Controls on Bacterial Cell Envelope Sulfhydryl Site ... - ACS Publications

Jun 12, 2017 - Controls on Bacterial Cell Envelope Sulfhydryl Site Concentrations: The Effect of Glucose Concentration During Growth. Qiang Yu* and Je...
0 downloads 0 Views 668KB Size
Download PDF
Article pubs.acs.org/est

Controls on Bacterial Cell Envelope Sulfhydryl Site Concentrations: The Effect of Glucose Concentration During Growth Qiang Yu* and Jeremy B. Fein Department of Civil & Environmental Engineering & Earth Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United States S Supporting Information *

ABSTRACT: Bacterial sulfhydryl sites can form strong complexes with chalcophilic metals such as Hg and Cd, thereby affecting the fate, transport, and bioavailability of these metals in both natural and engineered systems. In this study, five bacterial species were cultured in M9 minimal media containing a range of glucose concentrations as carbon source and in a high-nutrient TSB medium enriched with 50 g/L of glucose, and the sulfhydryl site concentrations of the obtained biomass samples were determined through selective sulfhydryl site-blocking, potentiometric titrations, and surface complexation modeling. The experimental results show that the glucose concentration in the M9 minimal media strongly affects the concentration of sulfhydryl sites that are present on the bacteria, with higher glucose concentrations yielding higher bacterial sulfhydryl site concentrations for each species studied. In contrast, although adding 50 g/L of glucose to the TSB medium significantly increases the sulfhydryl site concentrations for the three Bacillus species studied, the elevated glucose concentration does not significantly affect sulfhydryl site concentrations for S. oneidensis and P. putida samples when grown in the TSB medium. Our results suggest that bacterial sulfhydryl site concentrations in natural systems are likely affected by the composition of the bacterial community and by the available nutrients, and that these factors must be considered in order to determine and model the effects of bacterial cells on metal cycling and metal bioavailability in the environment.

1. INTRODUCTION A number of chacophilic metals, such as Hg, As, Cd, and Pb, are common worldwide pollutants of surface and ground waters, soils, and sediments.1 Serious human health effects (e.g., cancer and kidney damage) may occur with long-term intake of waters contaminated by even low concentrations of these metals.2,3 In order to model the environmental fate of these metals and to develop efficient and effective remediation strategies, the controls on the fate and transport of these metals in geologic systems must be determined. Bacteria are ubiquitous in both natural and engineered systems. The adsorption of metals onto bacterial cells, not only directly changes the mobility of the metals, but also promotes other metal-bacteria interactions by making the metals bioavailable.4 Most previous studies of metal adsorption onto bacteria were conducted under relatively high metal loading conditions (>10 μmol of aqueous metal/gram of wet biomass),5−9 and carboxyl, phosphoryl, and amino groups were identified as the major binding sites for metals within cell envelopes.10−12 However, recent studies have suggested that sulfhydryl sites dominate the adsorption of chacophilic metals onto bacterial cells when the ratios of aqueous metal/wet biomass are less than approximately 5 μmol/g.13−17 Owing to the strong interaction between sulfur and chacophiles, sulfhydryl sites exhibit particularly high affinities for binding chalcophilic metals.15,18,19 For example, the stability constant of Cd-sulfhydryl complexes on S. oneidensis is about 3 orders of © XXXX American Chemical Society

magnitude higher than the stability constants for bacterial surface complexes between Cd and nonsulfhydryl sites, such as carboxyl and phosphoxyl sites.15 Therefore, although sulfhydryl sites account for only about 5−10% of the total binding sites within bacterial cell envelopes,20,21 they are capable of dominating the adsorption under low metal loading conditions.13−17 Because typical metal loading conditions in natural systems are low, sulfhydryl sites likely play a more important role in metal adsorption onto bacterial cells than other binding sites within cell envelopes. Despite the importance of sulfhydryl sites in controlling metal adsorption by bacteria, direct experimental determination of the concentration of sulfhydryl sites within bacterial cell envelopes is difficult. For example, Fourier transform infrared spectroscopy (FTIR) has been successfully used for the identification of carboxyl and phosphoxyl sites within cell envelopes,6,12 but previous studies using FTIR did not detect sulfhydryl sites, likely due to their low abundance relative to other site types. Extended X-ray fine structure (EXAFS) spectroscopy can be used to identify metal-sulfhydryl site binding,14,17,22 but it is not capable of yielding values for the sulfhydryl site concentrations within cell envelopes. Recently, Received: Revised: Accepted: Published: A

February 26, 2017 May 13, 2017 June 12, 2017 June 12, 2017 DOI: 10.1021/acs.est.7b01047 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology

oneidensis in the M9 minimal media, 24 h for all species in the TSB medium enriched with 50 g/L of glucose) in order for the bacterial cell suspensions to reach early stationary phase, as determined by bacterial growth curves (data not shown). The standard M9 minimal medium24 consists of 6.78 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, 1 g of NH4Cl, 0.01 g of CaCl2, and 0.24 g of MgSO4 per 1 L of ultrapure 18 MΩ water, with solution pH adjusted to 7.3−7.4 using a 1 M NaOH solution. MgSO4 is the major sulfur source in the M9 minimal medium, and its concentration in the medium is about 20−50 times higher than the concentration of sulfur present as sulfhydryl sites for previously studied bacterial species,20,21,23 so sulfur is present in these experiments in significant excess. Because there was negligible growth of S. oneidensis and B. subtilis in the standard M9 minimal medium, 10 mL of a vitamin solution (Supporting Information (SI) Table S1) and 10 mL of a trace element solution (SI Table S2), similar to those used by Lovley and Phillips,25 were added to 1 L of the standard M9 minimal medium to promote growth of these species. P. putida was grown in the standard M9 minimal medium without adding vitamins or trace element solution. Glucose was used as both the carbon source and the electron donor in the M9 minimal media, and we varied the concentration of glucose within the range of 4−50 g of glucose per L. The TSB medium contains 30 g of trypticase soy broth and 5 g of yeast extract per L of ultrapure 18 MΩ water, and the sulfhydryl site concentrations for several bacterial biomass samples grown in unamended TSB medium have been measured in previous studies.20,23 In order to determine the effect of glucose on sulfhydryl site concentrations for bacteria grown in the TSB medium, the same bacterial species were grown in the TSB medium enriched with 50 g/L of glucose in the present study. Prior to use, the media were filter sterilized using a Nalgene 0.22 μm nylon filtration membrane. After incubation, the bacterial cells were harvested by centrifugation at 10 970g for 5 min. The biomass pellets were rinsed with a 0.1 M NaCl solution, followed by centrifugation at 8100g for 5 min, and the same process was repeated three times. The biomass pellets were then transferred into preweighed test tubes and centrifuged for two 30 min intervals at 8100g. After decanting the supernatant, the wet weight of the cells was used to calculate the bacterial concentrations in the subsequent experiments, and the bacterial concentrations that are reported in this study are these wet weights. 2.2. Determination of Concentrations of Sulfhydryl Sites. The approach used to determine the sulfhydryl site concentrations within the bacterial cell envelopes was the same procedure that we developed in a previous study.20 We used potentiometric titrations and surface complexation modeling to determine the concentration of total binding sites present within suspensions of bacterial cells. The concentration of sulfhydryl sites was determined by measuring the decrease in the concentration of total binding sites after the sulfhydryl sites were selectively blocked using a molecule that itself does not protonate or deprotonate. In the present study, monobromo(trimethylammonio)bimane bromide (qBBr), purchased from Toronto Research Chemical, Inc., was used to selectively block the sulfhydryl sites that are present within the bacterial cell envelopes. qBBr effectively blocks the protonation ability of sulfhydryl sites, but does not react with other binding sites such as carboxyl or phosphoryl groups, and qBBr itself is not proton-active.20,21 Therefore, the decrease that we measure with the potentio-

selective labeling or blocking of sulfhydryl sites was used for determining the concentration of sulfhydryl sites within bacterial cell envelopes.20,21 These studies show that most investigated bacterial species have similar concentrations of sulfhydryl sites, ranging from 20 to 40 μmol/g.20,21,23 In each study that quantified bacterial cell envelope sulfhydryl site concentrations, the bacteria were grown in nutrient-rich media, such as trypticase soy broth (TSB), in order to maximize biomass production. However, nutrient-rich conditions are rare in natural systems. Therefore, if bacterial sulfhydryl site concentrations vary in response to nutrient availability during cell growth, then the concentration of sulfhydryl sites on biomass in real systems may be markedly different from the sulfhydryl concentrations that have been determined experimentally using nutrient-rich media, potentially leading to significant inaccuracies in prediced environmental behaviors of chacophilic metals. In this study, we determine if bacterial cell envelope sulfhydryl site concentrations are controlled by cell growth conditions, specifically the concentration of the carbon source and electron donor in the growth medium. Three bacterial species were grown in M9 minimal media24 with different concentrations of glucose, and five bacterial species were grown in nutrient-rich TSB medium enriched with 50 g/L of glucose. The concentrations of the sulfhydryl sites of these biomass samples were determined by coupling a selective site-blocking technique with potentiometric titrations and surface complexation modeling in an approach that we developed previously.20 We find that the sulfhydryl sites within cell envelopes of the three species that were grown in minimal media are strongly affected by the concentration of glucose in the growth medium, and that the sulfhydryl concentrations on the bacterial biomass samples grown in this minimal medium are different from one species to another. However, adding 50 g/L of glucose to the nutrient-rich TSB medium only affected the sulfhydryl site concentrations of the three Bacillus species, and it showed no significant influence on the sulfhydryl site concentration of the two Gram-negative species, Shewanella oneidensis and Pseudomonas putida. Therefore, our results indicate that both the composition of bacterial communities and the nutrient levels in which they grow can affect bacterial sulfhydryl site concentrations and hence metal cycling and metal bioavailability in bacteria-bearing systems.

2. MATERIALS AND METHODS 2.1. Bacterial Cell Preparation. Two Gram-negative (Pseudomonas putida and Shewanella oneidensis) and three Gram-positive (Bacillus subtilis, Bacillus licheniformis, and Bacillus cereus) bacterial species were studied. Bacillus subtilis, Pseudomonas putida and Shewanella oneidensis were grown in both the M9 minimal media with different concentrations of glucose and the TSB medium enriched with 50 g/L of glucose. Bacillus licheniformis and Bacillus cereus were only cultured in the TSB medium enriched with 50 g/L of glucose. The procedures for growth, washing, and weighing of the bacterial cells were similar to those described previously20 except that the M9 minimal medium or the TSB medium enriched with 50 g/L of glucose was used for bacterial growth. Briefly, bacteria were first cultured aerobically in 3 mL of TSB medium at 32 °C for 24 h. This initial culture was then transferred to 1 L of the desired medium and was cultured aerobically with constant gentle agitation at 32 °C for predetermined time periods (48 h for P. putida and B. subtilis in the M9 minimal media, 72 h for S. B

DOI: 10.1021/acs.est.7b01047 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology

Figure 1. Summary of the calculation procedure to determine the concentration of sulfhydryl sites within bacterial cell envelopes using B. subtilis biomass samples that were grown in the M9 minimal medium containing 50 g/L of glucose as an example. The calculation was based on three titrations for each type of biomass sample (with or without qBBr treatment). The graph in the upper left depicts one representative titration curve for untreated biomass and one curve for qBBr-treated biomass, each with their corresponding best-fit models. The table in the lower left shows the modeling results for these two titration curves; the table in the lower right shows the average total site concentrations from three separate titration curves for the untreated and treated biomass. We then apply the Student’s t test to these two sets of three titration results to yield the calculated concentration of the sulfhydryl sites for this growth condition, as shown in the box in the upper right. This is the result just for a representative growth condition; a complete listing of the modeling results for all of the biomass samples in this study can be found in SI Table S3 and Table S4.

adding HCl standard; and (2) a forward titration from pH 3.0 to pH 9.7 by adding NaOH standard. Only these forward titration data were used for the surface complexation modeling to calculate the total sulfhydryl site concentrations. The titrator was set to operate using a method in which the equilibration time for each step of the titration was controlled, and the volume of acid or base added at each step was recorded, with a minimum addition volume of 0.25 μL. New titrant was added after the signal drift reached a minimum stability of 0.01 mV/s, or after a maximum waiting time of 60 s was achieved. In preliminary experiments, we conducted down-pH titrations from pH 9.7 to pH 3.0 immediately following the forward titrations, and the obtained titration curves (not shown) matched well with their corresponding forward titrations, suggesting rapid reversibility of the protonation reactions and that no significant damage occurred to the cells during the forward titrations. In order to compare titration results from different experiments, the results were plotted in terms of a mass normalized net concentration of protons added to the system:

metric titrations in the total site concentration after qBBr treatment can be attributed to the blocking of sulfhydryl sites, and is equal to the concentration of sulfhydryl sites in the titrated sample. Although sulfhydryl sites typically comprise only approximately 5−10% of the total proton-active sites on bacteria,20,21 potentiometric titrations provide a reliable method for measuring their concentrations due to the precision associated with the total site concentration measurements that are yielded by the approach, as shown in Figure 1. In order to block sulfhydryl sites, the bacterial pellets were suspended in a freshly prepared qBBr solution in 0.1 M NaCl with pH buffered to 7.0 ± 0.1 using a 1.8 mM Na2HPO4/18.2 mM NaH2PO4 buffer, with a qBBr:biomass ratio of approximately 100 μmol/g, and the mixture was allowed to react for 2 h at room temperature under continuous shaking on a rotating plate at 60 rpm. Our previous study demonstrated that the qBBr adsorption reaction onto the cell envelope sulfhydryl sites is complete after 2 h of reaction.20 Potentiometric titrations were conducted using a T70 autotitrator from Mettler Toledo Inc. and using 1 M HCl or NaOH standards with predetermined concentrations purchased from Fluka Chemical Corp. Prior to each titration, the 0.1 M NaCl solution in which the titration was to be conducted was purged with N2 for at least 1 h in order to remove dissolved CO2, and the electrode for pH measurement was calibrated using NIST-derived standard buffer solutions with pH values of 1.68, 4.01, 7.00, and 10.01. Bacterial cells with or without qBBr treatment were then suspended in the degassed 0.1 M NaCl solution to achieve a homogeneous suspension with a bacterial concentration of approximately 30 g/L, and 10−11 mL of this suspension was used in each titration. All the titrations were conducted in a closed vessel under a N2 headspace, and each suspension was stirred continuously with a magnetic stir bar. For each titration, two steps were conducted: (1) acidifying the bacterial suspension from the original pH down to pH 3.0 by

[H+]net added = (Ca − C b )/ m

(1)

where Ca and Cb are the total concentrations of acid and base added to the system during the titration, respectively, with units of mmol/L of biomass suspension, and m is the bacterial concentration in the suspension, with units of g (wet biomass)/ L. In our surface complexation modeling approach, we assume that the proton-active functional groups within bacterial cell envelopes are discrete monoprotic acids, whose deprotonation reactions can be expressed using the following reaction:26 R − Ai H ◦ ↔ R − Ai− + H + C

(2) DOI: 10.1021/acs.est.7b01047 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology Csulf = C total,untreated − C total,qBBr − treated

where R denotes the bacterial envelope macromolecule to which the ith organic acid functional group, Ai, is attached. Note that although we write Reaction 2 for an organic acid functional group, the same approach applies to protonation of amino-type sites as long as they are proton-active. The acidity constant (Ka,i) and the total concentration (Ci) of the ith site can be expressed as Ka , i =

σsulf =

(3)

Ci = [R − Ai−] + [R − Ai H °]

A−i ]

(4)

where [R − and [R − AiH ] represent the concentrations of the deprotonated and protonated ith organic acid functional group on the bacterial cell envelope, respectively, and aH+ is the activity of H+ in the bulk solution. Based on proton mass balance, the concentration of protons added to the system at any point of the titration can be described as 0

Ca − C b = [H+] − TH0 − [OH‐] −

∑ [R − Ai−]

3. RESULTS 3.1. Bacterial Biomass Samples Grown in M9 Minimal Media. Varying the glucose concentration in the M9 minimal medium causes different effects on the total binding site concentrations within the cell envelopes of the three bacterial species (Figure 2a, SI Table S3). For B. subtilis, the total

(5)

TH0

where represents the initial proton concentration at the commencement of the titration, [X] represents the concentration of species X in the experimental system, including H+, OH− and all the deprotonated organic acid functional groups within the bacterial cell envelope. FITEQL27 was used as a modeling tool for optimization of TH0, Ka,i and Ci in eqs 3, 4, and 5 to best fit the titration data and to solve for these unknown parameters, following the approach described by Fein et al.26 Activity coefficients of aqueous species were computed from the Davies Equation within the FITEQL program.27 Although different correction approaches can be applied to the acidity constant calculations to account for bacterial surface electric field effects, the fits to the experimental titration data by the different models are similar,28,29 and most previous studies demonstrate only a weak ionic strength influence on proton adsorption onto bacteria.29−31 Therefore, we used a nonelectrostatic surface complexation model to fit titration data. In our previous studies,20 we attempted to use one-, two-, three-, and four-site models to fit the potentiometric titration data, finding that a four-site nonelectrostatic model yields the best fit to the titration data for each of the bacterial species considered, and the same model was used in the present study to fit the titration data and in order to determine total binding site concentrations. Therefore, the concentration of total binding sites (Ctotal) within bacterial cell envelopes can be calculated as, C total = C1 + C2 + C3 + C4

(8)

where σ1 and σ2 are the standard deviations associated with the total binding sites for biomass samples with and without qBBr treatment, respectively, and n1, n2 represent the number of titrations conducted for the corresponding samples. A P value >0.05 was taken to indicate no significant difference between the signals from the biomass samples with and without qBBr treatment. In these cases, the concentration of sulfhydryl sites in the biomass was too low to be detectable using our approach. A summary of the calculation procedure for the concentration of sulfhydryl sites within bacterial cell envelopes is shown in Figure 1, using the B. subtilis biomass samples that were grown in the M9 minimal medium containing 50 g/L of glucose as an example.

[R − Ai−]a H + [R − Ai H °]

σ12 σ2 + 2 n1 n2

(7)

Figure 2. Effect of varying glucose concentration in the M9 minimal medium on (a) the total binding site concentrations and (b) the sulfhydryl site concentrations for B. subtilis (BS), P. putida (PP), and S. oneidensis (SO). All the data for total sites are from titrations with biomass that was not treated with qBBr. Error bars represent 1σ values.

binding site concentration significantly increases from 386 ± 14 (1σ) μmol/g to 452 ± 22 μmol/g when the glucose concentration increases from 4 g/L to 10 g/L. Further increasing the glucose concentration from 10 g/L to 50 g/L only slightly increases the total binding site concentration. In contrast, a consistent trend was not observed for the total concentration of binding sites for P. putida samples as a function of glucose concentration in the growth medium. The highest concentration of total binding sites for P. putida was observed for the samples grown with 26 g/L of glucose. This data point appears anomalous, but represents the average of three separate titrations of P. putida samples, with a standard deviation for the three total site concentrations of only ±1 μmol/g. For the S. oneidensis samples, varying the glucose concentration in the M9 growth medium only has a slight effect on the total concentration of binding sites, with total site concentrations increasing from 242 ± 3 μmol/g with 4 g glucose/L in the medium to 267 ± 3 μmol/g with 50 g glucose/L in the medium.

(6)

where C1, C2, C3, C4 represent the total concentrations for each of the four site types described by the four-site model, respectively. In order to calculate the sulfhydryl site concentrations, three titration experiments were conducted for each biomass with or without qBBr treatment. We used the Student’s t test to determine if the concentration of total binding sites decreased significantly after qBBr treatment, with a P value 0.05, SI Table S3). However, the P. putida samples that were grown in the M9 minimal medium with 15 g/L of glucose contain 25 ± 6 μmol/ g of sulfhydryl sites, and the measured sulfhydryl sites increase to 41 ± 5 μmol/g when the glucose concentration increases to 50 g/L. For S. oneidensis, we only detected sulfhydryl sites for biomass samples that were grown in the M9 minimal medium with 50 g/L of glucose, and the measured sulfhydryl site concentration in this medium is much lower than the sulfhydryl concentrations measured for the other two bacterial species at the same glucose concentration. Although the three bacterial species that were grown in the M9 minimal medium exhibit significantly different concentrations of sulfhydryl sites, following the order of B. subtilis > P. putida > S. oneidensis, these bacterial species exhibit similar sulfhydryl site concentrations when they were grown in the TSB medium (Figure 3b, and Yu et al.20,23).

4. DISCUSSION 4.1. Controls on Bacterial Sulfhydryl Site Concentrations during Growth. Carbon accounts for about half of the dry weight of bacterial cells. Previous studies have shown that varying the concentration of the carbon source in culture media can change the composition of bacterial cell wall components and bacterial exudates.32−34 In the present study, we found that increasing the concentration of glucose, a common carbon source that can be utilized by a wide range of bacteria, in the M9 minimal medium significantly increases the concentration of sulfhydryl sites within the cell envelopes of the three bacterial species studied (Figure 2b), suggesting that carbon utilization is required for the synthesis of bacterial sulfhydryl sites. Although sulfhydryl sites themselves do not contain carbon, some previous studies suggest that sulfhydryl sites are predominantly present attached to proteins within cell envelopes,14,35,36 and carbon is an essential element in the framework of proteins. It is likely that the cells require a certain concentration of carbon source in order to have enough bioavailable carbon to construct sulfhydryl-containing proteins. Because glucose is both the carbon source and the electron donor, or energy source, in these experiments, another explanation for our observations involves glucose as an energy source. Sulfhydryl sites are composed of reduced sulfur, which under the aerobic growth conditions in these experiments is not the thermodynamically stable form of the element, and hence metabolic energy expenditure is required in order to create

Figure 3. Comparison of (a) total site concentrations and (b) sulfhydryl site concentrations within the cell envelopes of B. licheniformis (BL), B. cereus (BC), B. subtilis (BS), P. putida (PP), and S. oneidensis (SO) grown in the TSB medium with (TSB+Glu) and without (TSB) the addition of 50 g/L of glucose. All the data for total sites are from titrations with biomass that was not treated with qBBr. The data for biomass samples that were grown in TSB medium without glucose are from previous studies.20,23 Error bars represent 1σ values.

3.2. Bacterial Biomass Samples Grown in NutrientRich TSB Media. Adding 50 g/L of glucose to the TSB medium causes significant and varied influences on the total site concentrations for the five bacterial species studied (Figure 3a). The addition of glucose to the TSB medium leads to slight decreases in the measured total site concentrations for the biomass samples of the two Gram-negative bacterial species studied, S. oneidensis and P. putida. In contrast, two of the three Gram-positive species studied, B. licheniformis and B. cereus, contained significantly higher total site concentrations when they were grown in the TSB medium enriched with 50 g/L of E

DOI: 10.1021/acs.est.7b01047 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology

concentrations for bacterial species that exhibit a preference for glucose as carbon source during growth. The effects of glucose on total binding site concentrations within bacterial cell envelopes are different than the effects that we observed for the sulfhydryl site concentrations, and are highly species-dependent. Only B. subtilis exhibited a significant and consistent increase in the total concentration of binding sites when glucose concentrations in the M9 medium increased from 4 g/L to 50 g/L (Figure 2a). The concentration of total binding sites for the B. subtilis samples increased markedly when glucose concentration increased from 4 g/L to 10 g/L, but a further increase in the glucose concentration to 50 g/L had a much smaller effect. The dramatic increase between 4 and 10 g glucose/L cannot be ascribed fully to the observed increase in sulfhydryl site concentration, as the sulfhydryl site concentration increased only by 6 μmol/g and the total site concentration increased by 67 μmol/g. Moreover, although the addition of 50 g/L of glucose to the TSB medium resulted in much higher sulfhydryl site concentrations for the three Bacillus species in the present study, the three species do not exhibit a consistent trend in their total site concentrations upon addition of the glucose. The addition of 50 g/L of glucose to the TSB medium causes the total site concentration for B. licheniformis samples to increase markedly from 168 ± 16 to 386 ± 7 μmol/ g (Figure 3a). In contrast, we did not observe a significant increase in total site concentration for B. subtilis samples under the same change in growth medium composition. These findings suggest that the concentrations of other binding sites within bacterial cell envelopes, such as carboxyl and phosphoryl groups, also respond to the glucose level in the growth medium, and that the other site types likely have different responses to the increase of glucose than those observed for sulfhydryl sites. If the increase in sulfhydryl site concentration was offset by a decrease in the concentration of other binding sites, the total binding site concentration would not change with increasing glucose in the growth medium. Potentiometric titrations and surface complexation modeling coupled with qBBr selective blocking of sulfhydryl sites provides precise constraints on the concentration of sulfhydryl sites in a sample, but because there are no equivalent molecules to qBBr that would selectively block the other binding sites within bacterial cell envelopes, it is not currently possible to directly identify the effect of glucose concentration on the abundance of the other binding sites. 4.2. Environmental Implications. Sulfhydryl sites can play a more critical role than other binding sites within cell envelopes in the adsorption of chalcophilic metals onto bacterial cells under low metal loading conditions, and thus may control the fate and bioavailability of these metals in the environment.14−17 A previous study found that different bacterial species grown in a nutrient-rich TSB medium have similar concentrations of sulfhydryl sites within their cell envelopes,20 suggesting that nutrient-rich systems with different compositions of bacterial species may contain the same overall abundance of bacterial sulfhydryl sites. However, this study shows that adding additional glucose to the TSB medium significantly increases the sulfhydryl site concentrations within cell envelopes of Bacillus species, and thus results in different sulfhydryl site concentrations between Bacillus species and S. oneidensis or P. putida. Furthermore, we also show that different bacterial species can have markedly different concentrations of sulfhydryl sites when they are grown in a more nutrient-poor M9 medium, whose nutrient levels are more representative of natural systems than nutrient-rich media such as the TSB

reduced sulfur to form sulfhydryl sites. Our results suggest that the production of sulfhydryl sites within bacterial cell envelopes is limited by the concentration of carbon source and/or energy source in the growth medium, and that sulfhydryl sites can only be produced if sufficient carbon and energy stores are provided during growth. Our results also suggest that the type of carbon source in the growth medium affects the sulfhydryl site concentrations within bacterial cell envelopes. Glucose is a preferred carbon source for the growth of B. subtilis,37 but S. oneidensis grows more readily utilizing three-carbon or smaller carbohydrates as carbon sources,38,39 and P. putida grows more readily on some organic acids and amino acids than with glucose.40 These differences in carbon source preference likely resulted in the different sulfhydryl site concentrations that we observed when each species was grown in the M9 minimal media with glucose as the only carbon source available (Figure 2b). That is, because B. subtilis utilizes glucose more easily than the other two species, B. subtilis could produce the highest sulfhydryl site concentrations among the three bacterial species studied when each species was provided the same concentration of glucose during growth. Moreover, a previous study39 showed that S. oneidensis uses glucose primarily as an energy source and less as a carbon source for building biomass structures. Therefore, it appears that the addition of glucose to the S. oneidensis growth medium created conditions of the cells having a plentiful electron donor, but being limited in carbon for structural features. The low resulting concentration of sulfhydryl sites in the S. oneidensis samples suggests that carbon source and not energy supply controls the synthesis of sulfhydryl-containing proteins for the S. oneidensis samples that were grown in the M9 minimal media. Previous studies found that B. subtilis, P. putida, and S. oneidensis produced similar concentrations of sulfhydryl sites when they were cultured in nutrient-rich TSB medium (Figure 3b).20,23 The TSB medium contains multiple types of substances that can be used as a carbon source by the bacteria, including 17 g of tryptone, 3 g of soytone, 5 g of yeast extract, and 2.5 g of glucose per L of medium. Besides glucose, the other carbon sources in the TSB medium contain abundant amino acids and small carbohydrates, which are the preferred carbon sources for P. putida and S. oneidensis, respectively. With the help of carbon catabolite repression,37 bacteria are able to selectively metabolize their preferred carbon sources for their growth in a medium containing multiple types of carbon sources, likely explaining the observed similarity in concentrations of sulfhydryl sites for B. subtilis, S. oneidensis and P. putida when grown in the TSB medium. It is noteworthy that the addition of 50 g/L of glucose to the TSB medium strikingly increases the sulfhydryl site concentrations of B. subtilis samples from 23 ± 7 to 93 ± 8 μmol/g, but does not significantly change the sulfhydryl site concentrations of either S. oneidensis or P. putida (Figure 3b). We also measured the sulfhydryl concentrations of biomass samples for B. licheniformis and B. cereus, both of which grow more readily with glucose as a carbon source, and found that they behaved similar to B. subtilis in that their sulfhydryl site concentrations are significantly higher when they are grown in the TSB medium enriched with 50 g/L of glucose than when they are grown in the TSB medium without additional glucose (Figure 3b). These results indicate that varying the glucose concentration in the growth medium has the strongest influence on sulfhydryl site F

DOI: 10.1021/acs.est.7b01047 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

■

ACKNOWLEDGMENTS Funding for this project was provided by U.S. National Science Foundation grant EAR-1424950. The comments and suggestions of three journal reviewers were helpful in improving the presentation of this research and are appreciated.

medium. Based on these observations, the bacterial sulfhydryl sites that are present in a natural system likely are strongly affected by the composition of the bacterial community, the carbon preference of the bacteria in the system, as well as the nutrient conditions in which they grow. These factors must be considered in order to determine and model the effect of bacterial cells on metal cycling and metal bioavailability in the environment. Our results may also lead to applications in environmental engineering, such as biosorption. In recent years, much effort has been made to improve the performance of biosorbents in both their general sorption capacity and their selectivity, either via chemical modification or via genetic modification.41 Our results suggest that increasing the glucose concentration in the growth medium for biosorbent species may represent a relative simple and inexpensive alternative to enhance biosorbent performance. For example, biomass of B. subtilis that is cultured in a glucose-rich TSB medium may be used as an effective biosorbent that exhibits high selectivity for chalcophilic metals, such as Hg, Cd, and Pb, due to their high percentage (up to 34% of the total site concentration) of sulfhydryl sites relative to total binding sites within the cell envelopes (Figure 3). This study is the first to determine the concentration of bacterial sulfhydryl sites in minimal media, with a focus on the effect of the carbon source and electron donor concentration. Given the strong effect of glucose that we observed on the production of bacterial sulfhydryl sites, and because of the potentially controlling role that bacterial sulfhydryl site binding of metal can play, it is important to examine the influences that the concentration of other nutrients, such nitrogen, sulfur and other trace elements, may have on sulfhydryl site abundance. A better understanding of the controls on bacterial sulfhydryl site concentrations will yield more accurate predictions of the effect of bacterial cells on the fate and transport of toxic metals in the environment, and will aid the development of more effective and efficient bioremediation approaches for waters and soils contaminated by toxic metals.

■

■

REFERENCES

(1) Jarup, L. Hazards of heavy metal contamination. Brit Med. Bull. 2003, 68, 167−182. (2) Hughes, M. F. Arsenic toxicity and potential mechanisms of action. Toxicol. Lett. 2002, 133 (1), 1−16. (3) Nordberg, G. F. Historical perspectives on cadmium toxicology. Toxicol. Appl. Pharmacol. 2009, 238 (3), 192−200. (4) Fein, J. B. Advanced biotic ligand models: Using surface complexation modeling to quantify metal bioavailability to bacteria in geologic systems. Chem. Geol. 2016, 464, 127. (5) Ngwenya, B. T.; Sutherland, I. W.; Kennedy, L. Comparison of the acid-base behaviour and metal adsorption characteristics of a gramnegative bacterium with other strains. Appl. Geochem. 2003, 18 (4), 527−538. (6) Ha, J.; Gelabert, A.; Spormann, A. M.; Brown, G. E. Role of extracellular polymeric substances in metal ion complexation on Shewanella oneidensis: Batch uptake, thermodynamic modeling, ATRFTIR, and EXAFS study. Geochim. Cosmochim. Acta 2010, 74 (1), 1− 15. (7) Yee, N.; Fein, J. Cd adsorption onto bacterial surfaces: A universal adsorption edge? Geochim. Cosmochim. Acta 2001, 65 (13), 2037−2042. (8) Beveridge, T. J.; Murray, R. G. E. Uptake and Retention of Metals by Cell-Walls of Bacillus-Subtilis. J. Bacteriol. 1976, 127 (3), 1502− 1518. (9) Plette, A. C. C.; Benedetti, M. F.; vanRiemsdik, W. H. Competitive binding of protons, calcium, cadmium, and zinc to isolated cell walls of a gram-positive soil bacterium. Environ. Sci. Technol. 1996, 30 (6), 1902−1910. (10) Beveridge, T. J.; Murray, R. G. E. Sites of Metal-Deposition in the Cell-Wall of Bacillus-Subtilis. J. Bacteriol. 1980, 141 (2), 876−887. (11) Kelly, S. D.; Kemner, K. M.; Fein, J. B.; Fowle, D. A.; Boyanov, M. I.; Bunker, B. A.; Yee, N. X-ray absorption fine structure determination of pH-dependent U-bacterial cell wall interactions. Geochim. Cosmochim. Acta 2002, 66 (22), 3855−3871. (12) Jiang, W.; Saxena, A.; Song, B.; Ward, B. B.; Beveridge, T. J.; Myneni, S. C. B. Elucidation of functional groups on gram-positive and gram-negative bacterial surfaces using infrared spectroscopy. Langmuir 2004, 20 (26), 11433−11442. (13) Pokrovsky, O. S.; Pokrovski, G. S.; Shirokova, L. S.; Gonzalez, A. G.; Emnova, E. E.; Feurtet-Mazel, A. Chemical and structural status of copper associated with oxygenic and anoxygenic phototrophs and heterotrophs: possible evolutionary consequences. Geobiology 2012, 10 (2), 130−149. (14) Mishra, B.; Boyanov, M.; Bunker, B. A.; Kelly, S. D.; Kemner, K. M.; Fein, J. B. High- and low-affinity binding sites for Cd on the bacterial cell walls of Bacillus subtilis and Shewanella oneidensis. Geochim. Cosmochim. Acta 2010, 74 (15), 4219−4233. (15) Yu, Q.; Fein, J. B. The effect of metal loading on Cd adsorption onto Shewanella oneidensis bacterial cell envelopes: The role of sulfhydryl sites. Geochim. Cosmochim. Acta 2015, 167, 1−10. (16) Nell, R. M.; Fein, J. B. Influence of sulfhydryl sites on metal binding by bacteria. Geochim. Cosmochim. Acta 2017, 199, 210−221. (17) Guine, V.; Spadini, L.; Sarret, G.; Muris, M.; Delolme, C.; Gaudet, J. P.; Martins, J. M. F. Zinc sorption to three gram-negative bacteria: Combined titration, modeling, and EXAFS study. Environ. Sci. Technol. 2006, 40 (6), 1806−1813. (18) Liu, A. M.; Hidajat, K.; Kawi, S.; Zhao, D. Y. A new class of hybrid mesoporous materials with functionalized organic monolayers for selective adsorption of heavy metal ions. Chem. Commun. 2000, 13, 1145−1146.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b01047. Recipes of the vitamin solution and the trace element solution used in the M9 medium. Summary of binding site concentrations within the cell envelopes of P. putida (PP), S. oneidensis (SO), and B. subtilis (BS) biomass samples that were grown in the M9 minimal medium containing 4−50 g/L of glucose. Summary of binding site concentrations within the cell envelopes of B. licheniformis (BL), B. subtilis (BS), B. cereus (BC), P. putida (PP), and S. oneidensis (SO) biomass samples that were grown in the TSB medium enriched with 50 g/L of glucose (PDF)

■

Article

AUTHOR INFORMATION

Corresponding Author

*Phone: (574) 631-4534; fax: (574) 631-9236; e-mail: qyu@ nd.edu. Notes

The authors declare no competing financial interest. G

DOI: 10.1021/acs.est.7b01047 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology (19) Brown, J.; Mercier, L.; Pinnavaia, T. J. Selective adsorption of Hg2+ by thiol-functionalized nanoporous silica. Chem. Commun. 1999, 1, 69−70. (20) Yu, Q.; Szymanowski, J.; Myneni, S. C. B.; Fein, J. B. Characterization of sulfhydryl sites within bacterial cell envelopes using selective site-blocking and potentiometric titrations. Chem. Geol. 2014, 373, 50−58. (21) Joe-Wong, C.; Shoenfelt, E.; Hauser, E. J.; Crompton, N.; Myneni, S. C. B. Estimation of Reactive Thiol Concentrations in Dissolved Organic Matter and Bacterial Cell Membranes in Aquatic Systems. Environ. Sci. Technol. 2012, 46 (18), 9854−9861. (22) Sarret, G.; Manceau, A.; Spadini, L.; Roux, J. C.; Hazemann, J. L.; Soldo, Y.; Eybert-BÉ rard, L.; Menthonnex, J. J. Structural determination of Zn and Pb binding sites in Penicillium chrysogenum cell walls by EXAFS spectroscopy. Environ. Sci. Technol. 1998, 32 (11), 1648−1655. (23) Yu, Q.; Fein, J. B. Sulfhydryl Binding Sites within Bacterial Extracellular Polymeric Substances. Environ. Sci. Technol. 2016, 50 (11), 5498−5505. (24) M9 minimal medium (standard). Cold Spring Harbor Protocols 2010, 2010, (8), pdb.rec12295. (25) Lovley, D. R.; Phillips, E. J. P. Novel Mode of Microbial EnergyMetabolism - Organic-Carbon Oxidation Coupled to Dissimilatory Reduction of Iron or Manganese. Appl. Environ. Microb. 1988, 54 (6), 1472−1480. (26) Fein, J. B.; Boily, J. F.; Yee, N.; Gorman-Lewis, D.; Turner, B. F. Potentiometric titrations of Bacillus subtilis cells to low pH and a comparison of modeling approaches. Geochim. Cosmochim. Acta 2005, 69 (5), 1123−1132. (27) Westall, J. C., FITEQL, A Computer Program for Determination of Chemical Equilibrium Constants from Experimental Data. Version 2.0. Report 82-02; Dept. Chem., Oregon St. Univ., Corvallis, OR., 1982. (28) Fein, J. B.; Boily, J.-F.; Yee, N.; Gorman-Lewis, D.; Turner, B. F. Potentiometric titrations of Bacillus subtilis cells to low pH and a comparison of modeling approaches. Geochim. Cosmochim. Acta 2005, 69 (5), 1123−1132. (29) Borrok, D. M.; Fein, J. B. The impact of ionic strength on the adsorption of protons, Pb, Cd, and Sr onto the surfaces of Gram negative bacteria: testing non-electrostatic, diffuse, and triple-layer models. J. Colloid Interface Sci. 2005, 286 (1), 110−126. (30) Martinez, R. E.; Smith, D. S.; Kulczycki, E.; Ferris, F. G. Determination of intrinsic bacterial surface acidity constants using a donnan shell model and a continuous pK(a) distribution method. J. Colloid Interface Sci. 2002, 253 (1), 130−9. (31) Gonzalez, A. G.; Shirokova, L. S.; Pokrovsky, O. S.; Emnova, E. E.; Martinez, R. E.; Santana-Casiano, J. M.; Gonzalez-Davila, M.; Pokrovski, G. S. Adsorption of copper on Pseudomonas aureofaciens: Protective role of surface exopolysaccharides. J. Colloid Interface Sci. 2010, 350 (1), 305−314. (32) Ahuja, S. K.; Ferreira, G. M.; Moreira, A. R. Production of an endoglucanase by the shipworm bacterium, Teredinobacter turnirae. J. Ind. Microbiol. Biotechnol. 2004, 31 (1), 41−47. (33) Celik, G. Y.; Aslim, B.; Beyatli, Y. Characterization and production of the exopolysaccharide (EPS) from Pseudomonas aeruginosa G1 and Pseudomonas putida G12 strains. Carbohydr. Polym. 2008, 73 (1), 178−182. (34) Jain, R. M.; Mody, K.; Joshi, N.; Mishra, A.; Jha, B. Production and structural characterization of biosurfactant produced by an alkaliphilic bacterium, Klebsiella sp.: Evaluation of different carbon sources. Colloids Surf., B 2013, 108, 199−204. (35) Norrod, E. P.; Browne, S. L.; Feldweg, A.; Leonard, J. A dominant sulfhydryl-containing protein in the outer membrane of Neisseria gonorrhoeae. J. Bacteriol. 1993, 175 (4), 1173−5. (36) Michelon, D.; Abraham, S.; Ebel, B.; De Coninck, J.; Husson, F.; Feron, G.; Gervais, P.; Cachon, R. Contribution of exofacial thiol groups in the reducing activity of Lactococcus lactis. FEBS J. 2010, 277 (10), 2282−2290. (37) Deutscher, J. The mechanisms of carbon catabolite repression in bacteria. Curr. Opin. Microbiol. 2008, 11 (2), 87−93.

(38) Serres, M. H.; Riley, M. Genomic analysis of carbon source metabolism of Shewanella oneidensis MR-1: Predictions versus experiments. J. Bacteriol. 2006, 188 (13), 4601−4609. (39) Howard, E. C.; Hamdan, L. J.; Lizewski, S. E.; Ringeisen, B. R. High frequency of glucose-utilizing mutants in Shewanella oneidensis MR-1. FEMS Microbiol. Lett. 2012, 327 (1), 9−14. (40) Rojo, F. Carbon catabolite repression in Pseudomonas: optimizing metabolic versatility and interactions with the environment. Fems Microbiol Rev. 2010, 34 (5), 658−684. (41) Vijayaraghavan, K.; Yun, Y. S. Bacterial biosorbents and biosorption. Biotechnol. Adv. 2008, 26 (3), 266−291.

H

DOI: 10.1021/acs.est.7b01047 Environ. Sci. Technol. XXXX, XXX, XXX−XXX