Influence of Cations on Growth of Thermophilic Geobacillus spp. and Anoxybacillus flavithermus in Planktonic Culture

Abstract

Free ions of Na⁺, K⁺, Ca²⁺, and Mg²⁺ influenced the optical density of planktonic cultures of thermophilic bacilli. Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 (milk powder manufacturing plant isolates) and A. flavithermus DSM 2641 and G. thermoleovorans DSM 5366 were studied. Ca²⁺ and Mg²⁺ were associated with increases in optical density more so than Na⁺ and K⁺. Overall, it appeared that Ca²⁺ and/or Mg²⁺ was required for the production of protein in thermophilic bacilli, as shown by results obtained with A. flavithermus E16, which was selected for further study.

TEXT

The thermophilic bacilli Geobacillus spp. and Anoxybacillus flavithermus are the predominant bacteria within foulants of heated regions (50 to 70°C) of milk powder manufacturing plants (3). The manufacturing process used for the production of milk powder selects for the growth of these bacteria, which prosper at high temperatures (50 to 70°C) (3). Thermophilic bacilli are the predominant spoilage organisms in the final milk powder product, and their presence determines the product selling price (3). If their numbers exceed acceptable levels, this has a negative financial impact on the milk powder manufacturer (3). Furthermore, since thermophilic bacilli are biofilm and spore formers, they are adept at persistence in milk powder manufacturing plants (3).

The total free cation concentration in unprocessed milk is approximately 60 mM, consisting of free Na⁺, K⁺, Ca²⁺, and Mg²⁺ ions at concentrations of approximately 20, 34, 3.6, and 1.3 mM, respectively (9). Processing factors, such as heating and evaporation, change the equilibrium between free and complexed cations within milk (1). Thus, the external free cation compositions that thermophilic bacilli encounter vary through the milk powder manufacturing process as chilled, unprocessed milk is heated and evaporated to produce milk powder and as products with different specifications are manufactured between runs (1). Furthermore, minerals and ions in milk have the potential to migrate from the bulk flow and concentrate within surface-conditioning layers, foulants, and biofilms in milk powder manufacturing plants (15).

A number of bacterial species respond uniquely to different external cation concentrations, both in a planktonic form and in a biofilm form (10, 16, 22). In our study, optical density was used to analyze indirectly the requirement for and the influence of a range of Na⁺, K⁺, Ca²⁺, and Mg²⁺ concentrations and proportions on thermophilic bacilli in planktonic culture, as a preliminary screening experiment leading into a study of the influence of cations on thermophilic bacilli propagated as a biofilm. Four bacterial isolates were studied; two Geobacillus sp. and two A. flavithermus isolates were intentionally selected so that each genus pair would include an isolate derived from a milk powder manufacturing plant and a type (DSMZ collection) strain.

Evaluation of the response of Geobacillus spp. and A. flavithermus to Na⁺, K⁺, Ca²⁺, and Mg²⁺ in planktonic culture.

Casein digest medium (1 g/liter) (Difco, BD Biosciences, Sparks, MD) was used to reconstitute NaCl, KCl, CaCl₂ · 2H₂O, and MgCl₂ · 6H₂O (Merck, Darmstadt, Germany) powders. Into each well of a Falcon Microtest 96-well culture plate (BD, Franklin Lakes, NJ) 50 μl of casein digest medium (1 g/liter) was dispensed, with various cation supplementation concentrations and proportions (Table 1), as was 50 μl of bacterial inoculum (approximately 1 × 10⁴ CFU/ml), which was previously grown in casein digest medium (1 g/liter) to early stationary phase (9 h). The background concentrations of Na⁺, K⁺, Ca²⁺, and Mg²⁺ in casein digest medium (1 g/liter) unsupplemented with cations were approximately 1.0, 0.03, 0.004, and 0.002 mM, respectively, as estimated by BD Biosciences (2). The microtiter plates were incubated at 55°C for 10 h. The optical densities of the cultures were measured for triplicate wells, using a wavelength of 600 nm, in an OptiMax tunable microplate reader (Molecular Devices, Sunnyvale, CA).

TABLE 1.

Cation proportions used to supplement a casein digest medium (1 g/liter)^a

Cation supplementation type	Free-cation proportion
	Na+	K+	Ca2+	Mg2+
Na	1	0	0	0
K	0	1	0	0
Ca	0	0	1	0
Mg	0	0	0	1
Na/K	0.5	0.5	0	0
Na/Ca	0.5	0	0.5	0
Na/Mg	0.5	0	0	0.5
Ca/Mg	0	0	0.5	0.5
K/Ca	0	0.5	0.5	0
K/Mg	0	0.5	0	0.5
Ca/Mg (1:5)	0	0	0.17	0.83
Na/K/Ca (1:1:2)	0.25	0.25	0.5	0
Na/K/Ca/Mg (1:1:1:1)	0.25	0.25	0.25	0.25

^aNa, K, Ca, and Mg designate free Na⁺, K⁺, Ca²⁺, and Mg²⁺, respectively.

The experiment was carried out on two separate occasions. The optical density data set from the 10-h time point was further analyzed using SAS software, with a population standard error and 95% confidence intervals (P ≤ 0.05) being calculated. The 10-h time point was chosen as this was when the bacteria reached late-exponential phase and sufficient growth had occurred such that apparent differences in optical density among the cultures could be analyzed. The resultant data and statistics were represented graphically; the zero values on the y axes denote the optical density of the respective bacterial isolates grown in casein digest medium (1 g/liter) unsupplemented with cations (baseline control), and the optical densities of cultures that were supplemented with cations are reported relative to the baseline control.

Effect of cation type.

The response of the thermophilic bacilli to Ca²⁺ and Mg²⁺ was predominantly responsible for an increase in the optical density of the cultures, whereas Na⁺ and K⁺ acted cooperatively with Ca²⁺ and Mg²⁺ to increase the optical density (Fig. 1). The extent of the differences in optical densities was unique for each bacterial isolate studied (Fig. 1).

Fig 1.

Optical densities of A. flavithermus E16 (A), A. flavithermus DSM 2641 (B), Geobacillus sp. F75 (C), and G. thermoleovorans DSM 5336 (D) planktonic cultures grown in casein digest medium (1 g/liter) supplemented with a total cation concentration of 2 mM with varied proportions of Na⁺, K⁺, Ca²⁺, and Mg²⁺, relative to the optical density of the respective bacterial isolate grown in casein digest medium (1 g/liter) unsupplemented with cations (baseline control). N, K, C, and M designate free Na⁺, K⁺, Ca²⁺, and Mg²⁺, respectively. CM1:5 refers to a Ca²⁺-Mg²⁺ ratio of 1:5, NKC refers to a Na⁺-K⁺-Ca²⁺ ratio of 1:1:2, and all other treatments have equal proportions of each supplemented cation. The cultures were incubated at 55°C for 10 h. Two replicates were done, and each replicate consisted of an average of triplicate cultures. The error bars represent 95% confidence intervals (P ≤ 0.05), which were determined using SAS statistical analysis software.

After 10 h of growth, optical density readings for A. flavithermus E16, A. flavithermus DSM 2641, and Geobacillus thermoleovorans DSM 5366 cultures increased when supplemented solely with Ca²⁺ (Fig. 1A, B, and D) and/or Mg²⁺ (Fig. 1B and D), relative to the baseline control. Na⁺, and to a greater extent K⁺, acted cooperatively with Ca²⁺ or Mg²⁺ to induce an increase in optical density readings in A. flavithermus E16 and G. thermoleovorans DSM 5366 cultures (Fig. 1A and D).

In contrast, there was no significant difference in the optical densities of Geobacillus sp. strain F75 cultures supplemented with Ca²⁺ or Mg²⁺ (Fig. 1C) relative to the baseline control, and the cooperative effect of Na⁺ or K⁺ in A. flavithermus DSM 2641 and Geobacillus sp. F75 cultures was either minimal or unapparent (Fig. 1B and C).

Other studies with different species have shown that Ca²⁺ and/or Mg²⁺ increases the optical density of planktonic bacterial cultures (4, 22). Furthermore, Ca²⁺ and Mg²⁺ have important physiological roles in the cell envelope of bacteria, where they have been shown to stimulate extracellular matrix production by bacteria (5, 16, 20); they are required to optimize enzyme functionality, particularly for enzymes involved in the biosynthesis of cell wall polymers (12), and have an important role in maintaining the structural integrity of the cell envelope (19). Na⁺ and K⁺ have largely intracellular physiological roles, such as in the optimization of enzyme functionality and in osmotic pressure and pH homeostasis (7, 14). Similarly to our study, Caldwell and Arcand (4) found that the monovalent cations Na⁺ and K⁺ have a crucial role in the planktonic growth of Bacteroides spp. The optical density of a bacterial culture can depend on a range of factors, such as culture biomass, which is determined by the concentration of both bacterial cells and bacterium-derived extracellular polymers, and the size, shape, and optical properties of particles within the culture (11). Since it was found that predominantly Ca²⁺ and Mg²⁺ caused an increase in thermophilic bacillus culture optical densities and other research has shown that Ca²⁺ and Mg²⁺ have important physiological roles in the cell envelope of bacteria, it is proposed that changes in the cell envelope of the thermophilic bacilli were responsible for eliciting differential culture optical densities in response to cation supplementation.

Additionally, the needs of thermophilic bacilli may be satisfied more easily by the background Na⁺ and K⁺ concentrations in casein digest medium (1 g/liter) (1.0 and 0.03 mM, respectively) than the lower background Ca²⁺ and Mg²⁺ concentrations (0.004 and 0.002 mM, respectively), therefore nullifying the observed effect of Na⁺ and K⁺ supplementation but allowing for supplementation effects of Ca²⁺ and Mg²⁺ to be observed. K⁺ tended to have a greater cooperative effect than Na⁺, which also suggested that there is a greater requirement by thermophilic bacilli for K⁺ than Na⁺ to increase the optical density of the culture. However, this observed difference may have been due to the higher background concentration of Na⁺ than K⁺ in casein digest medium (1 g/liter). Supplementing cultures with Na⁺ beyond 1 mM may have had no further increasing influence on their optical density. A Na⁺ concentration of 1 mM, as present in casein digest medium (1 g/liter), may have been close to the minimum threshold Na⁺ requirement of these bacteria.

Effect of cation concentration.

When all four cation types (Na⁺, K⁺, Ca²⁺, and Mg²⁺) were used to supplement cultures, the optical densities of the milk powder manufacturing plant isolates (A. flavithermus E16 and Geobacillus sp. F75 cultures) increased as the total cation concentration increased between 2 and 125 mM (Fig. 2A and C). A similar trend was not apparent for the DSM strains (Fig. 2B and D). This trend was indicative of the potential of thermophilic bacilli in milk powder manufacturing plants to be influenced as cation concentrations vary during dairy processing.

Fig 2.

Optical densities of A. flavithermus E16 (A), A. flavithermus DSM 2641 (B), Geobacillus sp. F75 (C), and G. thermoleovorans DSM 5336 (D) planktonic cultures grown in casein digest medium (1 g/liter) supplemented with equal proportions of Na⁺, K⁺, Ca²⁺, and Mg²⁺ at total cation concentrations of between 2 and 250 mM, relative to the optical density of the respective bacterial isolate grown in casein digest medium (1 g/liter) unsupplemented with cations (baseline control). The cultures were incubated at 55°C for 10 h. Two replicates were done, and each replicate consisted of an average of triplicate cultures. The error bars represent 95% confidence intervals (P ≤ 0.05), which were determined using SAS statistical analysis software.

Total viable cell and spore counts of A. flavithermus E16 cultures.

To determine the factors that influenced the optical densities of the cultures in our study, A. flavithermus E16 cultures supplemented with three different cation concentrations of 0, 2, and 125 mM (consisting of equal proportions of Na⁺, K⁺, Ca²⁺, and Mg²⁺) were further analyzed, as these cultures had significantly different optical densities after 10 h of growth.

Initially, the total viable cell and spore counts were measured to investigate their potential influence on culture optical densities. One-hundred-milliliter casein digest medium (1 g/liter) aliquots, supplemented with cation concentrations of 0, 2, or 125 mM (consisting of equal proportions of Na⁺, K⁺, Ca²⁺, and Mg²⁺), were inoculated with approximately 1 × 10⁴ CFU/ml of A. flavithermus E16 which was previously grown in casein digest medium (1 g/liter) to early stationary phase (9 h). The cultures were incubated at 55°C for 10 h, and total viable cell counts were determined using standard microbiological plating techniques on milk plate count agar (MPCA; Oxoid, Basingstoke, United Kingdom) at 55°C for 48 h. Spores reportedly have a greater potential than vegetative cells to influence the optical density of a suspension, as they have refractory properties (17). To determine the spore count in the cultures, 12 ml of each culture was sampled and heated at 100°C for 35 min (18). Standard microbiological plating techniques were used to determine the spore count in the heat-treated cultures on MPCA supplemented with starch (2 g/liter) (13). To obtain a lower spore detection limit of <0.5 log CFU/ml, 10 1-ml aliquots of each of the heat-treated cultures were spread plated. The total viable cell and spore counts were determined on three separate occasions.

The average total viable cell counts of the cultures supplemented with 0, 2, and 125 mM cations were similar, at 6.6, 6.2, and 6.4 log CFU/ml, respectively. Higher spore counts were determined in cultures supplemented with cation concentrations of 2 and 125 mM (5.3 and 4.8 log CFU/ml, respectively) than in the unsupplemented culture (<0.5 log CFU/ml). The total viable cell and spore count standard deviations (σ_n−1) were <1.0 log and ≤1.1 log, respectively. Neither the total viable cell nor the spore counts correlated with the differences in optical densities seen among the three cultures. Furthermore, phase-contrast light microscopy showed that there was a consistent cell size, shape, and appearance among the cultures and that the bacteria did not aggregate. Thus, it was concluded that total viable cell or spore counts and cell size, shape, and coaggregation were not factors that influenced the optical densities of these cultures.

Quantification of bacterial surface protein and polysaccharide in A. flavithermus E16 culture.

The amounts of bacterial protein and polysaccharide in cultures of A. flavithermus E16 containing 0, 2, or 125 mM cations were quantified to investigate their contribution toward the optical densities of the planktonic cultures.

A 10-ml aliquot of bacterial inoculum, grown as described above, was used to inoculate 1,000 ml of casein digest medium (1 g/liter), supplemented with a cation concentration of 0, 2, or 125 mM (consisting of equal proportions of Na⁺, K⁺, Ca²⁺, and Mg²⁺). The cultures were incubated at 55°C for 10 h. Approximately 900 ml of the culture was centrifuged at 11,800 × g for 10 min. The supernatant of the culture was discarded, and the pellet was washed once in 450 ml of distilled water and then resuspended in 5 ml of distilled water. The total viable cell counts in both the original 1,000-ml cultures and the resulting 5-ml culture concentrates were determined using standard microbiological plating techniques, as described above.

To quantify the amount of protein produced by A. flavithermus E16 in culture, the following protocol was used. A 1:10 dilution (800 μl) of the 5-ml culture concentrate was mixed with 200 μl of Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, Inc., Hercules, CA), and the absorbance of the mixture was read using a spectrophotometer (595 nm), in which the reference was set against a solution containing 800 μl of distilled water mixed with 200 μl of Bio-Rad protein assay dye reagent concentrate. Bovine serum albumin (Sigma-Aldrich, St. Louis, MO) was used to generate a standard curve (1 to 100 μg/ml).

To quantify the amount of extracellular polysaccharide produced by A. flavithermus E16 in culture, the following protocol was used, as modified from the protocol detailed by Dall and Herndon (6). One milliliter of the 5-ml culture concentrate was added dropwise to 8 ml of approximately 100% ethanol; the solution was incubated at 4°C for 18 h and then centrifuged at 10,000 × g for 20 min. The supernatant was discarded, and the pellet was resuspended by vortex mixing in 1 ml of distilled water. To the resuspended pellet suspension was added 7 ml of sulfuric acid (77%, vol/vol) and then 1 ml of l-tryptophan (10 g/liter) (BDH, Poole, England). Each suspension was thoroughly vortex mixed, dispensed into a glass test tube, and then heated for 20 min at 100°C. Each suspension was vortex mixed, and the absorbance was read using a spectrophotometer (500 nm), in which the reference was set against a solution containing 1 ml of distilled water mixed with 7 ml of sulfuric acid (77%, vol/vol) and 1 ml of l-tryptophan (10 g/liter), which had also been subjected to the heat treatment. Dextran (Sigma-Aldrich) was used to generate a standard curve (10 to 200 μg/ml).

The bacterial protein and polysaccharide assays and their associated standard curves were carried out on three separate occasions, and the results are quoted as averages ± 1 standard deviation (σ_n−1).

After high-speed centrifugation, the amount of protein and to a lesser extent the amount of polysaccharide in A. flavithermus E16 cultures, per CFU, increased with increasing concentration of cation supplementation (consisting of equal proportions of Na⁺, K⁺, Ca²⁺, and Mg²⁺) (Fig. 3).

Fig 3.

Amounts of polysaccharide (A) and protein (B), associated with the pellet after centrifugation at 11,800 × g, per CFU of A. flavithermus E16 culture after a 10-h incubation at 55°C, grown in casein digest medium (1 g/liter) supplemented with a total cation concentration of 0, 2, or 125 mM (consisting of equal proportions of Na⁺, K⁺, Ca²⁺, and Mg²⁺) (n = 3). Error bars represent ±1 standard deviation (σ_n−1).

Thus, it appeared that the predominant factor that influenced the optical densities of the cultures was the amount of bacterial protein produced. An increase in protein on the surface of A. flavithermus E16 may have increased the optical density of the culture either by increasing the culture biomass or by increasing the refraction of light due to changes in the optical properties of the cell surfaces (11).

It is known that cations create an environment that is favorable for optimal enzyme functionality; therefore, the scope of the metabolic diversity of the bacteria in our study may have widened, and a greater amount of enzyme may have been produced in response to the increase in external cation concentration (8). The bacteria may also have produced a greater amount of structural protein (8, 21).

Overall, it can be postulated that Ca²⁺ and/or Mg²⁺ was required for or stimulated the production of protein in thermophilic bacillus planktonic cultures. Although the cellular location of the protein that increased in response to varied external cation concentrations was not determined, it could be hypothesized that the protein was located in the cell envelope, as Ca²⁺ and Mg²⁺ have physiological roles focused at the cell envelope (12, 19).

It was observed that as the collective concentration of all four cation types (Na⁺, K⁺, Ca²⁺, and Mg²⁺) fluctuated between concentrations typically present in milk products during their manufacture (2 to 125 mM), the optical densities of the milk powder manufacturing plant isolates (A. flavithermus E16 and Geobacillus sp. F75) increased as the total cation concentration increased. This indicated that there is the potential that external free cation concentrations may influence the metabolic and physiological state of thermophilic bacilli, which may influence their proliferation during the manufacture of milk powders, for example, during biofilm formation. This hypothesis is currently being studied.