corresponds to the production of a critical amount of induction factor that binds to its receptor for the initiation of curvacin A production (12). The induction of bacteriocin production is dependent on both temperature and salt (4, 12). Oxidative stress caused by aeration of the growth medium seemed to be a stimulus for increased specific curvacin A production. LAB exhibit an inducible oxidative stress response when exposed to sublethal levels of oxygen intermediates (6). The induced protective system consists of a set of stress proteins which protect cells. It is possible that in some bacteria, oxidative stress also induces an increase in the production of defensive molecules, such as bacteriocins. The extra ATP which will have been generated as a consequence of the metabolic shift toward acetic acid production under aerobic conditions may explain the higher level of bacteriocin production by L. curvatus LTH 1174, since it did not produce a higher biomass concentration. Aeration also increased specific nisin Z production when the initial level of air saturation was 60% or higher (2). Increasing the oxygen saturation percentage caused significant increases in nisin A production and amylovorin production by L. lactis subsp. lactis IIM Lb. 1.13 (5) and Lactobacillus amylovorus DCE 471 (10), respectively. On the other hand, when a culture of L. sakei L45 was aerated during growth, only very low levels of lactocin S were detected compared to those obtained in a fermentation under anaerobic conditions, possibly due to an alteration of the chemical structure, such as oxidation of a methionine residue (33).

In the presence of sodium nitrite, L. curvatus LTH 1174 displayed the highest volumetric bacteriocin titers under anaerobic conditions, as a result of both increased biomass production and increased specific bacteriocin production. Since the slowing down of lactic acid production was never seen under anaerobic conditions, the inhibition of growth in the presence of sodium nitrite under aerobic conditions was due to the curing agent and not, for instance, to nutrient limitation. In view of the anaerobic conditions encountered in the actual sausage environment, the higher curvacin A activities under these conditions are an interesting feature. Furthermore, these conditions seem to partially reduce the negative effect of nitrite on the growth and metabolic activity of L. curvatus LTH 1174. Dry sausages made only with common salt quickly develop grey discolorations because of the comminution process and the influence of oxygen. Fat breakdown also takes place more rapidly, and the shelf life is limited (24). During chopping, oxygen from the air is mixed into the raw sausage batter, resulting in a relatively high Eh (25). The initial microflora of fresh meat consists largely of gram-negative aerobic rods, particularly Pseudomonas spp. and Brochothrix thermosphacta (23, 29). The oxygen present within the mixture is rapidly consumed by these bacteria. The rapid breakdown of oxygen in freshly made sausages is delayed by the presence of nitrite (35). The latter is more effective as a bactericidal substance when the Eh is low (25). Additionally, under aerobic conditions, these spoilage bacteria are relatively insensitive to lactic acid.

In conclusion, the use of L. curvatus LTH 1174 as a bacteriocin-producing starter culture in European sausage fermentation is promising because the process conditions match the temperature and pH requirements of this strain (31). While salt concentrations used for sausage fermentation reduce bacteriocin production, the anaerobic environment of fermented sausages masks the inhibitory effect of the curing agent, sodium nitrite. Although all experiments were conducted in a model system, these findings will aid in the industrial implementation of new, bacteriocin-producing starter cultures of L. curvatus and L. sakei in fermented meat production. These bacteriocinogenic meat starter cultures for sausage fermentations will certainly contribute to a safer end product.


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ACKNOWLEDGMENTS
 
We acknowledge financial support from the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), in particular, the STWW project "Functionality of Novel Starter Cultures in Traditional Fermentation Processes." Also, this work was supported by the Research Council of the Vrije Universiteit Brussel, the Fund for Scientific Research—Flanders, and various food companies.

The technical assistance of Vincent Schrijvers is greatly appreciated. L. curvatus LTH 1174 was kindly provided by W. P. Hammes (Institut für Lebensmitteltechnologie, Universität Hohenheim, Stuttgart, Germany).


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FOOTNOTES
 
* Corresponding author. Mailing address: Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing, Department of Applied Biological Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. Phone: 32-2-6293245. Fax: 32-2-6292720. E-mail: ldvuyst{at}vub.ac.be. Back


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Applied and Environmental Microbiology, July 2003, p. 3833-3839, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.3833-3839.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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