Changes in Sodium, Calcium, and Magnesium Ion Concentrations That Inhibit Geobacillus Biofilms Have No Effect on Anoxybacillus flavithermus Biofilms

Abstract

This study investigated the effects of varied sodium, calcium, and magnesium concentrations in specialty milk formulations on biofilm formation by Geobacillus spp. and Anoxybacillus flavithermus. The numbers of attached viable cells (log CFU per square centimeter) after 6 to 18 h of biofilm formation by three dairy-derived strains of Geobacillus and three dairy-derived strains of A. flavithermus were compared in two commercial milk formulations. Milk formulation B had relatively high sodium and low calcium and magnesium concentrations compared with those of milk formulation A, but the two formulations had comparable fat, protein, and lactose concentrations. Biofilm formation by the three Geobacillus isolates was up to 4 log CFU cm⁻² lower in milk formulation B than in milk formulation A after 6 to 18 h, and the difference was often significant (P ≤ 0.05). However, no significant differences (P ≤ 0.05) were found when biofilm formations by the three A. flavithermus isolates were compared in milk formulations A and B. Supplementation of milk formulation A with 100 mM NaCl significantly decreased (P ≤ 0.05) Geobacillus biofilm formation after 6 to 10 h. Furthermore, supplementation of milk formulation B with 2 mM CaCl₂ or 2 mM MgCl₂ significantly increased (P ≤ 0.05) Geobacillus biofilm formation after 10 to 18 h. It was concluded that relatively high free Na⁺ and low free Ca²⁺ and Mg²⁺ concentrations in milk formulations are collectively required to inhibit biofilm formation by Geobacillus spp., whereas biofilm formation by A. flavithermus is not impacted by typical cation concentration differences of milk formulations.

INTRODUCTION

Thermophilic bacilli belonging to the Geobacillus spp. and Anoxybacillus flavithermus groups are the predominant spoilage bacteria that may contaminate milk during its manufacture into milk powder (1, 2). The number of thermophilic bacilli in milk powder is of major importance because it is a measure of its quality and determines its market selling price (1, 2). Geobacillus spp. and A. flavithermus grow as biofilms on product contact surfaces in regions of milk powder manufacturing plants, such as in plate heat exchangers and evaporators, that are held at high temperatures (up to 70°C) (1, 2). It is postulated that these biofilms act as a reservoir of cells that slough off and disperse into milk as it transits through the plant (1, 2). The majority of thermophilic bacilli that appear in milk powder originate from biofilms on product contact surfaces (2).

The concentrations and ratios of free cations in the aqueous phase that immerses a biofilm can influence biofilms in many ways. Electrostatic interactions of free cations with bacterial polymers in a biofilm matrix can influence the structural integrity and cohesion of a biofilm (3, 4). In addition, bacteria respond to fluctuations in free cation concentrations by adapting their physiology, which may influence biofilm formation. For example, Kara et al. (5) showed that increasing Na⁺ concentrations increased the proportion of negatively charged, hydrophilic extracellular polymers, causing a decrease in the cohesion of a biofilm Conversely, Patrauchan et al. (6) showed how Ca²⁺ stimulates a Pseudoalteromonas sp. to increase both the amount and the composition of extracellular proteins it expresses, which primes the bacteria for biofilm formation. Additionally, Song and Leff (7) proposed that Mg²⁺ may enhance biofilm formation by Pseudomonas fluorescens by influencing the production of flagella and fimbriae or the production and structure of exopolysaccharide.

Furthermore, Na⁺, Ca²⁺, and Mg²⁺ have important roles in bacterial homeostasis and are required as nutrient sources (8–10). High Na⁺ concentrations can have a toxic effect on bacteria (8). Ca²⁺ and Mg²⁺ are required for the optimal functioning of many bacterial proteins, including enzymes (11–13), and localized fluxes of Ca²⁺ have an integral role in the regulation of important bacterial cellular processes, such as the cell growth cycle and cell division (12).

In the dairy industry, many different milk formulations are processed into milk powder. Milk formulations have a range of cation concentrations, and the cation concentrations of some milk formulations differ from those of unprocessed milk. Typical total (sum of bound and free) sodium, potassium, calcium, and magnesium concentrations in unprocessed milk are 22, 37, 30, and 5 mM, respectively (13). However, in some specialty milk formulations, total sodium concentrations can reach as high as 100 mM and total calcium concentrations can reach as low as 7 mM (Table 1). There is potential for different cation concentrations and ratios in milk formulations to differentially influence biofilm formation and proliferation by Geobacillus spp. and A. flavithermus during milk powder manufacture. To understand how varied cation concentrations in milk formulations differentially impact biofilm formation by Geobacillus spp. and A. flavithermus, we investigated the influence that different sodium, calcium, and magnesium concentrations in milk formulations had on the number of bacteria attached per square centimeter to 316 stainless steel coupons.

TABLE 1.

Total (sum of bound and free) cation concentrations of milk formulations A and B (10 g 90 ml⁻¹), including those supplemented with cation chlorides

MFa and cation supplementation	Concn (mM) of:
	Na+	K+	Ca2+	Mg2+
MF A	45	6	35	3
MF B	101	2	7	1
MF A + 50 mM NaCl	95	6	35	3
MF A + 100 mM NaCl	145	6	35	3
MF B + 2 mM CaCl2	101	2	9	1
MF B + 2 mM MgCl2	101	2	7	3

^aMF, milk formulation.

MATERIALS AND METHODS

Bacterial isolates.

A. flavithermus E16 and Geobacillus sp. strain F75 were isolated from product contact surfaces at milk powder manufacturing plant 1. A. flavithermus 136 and Geobacillus sp. strain 183 were isolated from milk powders manufactured at plant 1. A. flavithermus TRb and Geobacillus sp. strain TRa were isolated from product contact surfaces at milk powder manufacturing plant 2. Plant 1 is situated on the South Island and plant 2 is situated on the North Island of New Zealand.

Growth media.

Casein digest medium (1 g liter⁻¹) (Difco, BD Biosciences) and bovine, commercial, specialty milk formulations A and B (10 g 90 ml⁻¹) (Fonterra) (Table 1) were used as bacterial growth media. Milk powders were reconstituted with water that had been deionized by reverse osmosis and autoclaved (121°C for 15 min) to sterilize. Milk formulations A and B were derived from the same respective batches throughout experimentation. Milk formulations A and B had similar fat, protein, and lactose concentrations: 1.5, 81.7, and 3.9 g liter⁻¹, respectively, in milk formulation A and 1.7, 81.7, and 3.9 g liter⁻¹, respectively, in milk formulation B. However, milk formulations A and B had different total (sum of bound and free) sodium, calcium, and magnesium concentrations (Table 1). Milk formulation A was supplemented with analytical-grade NaCl (Merck) powder, and milk formulation B was supplemented with analytical-grade CaCl₂·2H₂O (Merck) or MgCl₂·6H₂O (Merck) powder, which was achieved by dissolving the NaCl, CaCl₂·2H₂O, or MgCl₂·6H₂O powder in deionized water prior to dissolving the milk powder. Milk formulations A and B were used as representatives of functional milk formulations manufactured by the dairy industry. Prior to reconstitution, the milk powders were gamma irradiated (25,000 Gy) to inactivate any contaminating microorganisms present, so that growth and analysis of the inoculated bacteria of interest were unimpeded.

Culture storage.

The bacterial isolates were grown in tryptic soy broth (Merck) to mid-log phase and were stored with the addition of glycerol (10%, vol/vol) (Merck) at −80°C.

Inoculum preparation.

To propagate bacteria for use in the biofilm formation assay, 1 ml of a thawed bacterial culture was used to inoculate 100 ml of casein digest medium (1 g liter⁻¹). The inoculated medium was incubated at 55°C for 9 h, which was sufficient time for the bacteria to reach stationary growth phase. Bacteria were propagated in casein digest medium (reconstituted at the low concentration of 1 g liter⁻¹ with deionized water) prior to the initiation of biofilm formation to simulate the nutrient-starved condition likely to exist on the surface of a milk powder manufacturing plant following cleaning.

Biofilm formation assay.

Stainless steel coupons were cleaned and passivated prior to use in the biofilm formation assay as previously described by Flint et al. (14). Bacteria were diluted in either milk formulation A or B to 4.5 log CFU ml⁻¹, and 1.5 ml was added to each well of a 24-well culture plate (Becton Dickinson). One 316 stainless steel coupon (part RD128-316; Biosurface Technologies Corporation), with a surface area of approximately 4 cm², and sterilized by autoclaving, was added, using sterile forceps, to each inoculum-containing well so that it was fully submerged and horizontal. The plate, placed in a plastic bag to prevent evaporation, was incubated at 55°C for 6, 10, 14, or 18 h under static conditions. Biofilm formation was investigated for up to 18 h to simulate the duration of a typical milk powder manufacturing run (1). Biofilm formation was investigated on coupons made from 316 stainless steel, as this is the grade of stainless steel that typically comprises product contact surfaces in milk powder manufacturing plants (1).

Cell enumeration.

The following protocol was used to enumerate the attached viable cells per square centimeter on the coupons. The coupons were removed from the cultures using sterile forceps, dipped and rinsed three times in approximately 50 ml of deionized water to displace any loosely attached cells, and placed into a 35-ml plastic container (item code LBS3722W; Thermo Fisher Scientific) with 5 ml of fresh casein digest medium (1 g liter⁻¹) and 12 g of glass beads with a mean diameter of 6.35 mm (catalogue number 11079635; Biospec Products, Inc.). The plastic containers were vortex mixed vigorously for 2 min to dislodge the attached cells into the surrounding medium. Standard microbiological plate counting techniques were used to enumerate the bacteria in the dislodged cell suspension, using casein digest medium (1 g liter⁻¹) as the diluent and milk plate count agar (Oxoid), incubated at 55°C 18 h. The number of attached viable bacteria per square centimeter was determined.

Statistical analysis.

The experiments were carried out on three separate occasions, and mean numbers of attached viable cells (CFU per square centimeter) ± 1 standard deviation are reported. Minitab software (Minitab Pty Ltd.) was used to calculate the population standard error, and 95% confidence intervals (P ≤ 0.05) were used to determine significant differences among the mean values.

RESULTS AND DISCUSSION

Milk formulations A and B.

The total calcium concentrations in milk formulations A and B differed (Table 1). The free Ca²⁺ concentration was determined to be 0.4 mM in both milk formulations (determined by a calcium-selective electrode). However, both milk formulation A and milk formulation B had a high protein concentration, 81.7 g liter⁻¹.

Comparison of Geobacillus biofilm formation in milk formulations A and B.

Biofilm formation, as determined by the number of viable cells per square centimeter, by all three of the Geobacillus isolates was significantly lower (P ≤ 0.05) in milk formulation B than in milk formulation A at all four time points (6, 10, 14, and 18 h) (Fig. 1a and d, 2a and d, and 3a and d). The extent of the difference in biofilm formation by the three Geobacillus isolates in milk formulation A compared with milk formulation B ranged between 1 and 4 log CFU cm⁻² (Fig. 1a and d, 2a and d, and 3a and d). The one exception, when there was no significant difference (P ≤ 0.05) in biofilm formation in milk formulations A and B, was for Geobacillus sp. TRa after 18 h (Fig. 2a and d).

FIG 1.

Biofilm formation, after 6 to 18 h of incubation at 55°C, by viable Geobacillus sp. F75 cells on stainless steel coupons fully submerged in milk formulation A (a), milk formulation A supplemented with 50 mM NaCl (b), milk formulation A supplemented with 100 mM NaCl (c), milk formulation B (d), milk formulation B supplemented with 2 mM CaCl₂ (e), and milk formulation B supplemented with 2 mM MgCl₂ (f). Experiments were repeated as triplicates, and error bars represent ±1 standard deviation. An asterisk indicates a significant difference (P ≤ 0.05) between cation-supplemented and unsupplemented milk formulations for the respective milk formulation and time point.

FIG 2.

Biofilm formation, after 6 to 18 h of incubation at 55°C, by viable Geobacillus sp. TRa cells on stainless steel coupons fully submerged in milk formulation A (a), milk formulation A supplemented with 50 mM NaCl (b), milk formulation A supplemented with 100 mM NaCl (c), milk formulation B (d), milk formulation B supplemented with 2 mM CaCl₂ (e), and milk formulation B supplemented with 2 mM MgCl₂ (f). Experiments were repeated as triplicates, and error bars represent ±1 standard deviation. An asterisk indicates a significant difference (P ≤ 0.05) between cation-supplemented and unsupplemented milk formulations for the respective milk formulation and time point.

FIG 3.

Biofilm formation, after 6 to 18 h of incubation at 55°C, by viable Geobacillus sp. 183 cells on stainless steel coupons fully submerged in milk formulation A (a), milk formulation A supplemented with 50 mM NaCl (b), milk formulation A supplemented with 100 mM NaCl (c), milk formulation B (d), milk formulation B supplemented with 2 mM CaCl₂ (e), and milk formulation B supplemented with 2 mM MgCl₂ (f). Experiments were repeated as triplicates, and error bars represent ±1 standard deviation. An asterisk indicates a significant difference (P ≤ 0.05) between cation-supplemented and unsupplemented milk formulations for the respective milk formulation and time point.

It is proposed that biofilm formation by a large proportion of the dairy Geobacillus population is inhibited throughout the duration of a manufacturing run when milk formulations with high sodium and low calcium and magnesium concentrations are processed. As the Geobacillus isolates used in this study were isolated from different geographical regions (i.e., two different manufacturing plants) and different sources (i.e., product contact surfaces and milk powders), they were likely to represent a range of dairy Geobacillus phenotypes. Furthermore, a substantial proportion of thermophilic bacilli that contaminate milk powder manufacturing plants belong to Geobacillus (1, 2). Thus, there is potential for the count of thermophilic bacilli in the final milk powder to decrease markedly when a specialty milk formulation with relatively high sodium and low calcium and magnesium concentrations is processed.

Characterization of the effects of sodium, calcium, and magnesium on Geobacillus biofilm formation.

To characterize the roles that high sodium and low calcium and magnesium concentrations have in the inhibition of biofilm formation by Geobacillus spp., we investigated the influence of supplementation of milk formulation A with 50 or 100 mM NaCl and supplementation of milk formulation B with 2 mM CaCl₂ or 2 mM MgCl₂ on biofilm formation by three dairy Geobacillus isolates.

Relative to unsupplemented milk formulation A, supplementation of milk formulation A with 100 mM NaCl significantly decreased (P ≤ 0.05) biofilm formation by Geobacillus isolates F75 and TRa at 6 and 10 h by between 1.4 and 2.8 log CFU cm⁻² (Fig. 1a and c and 2a and c) and significantly decreased (P ≤ 0.05) biofilm formation by Geobacillus sp. 183 at all time points (6 to 18 h) by between 2.2 and 3.8 log CFU cm⁻² (Fig. 3a and c). Supplementation of milk formulation A with 50 mM NaCl did not inhibit biofilm formation by the Geobacillus isolates studied as greatly as supplementation with 100 mM NaCl. Relative to that in unsupplemented milk formulation A, supplementation of milk formulation A with 50 mM NaCl did not significantly decrease (P ≤ 0.05) biofilm formation by Geobacillus sp. F75 at any of the time points studied (Fig. 1a and b) but significantly decreased (P ≤ 0.05) biofilm formation by Geobacillus isolates TRa and 183 after 6 h, by 1.2 and 1.7 log CFU cm⁻², respectively (Fig. 2a and b and 3a and b).

Supplementation of milk formulation B with either 2 mM CaCl₂ or 2 mM MgCl₂ significantly increased (P ≤ 0.05) biofilm formation by all three Geobacillus isolates at 10, 14, and 18 h, by up to 4 log CFU cm⁻² (Fig. 1d to f, 2d to f, and 3d to f).

These results indicated that high sodium, low calcium, and low magnesium concentrations each inhibit Geobacillus biofilm formation and that all three are required to maximize the inhibition of Geobacillus biofilm formation over an 18-h period.

It is likely that the free (ionized) form of the cations influenced biofilm formation by the Geobacillus isolates, because it is the free form of cations that is biologically active and has the potential to interact with and influence bacteria (15). In any given milk formulation, approximately 10% of the total calcium and magnesium and approximately 90% of the total sodium exist in the free form (13, 16). Thus, the free Na⁺, Ca²⁺, and Mg²⁺ concentrations in milk formulation B were estimated to be 91, 0.7, and 0.1 mM, respectively. It is proposed that differences in the free Na⁺, Ca²⁺, and Mg²⁺ concentrations in the milk formulations (both intrinsic to the milk formulations and from supplementation) caused the differences in biofilm formation by the Geobacillus isolates.

Supplementation of milk formulation A with 50 or 100 mM NaCl did not inhibit biofilm formation by the Geobacillus isolates as greatly as for milk formulation B (Fig. 1b to d, 2b to d, and 3b to d), even though the total sodium concentration of milk formulation A supplemented with 50 or 100 mM NaCl was close to or higher than that of milk formulation B (Table 1). The supplementation of any given milk system with NaCl (or CaCl₂ or MgCl₂) causes bound divalent cations to dissociate and increases the concentrations of free Ca²⁺ and Mg²⁺ (16, 17). Thus, supplementing milk formulation A with NaCl would have increased the free Ca²⁺ and Mg²⁺ concentrations. Although supplementing milk formulation A with 50 or 100 mM NaCl would have increased the free Na⁺ concentration, and caused the observed decrease in biofilm formation by the Geobacillus isolates, the slight increase in free Ca²⁺ and Mg²⁺ concentrations (due to NaCl supplementation) may have increased biofilm formation relative to that observed in milk formulation B.

When milk formulations are supplemented with CaCl₂ or MgCl₂, only part of the supplemented calcium or magnesium exists in the free form. For example, Philippe et al. (18) found that the addition of 4.5 mM CaCl₂ to skim milk increased the free Ca²⁺ concentration from 1.56 to 2.86 mM. Thus, it was predicted that when milk formulation B was supplemented with 2 mM CaCl₂ or MgCl₂, the free Ca²⁺ or Mg²⁺ concentration would have increased by less than 2 mM. However, this increase was enough to elicit the observed increase in biofilm formation by the Geobacillus isolates. This observation is feasible given that the estimated intrinsic free Ca²⁺ and Mg²⁺ concentrations in milk formulation B were low.

Comparison of the effects of calcium and magnesium on Geobacillus biofilm formation.

There was no significant difference (P ≤ 0.05) found when the effects of Ca²⁺ and Mg²⁺ on biofilm formation by the three Geobacillus isolates studied were compared (Fig. 1e and f, 2e and f, and 3e and f). Divalent cations, such as Ca²⁺ and Mg²⁺, have structural, functional, and regulatory interactions with many bacterial polymers, enzymes, and regulatory proteins (17–19). Often the interaction is specific to a particular cation species, or the interaction of one cation species is more efficacious than that of other cation species (19–21). This study was unable to conclude whether the effect of Ca²⁺ and Mg²⁺ on biofilm formation by Geobacillus isolates was specific or nonspecific in regard to each of these cation species.

Proposed mechanisms for cation inhibition of Geobacillus biofilm formation.

Two mechanisms can be proposed to explain the observed inhibition of Geobacillus biofilm formation by relatively high sodium and low calcium and magnesium concentrations. First, this combination of cation concentrations may have compromised the structural integrity of the biofilm. Bacterial cell wall and extracellular matrix polymers are often composed of an abundance of negatively charged functional groups, such as phosphate and carboxyl groups (22). Free cations associate with negatively charged functional groups on polymers and enhance the overall stability and cohesion of the cell wall and the extracellular matrix (20, 23). Furthermore, Ca²⁺ and Mg²⁺ have greater cohesive properties than Na⁺, because divalent cations have a higher charge density and a greater capacity to neutralize negatively charged functional groups and may form divalent cation bridges (4, 5). Although Ca²⁺ and Mg²⁺ have greater binding affinities than Na⁺, as the Na⁺ concentration surrounding bacteria and biofilms increases, Na⁺ can displace Ca²⁺ and Mg²⁺ within the biofilm matrix and consequently decrease the cohesion and structural integrity of the biofilm (20, 24). There was a stark contrast in the estimated free Na⁺ concentration relative to the free Ca²⁺ and Mg²⁺ concentrations in milk formulation B, such that it had a very high ratio of monovalent to divalent cations. This may have sufficiently decreased the electrostatic forces within the biofilms formed by the Geobacillus isolates and inhibited biofilm formation. This hypothesis agrees with results for wastewater sludges, in which high ratios of monovalent to divalent cations compromised the structural integrity of wastewater sludge biofilms (24).

The second proposed mechanism is that the combination of cation concentrations may have influenced regulatory pathways that altered the metabolism or physiology of the Geobacillus species isolates and compromised the growth or structural integrity of the biofilms. Elevated extracellular Na⁺ concentrations have been shown to stimulate an increase in the proportion of negatively charged, hydrophilic bacterial cell wall polymers in a wastewater sludge, which decreases its structural integrity (5). Conversely, Ca²⁺ and Mg²⁺ have the potential to enhance biofilm formation by binding to regulatory proteins, such as response regulators and secreted proteins, which have implications in biofilm formation (7, 11, 12, 25). In addition, Ca²⁺ has been shown to influence the morphology and proteome expression of bacteria, which consequently enhances their biofilm formation (6, 26).

Na⁺ competes with Ca²⁺ and Mg²⁺ for assimilation into the cell wall and accumulation at the cell wall-cytoplasmic membrane interface (9, 20, 22). When the Geobacillus isolates formed biofilms in milk formulation B, relatively high concentrations of Na⁺ and low concentrations of Ca²⁺ and Mg²⁺ may have accumulated at the cell wall-cytoplasmic membrane interface. The high Na⁺ concentrations may have directly stimulated the Geobacillus isolates to enter a metabolic or physiological state that decreased their ability to grow as a biofilm. Alternatively, the low Ca²⁺ and Mg²⁺ concentrations may have resulted in inadequate stimulation of regulatory proteins that recognize Ca²⁺ or Mg²⁺ as a stimulus. The lack of stimulation by Ca²⁺ or Mg²⁺ may have decreased the ability of the Geobacillus isolates to grow as a biofilm (6, 7).

Comparison of A. flavithermus biofilm formation in milk formulations A and B.

There was no significant difference (P ≤ 0.05) in biofilm formation after 6 to 18 h by any of the three A. flavithermus isolates studied when growth in milk formulations A and B at each time point was compared (Fig. 4). It is proposed that A. flavithermus is more adept than Geobacillus spp. at tolerating the high sodium and low calcium and magnesium concentrations that existed in milk formulation B. In contrast, there was inhibition of biofilm formation of all Geobacillus spp. isolated from the dairy industry in milk formulations A and B, although the degree of inhibition varied between strains. Strain variation in dairy isolates of Geobacillus has been reported (27) and is to be expected.

FIG 4.

Biofilm formation, after 6 to 18 h of incubation at 55°C, by viable Anoxybacillus flavithermus E16 (a and b), A. flavithermus TRb (c and d), and A. flavithermus 136 (e and f) cells on stainless steel coupons fully submerged in milk formulation A (a, c, and e) and milk formulation B (b, d, and f). Experiments were repeated as triplicates, and error bars represent ±1 standard deviation.

Planktonic growth of Geobacillus species in low calcium and magnesium solutions.

The growth of Geobacillus in planktonic culture was influenced by varied cation (including Ca and Mg) ion concentrations (see the supplemental material).

Conclusions.

Geobacillus biofilm formation was inhibited for up to 18 h in a milk formulation that had relatively high sodium and low calcium and magnesium concentrations. High sodium, low calcium, and low magnesium concentrations were collectively required for Geobacillus biofilm formation to be maximally inhibited. In contrast, biofilm formation by A. flavithermus was not inhibited in the milk formulation with relatively high sodium and low calcium and magnesium concentrations. High free Na⁺ and low free Ca²⁺ and Mg²⁺ concentrations may have inhibited Geobacillus biofilm formation, either by decreasing electrostatic forces and consequently compromising the structural integrity of the biofilm or by influencing the metabolism or physiology of the Geobacillus spp. As a substantial proportion of thermophilic bacilli that may contaminate milk powder manufacturing plants and milk powders belong to the genus Geobacillus, these findings indicate that milk powders derived from milk formulations that collectively have high sodium and low calcium and magnesium concentrations may have markedly decreased counts of thermophilic bacilli, may have superior quality, and may fetch higher selling prices.