The Role of Dissolved Silica on the Biodegradation of Octylamine

A study was conducted to evaluate the effect of dissolved silica on the rates of biodegradation of a cationic surfactant, octylamine, by Rhodococcus e...
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Environ. Sci. Technol. 1999, 33, 3723-3729

The Role of Dissolved Silica on the Biodegradation of Octylamine HILDEGARDE SELIG, KIM F. HAYES,* AND PETER ADRIAENS Environmental and Water Resources Engineering, The University of Michigan, Ann Arbor, Michigan 48109

Dissolution of aquifer-associated mineral solids such as silica may affect the natural bioattenuation of organic compounds by altering their chemical speciation and thus their biodegradability. A study was conducted to evaluate the effect of dissolved silica on the rates of biodegradation of a cationic surfactant, octylamine, by Rhodococcus erythropolis. The presence of dissolved silica or a surrogate added by dissolving sodium metasilicate significantly enhanced the biodegradation rates of octylamine. Microbial kinetic studies based on the Monod/Haldane equation indicated that the rate enhancement was caused by a decrease in the inhibitory properties of octylamine, rather than by an improvement in buffering of the medium. In the presence of dissolved silica, µmax and Ks remain virtually unaffected, but the inhibition constant, Ki, increased several orders of magnitude (from Ki ) 1.32 mM in the silicafree system to Ki > 106 mM in the silica system). Surface tension studies suggested that the presence of dissolved silica significantly decreased the surface-activity of octylamine.

Introduction The mineral fraction of soils and sediments may play an important role in determining the fate of toxic organic chemicals in low organic content environments such as subsurface aquifers (1). It has been well documented in the literature that the sorption of organic compounds onto mineral particles can influence the bioavailability of these compounds to microbial populations and significantly increase their persistence in the environment (2, 3). Mineral surfaces, however, may also influence biodegradation through dissolution of chemicals into the aqueous phase, which could affect the pH and buffering capacity of the medium (4-6) or by reacting with organic substrates (7, 8). Complexes formed between organic pollutants and organic and inorganic ligands in solution can change their physicochemical properties and thus alter their biochemical interactions with the cell membrane and consequent bioavailability (9-14). This study evaluates the effect of dissolved silica on the biodegradation and inhibitory properties of the cationic surfactant octylamine. Alkylamines have been shown to be readily biodegradable in pure culture (15, 16) and mixed culture (17, 18) studies at low concentrations but are inhibitory at concentrations higher than a certain threshold (16). Complexation reactions of cationic surfactants with dissolved organic matter (19, 20) and anionic surfactants (21) have been reported to reduce the toxicity of cationic surfactants to aquatic organisms. * Corresponding author phone: (734)763-9661; fax: (734)763-2275; e-mail: [email protected]. 10.1021/es9808595 CCC: $18.00 Published on Web 09/14/1999

 1999 American Chemical Society

Groundwater aquifers are often predominantly composed of sand, comprised mostly of quartz (SiO2) and feldspar (such as KAlSi3O8) (22). The sorptive properties of SiO2 minerals have been well characterized (23-25). Due to the protonation of the amino group, alkylamines will bind strongly to silica surfaces at pH values above 2, the point of zero charge for most silica polymorphs (25). Adsorption of cationic surfactants to silica surfaces has been extensively studied (26). A decrease in the bioavailability of alkylamines (15) and benzylamines (27) sorbed to clay minerals has been observed. However, no direct evidence is available in the literature to support the involvement of dissolved silica in the biodegradation of organic compounds. Considering the potential for an extensive increase of dissolved silica in hydrocarboncontaminated aquifers (28, 29), the interactions of dissolved silica with organic compounds deserve further investigation. The aqueous chemistry of silica is regulated by a number of coupled processes such as dissolution/precipitation and complexation to ions in the aqueous phase or at the solidwater interface (30). Silica predominantly dissolves into neutral monomeric silicic acid H4SiO4 (31); the concentration of silica dimers may be significant when amorphous silica controls the equilibrium solubility (32). At high pHs, silicate anions are present (pKa ) 9.47) and diverse polysilicates are formed (33). The equilibrium concentration of dissolved silica is a function of the silica phase, pH, temperature, and solution species (32). The solubility of silica phases at 25 °C has been reported between 0.1 mM for quartz (34) and 1.9 mM for amorphous silica (35). Dissolved silica concentrations in groundwaters range between 85 and 5 ppm, with a median concentration of 17 ppm (0.3 mM) (36). Contaminated groundwaters have been reported supersaturated with respect to quartz and approaching equilibrium with amorphous silica (37). Silicic acid has been traditionally characterized as having relatively simple aqueous speciation chemistry, the exception being the self-polymerization reaction that occurs at relatively high concentrations (31). At basic pH values, cationic surfactant complexes with aqueous silicates are the basis for the synthesis of custom-tailored zeolite catalysts (38, 39). At neutral to acid pH values, conditions more relevant to this research, some reactions of silicic acid with organic compounds have been identified. Increased dissolution of quartz in organic-rich aqueous environments has been attributed to aqueous complexation reactions between dissolved silica and various organic acids (40). Aqueous complexes of silicic acid with substituted catechols (41-43), including catecholamines (44) have been reported to occur over a wide pH range. Amines and polyamines have been recently reported as catalysts for the polymerization of silicic acid; hydrogen bonding between NH3+ groups and dissolved silica was suggested (45). For this study, a quartz sand and a pure bacterial culture were used to evaluate the impact of dissolved silica on the biodegradation of the cationic surfactant octylamine, an inhibitory organic compound. Biodegradation rate and kinetic experiments were performed as a function of medium buffering capacity, concentration of dissolved silica or sodium metasilicate solutions, and cell density. Reduction of the octylamine surface activity, supported by surface tension measurements, is proposed to explain the results of the kinetic studies.

Materials and Methods Materials. The mineral media used for bacterial growth studies was designed with the aid of a chemical equilibrium VOL. 33, NO. 21, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Characteristic of the Silica Particles Used in Biological Experiments type of silica

SA (m2/g)

concn concn of silanol (g/L) groups (mM)c

concn of adsorption sites (mM)d

Si-100 0.0182 ( 0.003a 1000 0.0303 ( 0.005 0.00164 ( 0.0003 Si-1 1.1b 100 0.216 0.0117 a SA, surface area, determined by assuming spherical particles (SA ) 6/FD), with diameter D between 150 and 106 µm (sieves no. 140 and no. 100) and a density F of 2.65. b SA determined by nitrogen adsorption (BET method, Autosorb 1, Quantachrome, Syosset, NY). c Calculated assuming 5 sites/nm2 (25). d Calculated assuming only 0.27 sites/nm2 dissociated silanol sites (SiO-), at pH conditions of experiment (60).

speciation program (HYDRAQL (46)) to minimize nutrient precipitation and to provide good buffering capacity (buffering capacity, β ) 11.8 mM) at the designed pH (pH ) 7.0 ( 0.2). The composition of the basic mineral medium (MM-1) is as follows: 100 mg/L of NH4Cl, 700 mg/L of KH2PO4, 1 g/L of K2HPO4, 10 mg/L of NaCl, 5 mg/L of CaCl2, 10 mg/L of MgCl2, 0.0392 mg/L of CuCl2‚2H2O, 0.136 mg/L of ZnCl2, 0.013 mg/L of NiCl2, 0.702 mg/L of FeCl2‚4H2O, 0.111 mg/L AlCl3, 0.281 mg/L of MnCl2‚4H2O, 0.0382 mg/L of CoCl2‚6H2O, 0.0254 mg/L of Na2MoO4‚2H2O, 0.0618 of H3BO3, and 0.142 mg/L of Na2SO4. Two media were derived from the basic mineral medium: (1) MM-2, a 1:100 dilution of the basic medium with the addition of phosphate buffer (1.0 mM of PO4, β ) 1.3 mM), and (2) MM-3, a 1:100 dilution of the basic medium (β ) 0.12 mM). Reagent-grade octylamine (99% purity, Fluka AG, Switzerland) was used as the organic substrate. 14C-Octylamine (NH214CH2(CH2)6CH3) was customsynthesized (10 mCi/mmol, 98% radiochemical purity, Sigma Chemical Co., St. Louis, MO). Two different size fractions of nonporous silica particles were used. A large particle size fraction, Si-100, was obtained by sieving Ottawa sand (“Crystal”, 99.8% SiO2, U.S. Silica, Ottawa, IL). Si-100 was the fraction that passed sieve no. 100 but was retained by sieve no. 140 (giving a range of 106-150 µm with an average diameter of 100 µm). A smaller silica particle size fraction, Si-1, was obtained by grinding Ottawa sand (Sil-Co-Sil-40, 99.8% SiO2, U.S. Silica, Ottawa, IL) resulting in a size range of 1-15 µm, with a median diameter of 5.1 µm (47). Both size fractions were thoroughly washed with hydrochloric acid and hydrogen peroxide to remove possible organic and inorganic impurities and rinsed with water until neutral pH was attained (48). Since the particles were not specifically pretreated to remove silica coatings to expose the quartz substrate, a surface layer of amorphous silica likely remained on the particles following the above treatments. Measured values of dissolved silica concentrations approaching that expected for amorphous silica and greater than that of quartz support this. As such, the particles in this study are referred to and operationally defined as silica particles. The specific surface area of the particles, the concentration used in most experiments, as well as the estimated concentration of adsorption sites are presented in Table 1. A pure bacterial culture was isolated from Ann Arbor topsoil using octylamine as the sole source of carbon and energy. The culture was a small gram-positive coccus identified as Rhodococcus erythropolis (Microbial ID Inc., Newark, DE) based on fatty acid methyl ester analysis. Initial bacterial concentrations were measured by the standard serial dilution and plate count method (49). Tryptic Soy Agar (Bacto, Fisher Scientific, Pittsburgh, PA) was used as the solid medium, and phosphate buffer in prefilled sterile dilution bottles (Fisherbrand, Fisher) was used as the dilution medium. Cell counts were translated to cell mass by a factor of 6.8 × 10-8 to convert cells/mL to mg/L of bacteria. This 3724

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factor was based on the reasonable assumption that bacteria are 0.5 µm spheres (from microscopic observations) with a specific gravity of 1.04 (50). Cell yield, mass of cells/mass of organic, was obtained by determining the dry mass of cells (51) after consumption of known concentrations of substrate. Sampling and Analytical Techniques. Mineralization of octylamine was measured by determining the removal of 14 C-octylamine from solution. Acid and compressed air sparging were used to remove dissolved 14CO2; thereafter scintillation cocktail was added (ECOLUME, ICN Biomedicals, Costa Mesa, CA) and the solution counted (1219 RacBeta, LKB Wallac, Finland). Particle separation was performed prior to 14C detection by gravity settling for the samples containing large Si-100 particles and by centrifugation in high-speed microcentrifuge (15 700 RCF, 15 min, MC-140 Tomy, Tomy Tech U.S.A., Inc. Palo Alto, CA) for those with the smaller Si-1 particles. Total aqueous silica was measured using a Graphite Furnace Atomic Adsorption Spectrometer (GF-AA) (1100B AA Spectrophotometer, HGA 700 Graphite Furnace, PerkinElmer, Norwalk). Silicon standards were prepared by diluting a commercial silicon standard for atomic adsorption (1000 µg/mL) (Fisher Scientific, Fairlawn, NJ) in Milli-Q-water. Silica suspension samples were centrifuged (2000 RCF, GS-6 Beckman, VWR Scientific, Palo Alto, CA) and/or filtered through a 0.2 µm syringe filter (Gelman Science, Ann Arbor, MI) prior to silicon measurements. Comparative Rate and Kinetic Studies. Biodegradation rates of octylamine in the presence and absence of silica particles were studied in systems in which octylamine losses due to sorption onto the silica particles were negligible. This was accomplished by keeping the amount of surface area for sorption in the particle suspensions relatively small compared to octylamine concentrations. Consequently, no significant reduction in the concentration of octylamine in solution occurred. Three sets of samples were considered: (i) silica-free control samples, which were used to establish background degradation rates at specific culturing conditions, (ii) silicacontaining samples, in which bacteria were cultured in the presence of solid and dissolved silica, and (iii) supernatant samples, in which bacteria were cultured in a supernatant medium that had been previously contacted with silica particles for a set amount of time. Some experiments included sodium metasilicate as a surrogate for dissolved silica to determine if the source of dissolved silica was a factor. Sterile samples containing mineral media, octylamine, and silica particles were in contact for at least a week prior to addition of the cell inoculum. At this point, samples were analyzed for dissolved silica and initial octylamine concentration. Dissolved silica concentrations were also measured as a function of time for Si-100 particles in MM-2 medium and Si-1 particles in both MM-1 and MM-2 media (data not shown) verifying that equilibrium with respect to dissolved silica was not reached in any of the conditions tested. Three types of experiments were conducted to evaluate the effect of dissolved silica on octylamine biodegradation: (i) nutrient concentration and buffering capacity of the media, (ii) the concentration and source of dissolved silica, and (iii) the initial concentration of bacteria. The experimental conditions of each study are presented in Table 2. In these experiments, mineralization of inhibitory levels of octylamine was evaluated over time in the three different media described before. Preliminary kinetic studies of octylamine mineralization over a wide range of substrate concentrations (not shown) indicated that the inhibitory threshold of octylamine is about 0.1 mM for the experimental conditions and cell densities used in these experiments. They also demonstrated that dissolved silica alone was not inhibitory to cells grown below the octylamine inhibitory threshold.

TABLE 2. Experimental Conditions of the Comparative Rate Experiments experiment

type of Si/[Si], g/L

dissolved Si, mM

[octylamine], mM

mineral medium

cell inocula cells/mL

1 Variable Media

Si-100/1000

nd 0.25 nd

0.5

MM-1 MM-2 MM-3

7.2 × 105 7.2 × 105 1.4 × 105

2 Variable Media

Si-1/500 Si-1/100 Si-1/100 Si-1/20 Na2SiO4a

0.78 ( 0.02 0.45 ( 0.04 0.45 ( 0.04 0.07 ( 0.008 0.80 0.55 0.20 0.06

0.5 0.5 0.1 0.1 0.5 0.5 0.1 0.1

MM-1

2.8 × 105

3 Variable Initial Cell Density

Si-1/100

0.45

0.5

MM-1

2.0 × 107 2.0 × 106 2.0 × 105 2.0 × 104

a

Na2SiO4 salt completely dissolved to concentration shown under dissolved silica.

Experiments were designed to obtain the Monod/Haldane kinetic parameters for the mineralization of octylamine in the presence and absence of silica particles. Silica samples containing 100 g/L of Si-1 were contacted for 7 days with MM-1 medium. Six different concentrations of octylamine were tested: 0.5, 0.25, 0.1, 0.05, 0.01, and 0.005 mM. Bacterial inoculum (2% v/v) consisted of a 36-h culture of R. erythropolis in a growth medium containing 0.24 mM octylamine. Surface Tension Measurements. Surface tension measurements of octylamine solutions with and without the addition of dissolved forms of silica were performed to identify possible changes of octylamine surface activity in the presence of dissolved silica. Surface tension was measured with the ring method using a Du Nou ¨ y tensiometer (Kruss K8 Interfacial Tensiometer, Hamburg, Germany). All glassware was thoroughly cleaned since surface tension is sensitive to trace quantities of impurities. To remove possible trace impurities introduced during sample preparation, the Pt-Ir ring of the tensiometer was immersed at least five times in the solution prior to surface tension measurements. This technique has been shown to remove impurities as evidenced by the consistency of the surface tension value after the procedure has been implemented. Octylamine concentrations between 6 and 2 mM were prepared in MM-1 medium. Sample pH was adjusted to 7 with 1 N HCl. Sodium metasilicate was added to half of the samples to a final concentration of 1.5 mM. Solutions were equilibrated for 48 h.

Data Analyses

(

ln So +

)

Xo Xo - S ) ln + µt Y Y

(2)

where X and S are the mass of cells and the substrate concentration, respectively, expressed in mg/L. Y is the yield coefficient in mg of X/mg of S. The subscript o denotes initial concentrations. Equation 2 results from combining the Monod relationship for logarithmic growth (dX/dt ) µX) and the basic relationship between substrate utilization and cell growth (-dS/dt ) 1/Y dX/dt). A plot of ln(So + Xo/Y - S) vs time should give a straight line with slope equal to µ during logarithmic growth. The Haldane modification of the Monod equation (53) was adopted to describe the inhibitory effect of octylamine on biodegradation rates which, when expressed in terms of substrate utilization, has the differential form

-

(

µmaxS Xo dS ) S + -S dt K + S + S2/K o Y s i

)

(3)

where µmax is defined as the maximum specific growth rate (1/h), Ks as the Monod affinity constant (mg/L), and Ki as Haldane inhibition constant (mg/L). Equation 3 reaches a maximum at a certain point (S*, µ*), where S* is considered the inhibitory threshold concentration and µ* the maximum attainable growth rate in the presence of the inhibitor. Equation 3 is combined with the following equation to account for the 14C concentration present in solution in compounds or phases different from the parent compound octylamine 14

Buffering Capacity. To quantify the capacity of media to maintain a stable pH, the buffering capacity index (β) was calculated. The buffering index quantifies the capacity of a system to withstand pH changes when strong acid is added to a solution and can be calculated based on the Minor Species Theorem (52). Since mineral media were defined at neutral pH, the β index was calculated as

β ) 2.3{[NH3] + [H2PO4-] + [H2S] + [H2CO3*] + [CO32-]} (1) Mineralization Rate Calculations and Kinetic Modeling. The specific growth rates, µ, the rate of cell growth normalized per unit mass of cells (1/h), were determined in batch cultures during the logarithmic growth phase using the following expression

C ) ζSo + (1 - ζ)S

(4)

This assumes that the ratio between 14CO2 and 14C in products, denoted ζ, remains constant during the experiment (54). Kinetic parameters µmax, Ks, and Ki and the fraction ζ were estimated by applying a nonlinear regression routine to sets of data randomly generated within the experimental observation values. This procedure is commonly employed to generate sufficient data sets from a limited set of data within a measured range (55, 56). The method allows the estimation of reliable confidence intervals of nonlinear regression problems. The kinetic modeling method and proofs of uniqueness of the solution are available as Supplementary Information.

Results and Discussion Effect of Nutrient Concentration and Buffering Capacity. The substrate mineralization curves over time in all media VOL. 33, NO. 21, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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observed due to the low concentration of surface sites (0.0016 mM) and the high octylamine concentration used (0.5 mM) (Table 1). Although enhanced bacterial respiration rates in the presence of clay and other minerals has been attributed to the increase in buffering capacity of the media due to mineral dissolution or exchange of basic cations to the medium (46), this was not the case here. In the present study, mineralization rates in the presence of solid or dissolved silica increased significantly in the three different buffered media tested, including the well-buffered medium (MM-1) (Table 3). In addition, the pH of the media at the end of the experiments similarly decreased (initial media pH ) 7.1) in both silica-containing and silica-free samples with decreasing buffer concentration (Table 3). Thus, solid or dissolved silica did not affect the ability of a given medium to maintain a constant pH. An important finding of this experiment is that octylamine mineralization rates in the particle-free supernatant samples are similar to those of the particle-containing samples (Table 3). Thus, biodegradation enhancement appears to be mediated by dissolved constituents from the silica particles, rather than processes such as bacterial attachment or sorption of toxic metabolites onto the silica surface. These results suggest that dissolved silica must play some role in causing biodegradation rate enhancement compared to the controlled rate studies in which dissolved silica is absent.

FIGURE 1. Mineralization of 0.5 mM octylamine in silica-free control samples, samples containing 1000 g/L of Si-100, and the particlefree supernatant of medium previously contacted with silica. Substrate concentrations, S, are normalized to So, the concentration of octylamine initially added to the system. Different plots compare the effect of media with different buffering capacity: (1) MM-1 (β ) 11.9 mM), (2) MM-2 (β ) 1.29), and (3) MM-3 (β ) 0.12 mM). tested clearly show that the mineralization of inhibitory concentrations of octylamine is enhanced in the presence of silica particles or in media that has been previously contacted with silica particles (Figure 1). An average 50% mineralization enhancement occurred in all media tested regardless of the buffering capacity and nutrient concentrations of the medium (Table 3). Removal of octylamine due to sorption onto silica particles was negligible; the aqueous phase concentration of octylamine was found to be unchanged from the initial concentration following solid-liquid separation. Enhancement of mineralization rates of inhibitory compounds in the presence of surfaces has been observed when sorption onto the surface decreases the concentration in solution of the inhibitory compound (18, 57). Since changes in the octylamine solution concentration due to adsorption onto silica were negligible in this study, adsorption cannot explain the increased rate at which octylamine is mineralized in the presence of silica. Significant removal of octylamine by sorption was not 3726

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Effect of Concentration and Source of Dissolved Silica. Biodegradation studies with variable concentrations of Si-1 particles and octylamine also resulted in a significant increase in octylamine mineralization in the presence of silica compared to silica-free samples. The higher concentration of surface sites in the Si-1 systems compared to Si-100 systems resulted in higher concentrations of dissolved silica (Tables 2 and 3). Figure 2 shows the substrate-time curve for silicafree samples, supernatant samples, and samples containing sodium metasilicate at 0.5 and 0.1 mM of octylamine. In general, a higher rate enhancement was observed in the more inhibitory systems (i.e., higher concentration of octylamine), suggesting a relationship between octylamine toxicity and the influence of dissolved silica (Table 4). Octylamine mineralization was also enhanced with the addition of the metasilicate salt, confirming that dissolved silica is the factor that affects the increase in octylamine mineralization rates (Figure 2 and Table 4). Apparently, reduction of octylamine toxicity occurs at the lowest dissolved silica concentration tested in each system ([Si] ) 0.45 mM for 0.5 mM octylamine and [Si] ) 0.07 mM for 0.1 mM octylamine) since the mineralization curves in both supernatant samples coincide regardless of dissolved silica concentration (Figure 2). Similarly, metasilicate samples with 0.5 mM of octylamine with different aqueous silica concentration (0.8 and 0.55 mM) resulted in similar mineralization rates. The degradation enhancement in metasilicate samples was similar to that observed in supernatant samples at 0.1 mM of octylamine. However, at high octylamine concentrations, the extent of the rate enhancement appears to be a function of the source of dissolved silica. In this case, the degradation enhancement in the supernatant samples dissolved from the silica solids was much higher than that from dissolved sodium metasilicate (Figure 2). Since supernatant samples were obtained by equilibrating growth medium with silica particles, surface interactions between octylamine and silica may have facilitated an association between dissolved silica and octylamine compared to the metasilicate system. Alternatively, the equilibration of the growth medium with the silica particles may have caused changes in the medium composition and speciation resulting in different types of octylamine-

TABLE 3. Specific Biodegradation Rates, µ, of 0.5 mm Octylamine (mg of Cells/mg of Octylamine-h) in Different Media in the Presence and Absence of Silica Particles and Supernatanta buffering and pH

silica-containing

supernatant

medium

β, mM

pH ( std

silica-free µ, 1/h

µ, 1/h

increase, %

µ, 1/h

increase, %

MM-1 MM-2 MM-3

11.9 1.29 0.12

6.9 ( 0.03 6.3 ( 0.05 5.5 ( 0.01

0.077 ( 0.008 0.071 ( 0.001 0.042 ( 0.0003

0.114 ( 0.0005 0.104 ( 0.008 0.062 ( 0.003

48 46 47

0.115 ( 0.0002 0.111 ( 0.003 0.071 ( 0.013

49 56 68

a The percent rate increase of silica-containing samples is calculated with respect to the corresponding silica-free control. The buffering capacity index, β, and the resulting average pH at the end of the experiments are included. No significant difference in pH was observed among silicacontaining samples compared to silica-free samples for each type of media as reflected by the standard deviations of the average pH shown.

FIGURE 2. Mineralization of 0.5 and 0.1 mM of octylamine in silicafree samples, the supernatant of samples previously contacted with silica particles, and samples with added sodium metasilicate. Different lines and symbols for supernatant and metasilicate samples correspond to different dissolved silica concentrations included in the legend. Absence of error bars indicates errors smaller or of the size of the symbol. dissolved silica associations, leading to the lack of a direct correlation with total dissolved silica between the two systems.

Comparative Kinetic Study. The inhibitory kinetics of octylamine mineralization was studied in the presence and absence of silica particles to evaluate the effect of dissolved silica on the Monod kinetic parameters µmax and Ks and on the Haldane inhibition constant Ki (Table 5). The values of the kinetic parameters µmax and Ks for the silica-free samples are comparable to those of the silica-containing samples. However, the inhibition constants are very different for these conditions. A significant inhibitory effect is evident in the silica-free samples (0.99 mM < Ki < 1.32 mM), while negligible inhibition is exhibited by the silica-containing samples (Ki . 106 mM). The plot of the Haldane equation (Figure 4) using the 95% C.I. of the parameters obtained illustrates the reduction of the inhibitory effect in the presence of silica. These results clearly support the hypothesis that dissolved silica reduces the inhibitory effects of octylamine as reflected in the higher mineralization rates. Effect of Inoculum Density. Experiments were performed to test the effect of initial cell density on the mineralization rate enhancement of dissolved silica at inhibitory levels of octylamine. In the studies discussed above as well as in other experiments not reported here, the rates of octylamine biodegradation and the extent of dissolved silica enhancement appeared to be a function of the cell inoculum density. For example, at initial cell density of 7.2 × 105 cells/mL, the mineralization rate of 0.5 mM of octylamine in the silica-free system was 0.077 1/h with a rate enhancement of 49% in supernatant samples. Mineralization rates of similar octylamine concentration were lower (0.057 1/h) and the rate enhancement in the supernatant samples higher (70-74%) at a lower initial cell density of 2.8 × 105 cells/mL. Decreased inhibitory effects of alkylamines with increasing cell inoculum concentration have been reported before (17). A comparison of octylamine mineralization rates for silicafree control and silica samples at variable initial cell densities (Table 6) clearly shows that µ decreases and the enhancement percent increases with decreasing initial cell concentration. Rates could not be determined in the systems with the lowest cell densities (2 × 104 cells/mL), since little removal of octylamine was observed during the 120-h experiment. At high cell densities (2.0 × 107 cells/mL), the presence of dissolved silica does not enhance the rate of octylamine biodegradation but rather decreases mineralization when compared to silica-free control samples. The same results were observed in other experiments (not shown) at initial cell densities higher than 107 cells/mL. At such high cell concentrations, the rates of octylamine removal are much faster and the same mechanisms causing the enhancement at lower cell concentrations may now negatively affect the biodegradation of octylamine. When cell inocula are diluted to the concentrations used in the experiments discussed (between 2.0 × 106 and 2.0 × 105 cells/mL), the enhancement of rates in the presence of dissolved silica is apparent. The decrease in rate enhancement from 85% at 2 × 105 cells/mL to 38% at 2 × 106 cells/mL is consistent with the results reported before. At very low cell concentrations (2.0 × 104 cells/mL), inhibitory effects appear VOL. 33, NO. 21, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 4. Specific Biodegradates Rates (µ, mg of Cells/mg of Octylamine-h) of All Samples and Percent Rate Increase in Dissolved Silica-Containing Samples Reflecting the Variations between Silica-Free Samples (0.5 and 0.1 mM Octylamine) and Silica Supernatant and Metasilicate Samples with Variable Dissolved Silica Concentrations [octylamine] ) 0.5 mM, silica-free µ ) 0.057 ( 0.005, 1/h supernatant

[octylamine] ) 0.1 mM, silica-free µ ) 0.043 ( 0.001, 1/h

metasilicate

supernatant

metasilicate

[Si], mM

µ, 1/h (% increase)

[Si], mM

µ, 1/h (% increase)

[Si], mM

µ, 1/h (% increase)

[Si], mM

µ, 1/h (% increase)

0.78 0.45

0.099 ( 0.00 (74%) 0.097 ( 0.002 (70%)

0.8 0.55

0.076 ( 0.002 (33%) 0.079 ( 0.002 (38%)

0.45 0.07

0.059 ( 0.004 (37%) 0.065 ( 0.000 (51%)

0.2 0.06

0.062 ( 0.002 (44%) 0.065 ( 0.000 (51%)

TABLE 5. Summary of Average Kinetic Parameters for Octylamine Mineralization in the Presence and Absence of Silica (Silica-Free Control and Silica-Containing Samples) and Their 95% Confidence Intervals silica-free control parameter µmax, mg of cells/ mg of S-h Ks, (× 102 mM) Ki, mM ζ, unitless S*, mM µ*, mg of cells/ mg of S-h a

av

95% CI

silica-containing av

95% CI

0.160 0.144-0.170 0.160 0.158-0.163 0.96 1.32 0.194 0.113 0.137

0.82-1.1 0.84 0.88-0.80 0.99-2.13 >106 nda 0.187-0.201 0.185 0.181-0.190 0.090-0.153 0.127-0.150

TABLE 6. Specific Biodegradation Rates (µ) of 0.5 mM Octylamine (mg of Cells/mg of Octylamine-h) with Variable Cell Inocula in the Presence and Absence of Dissolved Silica and the Percent Rate Increase of Silica-Containing Samples Respect to the Correspondent Silica-Free Control silica

initial cell concn Xo, cells/mL

silica-free control µ, 1/h

µ, 1/h

increase, %

2.0 × 107 2.0 × 106 2.0 × 105 2.0 × 104

0.091 ( 0.0004 0.070 ( 0.0003 0.040 ( 0.004 nda

0.053 ( 0.001 0.097 ( 0.0009 0.074 ( 0.003 nda

-42 38 85 nda

aNot

determined due to low octylamine removal: nd.

Not determined: nd.

FIGURE 3. Graphical representation of the kinetics of octylamine mineralization in the presence and absence of dissolved silica. Wide solid lines were calculated with the averaged values of the parameters and dashed lines with the upper and lower limits of the 95% C.I.

FIGURE 4. Surface tension measurements of octylamine solutions in MM-1 medium with and without the addition of 1.5 mM sodium metasilicate. Error bars are smaller than the size of the symbol. extensive and the presence of dissolved silica does not seem to affect octylamine mineralization. In this case, dissolved silica is not sufficient to reduce the inhibitory effects. Effect of Dissolved Silica on the Surface Tension of Octylamine Aqueous Solutions. Organic compounds, particularly surfactants, are known to adsorb at the air-water interface resulting in a reduction of the surface tension of water. Surface tension measurements of MM-1 solutions 3728

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containing 0-6 mM of octylamine showed the characteristic decreased in surface tension with increasing organic concentration (Figure 4). Interestingly, addition of 1.5 mM sodium metasilicate to half of the samples resulted in a significant increase in the surface tension of solutions with similar octylamine concentrations and ionic strength (Figure 4). These results suggest the presence of dissolved silica prevents some of the octylamine from adsorbing at the airliquid interface. This reduction in surface activity is indicative of the formation of a less surface active-octylamine moiety. The type of association between dissolved silica and octylamine, however, remains unclear. While at basic pHs the deprotonated silicic acid undergoes complexation reactions with organic and inorganic cations, at neutral pH, the experimental conditions of this study, few reactions have been observed. Reaction with oligomeric species of dissolved silica is also a possibility. Polymeric silicic acids undergo hydrogen bonding with many compounds, including amines (31). The formation of hydrogen bonds between the NH3+ groups of polyamines and silicic acid have been speculated as the mechanism for amine induced polymerization of silicic acid (45). Oligomeric forms of dissolved silica have been reported to have pKa values lower than that of the monomer (30, 31), which bring the possibility of ion-pair interactions with the cationic octylamine. However, based on UV spectrophotometric studies we have conducted but not reported here, we have found no evidence for an octylamine association which causes changes in the UV spectrum of dissolved silica alone. While possibilities exist for associations that may not involve UV sensitivity, including interactions between the hydrophobic alkyl portion of octylamine and dissolved silica or weak ion associations that do not disturb the primary solvation environment of dissolved silica, further studies will be necessary to characterize the cause of the surface activity reduction noted here. Although a strong association (e.g., covalent or ionic bonding) could render a substrate virtually unavailable to bacterial utilization, a weaker association as suspected here may aid the biodegradation of cationic substrates by fully or partially neutralizing or shielding their positive charge. The strong germicidal properties of cationic surfactants have been explained as a result of the adsorption of the chemical onto

the bacterial membrane favored by the electrostatic attraction between the positively charged surfactant and the negatively charged bacterial surface (21). Thus, neutralizing or shielding the charge of octylamine by its association with dissolved silica may render the molecule less toxic and more amenable for microbial utilization. This type of an association, although not fully characterized here, is consistent with the impact of dissolved silica on octylamine mineralization rates observed.

Acknowledgments This research was supported in part by NIEHS Grant no. 5-RO1-CA12345-03. The authors thank the three anonymous reviewers whose comments helped to improve the presentation of this material.

Supporting Information Available Modeling approach, parameter estimation methods, and uniqueness of solution information provided. This material is available free of charge via the Internet at http:// pubs.acs.org.

Literature Cited (1) Mingelgrin, U.; Prost, R. In Toxic Organic Chemicals in Porous Media; Gerstl, Z., Chen, Y., Mingelgrin, U., Yaron, B., Eds.; Springer-Velag: New York, 1989; pp 91-135. (2) Dashman, T.; Stotzky, G. Soil Biol. Biochem. 1986, 18, 5-14. (3) Knaebel, D. B.; Federle, T. W.; McAvoy, D. C.; Vestal, J. R. Appl. Environ. Microbiol. 1994, 60, 4500-4508. (4) Stotzky, G.; Rem, L. T. Can. J. Microbiol. 1966, 12, 547-563. (5) Stotzky, G. Can. J. Microbiol. 1966, 12, 831-848. (6) Stotzky, G. Can. J. Microbiol. 1966, 12, 1235-1246. (7) Barber, R. T. In Trace Metals and Metal-Organic Interaction in Natural Waters; Singer, P. C., Ed.; Ann Arbor Science: Ann Arbor, MI, 1973; pp 321-338. (8) Morel, F. M. M.; Hudson, R. J. M.; Price, N. M. Limnol. Oceanogr. 1991, 36, 1742-1755. (9) Anderson, D. A.; Morel, F. M. M. Limnol. Oceanogr. 1978, 23, 283-295. (10) Anderson, D. A.; Morel, F. M. M.; Guillard, R. R. L. Nature 1978, 276, 70-71. (11) Madsen, E. L.; Alexander, M. Appl. Environ. Microbiol. 1985, 50, 342-349. (12) Xun, L.; Reeder, R. B.; Plymale, A. E.; Girvin, D. C.; Bolton, H., Jr. Environ. Sci. Technol. 1996, 30, 1752-1755. (13) Bolton, H.; Girvin, D. C.; Plymale, A. E.; Harvey, S. D.; Workman, D. J. Environ. Sci. Technol. 1996, 30, 931-938. (14) Boudot, J. P.; Brahim, B. H.; Steiman, R.; Seigle-Murandi, F. Soil Biol. Biochem. 1989, 21, 961-966. (15) Wszolek, P.; Alexander, M. J. Agric. Food Chem. 1979, 27, 410414. (16) Yoshimura, K.; Mashida, S.; Masuda, F. JAOCS 1980, 57, 238241. (17) Ruiz Cruz, J.; Dobarganes Garcia, M. C. Grasas Aceites 1979, 30, 67-74. (18) van Ginkel, C. G. In Biodegradability of surfactants; Carsa, D. R., Ed.; Akcross Chemicals Eccles: Manchester, 1995; Chapter 6. (19) Lewis, M. A. Water Res. 1992, 26, 1013-1023. (20) Versteeg, D. J.; Shorter, S. J. Environ. Toxicol. Chem. 1992, 11, 571-580. (21) Fredell, D. L. In Cationic Surfactants. Analytical and Biological Evaluation; Cross, J., Singer, E. J., Eds.; Marcel Dekker: New York, 1994; pp 31-60. (22) Ainsworth, C. C.; Zachara, J. M. Selection of Subsoils, Saturated Zone Materials, and Related Sorbents for Research; Report no. DOE/ER-390; Office of Energy Research, U.S. Department of Energy: Washington, DC, 1991; 27 pp. (23) Grilllet, Y.; Llewellyn, P. L. In The Surface Properties of Silicas; Legrand, A. P., Ed.; John Wiley & Sons: West Sussex, England, 1998; pp 23-81. (24) Davis, J. A.; Kent, D. B. In Mineral-Water Interface Geochemistry; Hochella, M. F., White, A. F., Eds.; Reviews in Mineralogy 23, Mineralogical Society of America: Washington, DC, 1990; pp 177-260. (25) James, R. O.; Parks, G. A. Surface Colloid Sci. 1982, 12, 119-216. (26) Ingram, B. T.; Ottewil, R. H. In Cationic Surfactants: Physical Chemistry; Rubingh, D. N., Holland, P. M., Eds.; Marcel Dekker: New York, 1991; pp 87-140.

(27) Subba-Rao, R. V.; Alexander, M. Appl. Environ. Microbiol. 1982, 44, 659-668. (28) Bennett, P. C.; Siegel, D. I. Nature 1987, 326, 684-686. (29) Bennett, P. C.; Hasset, J. P.; Melcer, M. E.; Siegel, D. I. Geochim. Cosmochim. Acta 1988, 52, 1521-1530. (30) Sjoberg, S. J. Non-Crystalline Solids 1996, 196, 51-57. (31) Iler, R. The Chemistry of Silica; Wiley-Interscience: New York, 1979. (32) Dove, P. M.; Rimdtidt, J. D. In Silica: Physical Behavior, Geochemistry, and Materials Applications; Heaney, P. J., Prewitt, C. T., Gibbs, G. V., Eds.; Mineralogical Society of America: Washington, DC, p 263. (33) Sjoberg, S.; Ohman, L.-O.; Ingri, N. Acta Chem. Scand. A 1985, 39, 93-107. (34) Rimstidt, J. D.; Barnes, H. L. Geochim. Cosmochim. Acta 1980, 44, 1683-99. (35) Fournier, R. O. In Geology and Geochemistry for Epithermal Systems; Berger, B. R., Bethke, P. M., Eds.; Reviews in Econ. Geol. 2; Society of Economic Geologists: Socorro, NM, 1985; pp 45-61. (36) Langmuir, D. Aqueous Environmental Geochemistry; Prentice Hall: New Jersey, 1997; p 247. (37) Hiebert, F. K.; Bennett, P. C. Science 1992, 258, 278-281. (38) Monnier, A.; Schu ¨ th, F.; Huo, Q.; Kumar, D.; Margolese, D.; Maxwell, R. S.; Stucky, G. D.; Krishnamurty, M.; Petroff, P.; Firouzi, A.; Janicke, M.; Chmelka, B. F. Science 1993, 261, 12991303. (39) Tanev, P. T.; Pinnavaia, T. J. Science 1995, 267, 865. (40) Bennett, P. C. Geochim. Cosmochim. Acta 1991, 55, 1781-1797. (41) Evans, D. E.; Parr, J.; Coker, E. N. Polyhedron 1990, 6, 813-823. (42) Ohman, L.-O.; Nordin, A.; Fattahpour Sedeh, I.; Sjoberg, S. Acta Chem. Scand. 1991, 45, 335-341. (43) Fattahpour Sedeh, I.; Ohman, L.-O.; Sjoberg, S. Acta Chem. Scand. 1992, 46, 933-940. (44) Fattahpour Sedeh, I.; Sjoberg, S.; Ohman, L.-O. J. Inorg. Chem. 1993, 46, 119-132. (45) Mizutani, T.; Nagase, H.; Fujiware, N.; Ogoshi, H. Bull. Chem. Soc. Jpn. 1998, 71, 2017-1022. (46) Papelis, C.; Hayes, K. F.; Leckie, J. O. HYDRAQL: a Program for the Computation of Chemical Equilibrium Composition of Aqueous Batch Systems Including Surface-Complexation Modeling of Ion Adsorption at the Oxide/Solution Interface; Technical Report No. 306; Department of Civil Engineering, Stanford University: Palo Alto, CA, 1988. (47) Demond, A. H.; Desai, F. N.; Hayes, K. F. Water Resour. Res. 1994, 30, 333-342. (48) Kunze, G. W.; Dixon, J. B. In Methods of Soil Analysis, part 1, Physical and Mineralogical Methods; Klute, A., Ed.; American Society of Agronomy: Madison, WI, 1986; pp 91-100. (49) Standard Methods for the Examination of Water and Wastewater, 15th ed.; APHA, AWWA, and WPCF: Washington, DC, 1980; p 789. (50) Bratbak, G.; Dundas, I. Appl. Environ. Microbiol. 1984, 48, 755757. (51) Standard Methods for the Examination of Water and Wastewater, 15th ed.; APHA, AWWA, and WPCF: Washington, DC, 1980; p 94. (52) Morel, F. M. M. Principles of Aquatic Chemistry; WileyInterscience: New York, 1983; pp 300-308. (53) Andrews, J. F. Biotech. Bioeng. 1968, 10, 707-723. (54) Simkins, S.; Alexander, M. Appl. Environ. Microbiol. 1984, 47, 1299-1306. (55) Panikov, N. S. Microbial Growth Kinetics; Chapman & Hall: New York, 1996. (56) Cornish-Bowden, A. Analysis of Enzyme Kinetic Data; Oxford University Press: Oxford, 1995; pp 179-189. (57) Ehrhardt, H. M.; Rehm, H. J. Appl. Microbiol. Biotechnol. 1985, 21, 32-36. (58) Applin, K. R. Geochim. Cosmochim. Acta 1987, 51, 2127-2151. (59) Sjoberg, S.; Ingri, N.; Nenner, A.-M; Ohman, L.-O J. Inorg. Chem. 1985, 24, 267. (60) Bijstertoch, B. H. J. Colloid Interface Sci. 1974, 47, 186-198.

Received for review August 20, 1998. Revised manuscript received June 28, 1999. Accepted July 2, 1999. ES9808595 VOL. 33, NO. 21, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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