Adsorption and Biodegradation of Aromatic Chemicals by Bacteria

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Adsorption and biodegradation of aromatic chemicals by bacteria encapsulated in a hydrophobic silica gel Jonathan Konstantine Sakkos, Baris R Mutlu, Lawrence P. Wackett, and Alptekin Aksan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06791 • Publication Date (Web): 18 Jul 2017 Downloaded from http://pubs.acs.org on July 19, 2017

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ACS Applied Materials & Interfaces

Adsorption and biodegradation of aromatic chemicals by bacteria encapsulated in a hydrophobic silica gel Jonathan K. Sakkos a, Baris R. Mutlu a, Lawrence P. Wackett b,c, Alptekin Aksan a,c* a b

Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN 55455, USA

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA c

The BioTechnology Institute, University of Minnesota, St. Paul, MN 55108, USA

* Corresponding author (AA) Address: 111 Church St SE, Minneapolis, MN 55455 Phone: 612-626-6618 Email: [email protected]

Keywords: bioencapsulation, biodegradation, adsorption, sol-gel, ormosil

An adsorbent silica biogel material was developed via silica gel encapsulation of Pseudomonas sp. NCIB 9816-4, a bacterium that degrades a broad spectrum of aromatic pollutants. The adsorbent matrix was synthesized using silica precursors methyltrimethoxysilane (MTMS) and tetramethoxysilane (TMOS) to maximize the adsorption capacity of the matrix while maintaining a highly networked and porous microstructure. The encapsulated bacteria enhanced the removal rate and capacity of the matrix for an aromatic chemical mixture. Repeated use of the material over 4 cycles was conducted to demonstrate that the removal capacity could be maintained with combined adsorption and biodegradation. The silica biogel can thus be used extensively without the need for disposal, as a result of continuous biodegradation by the encapsulated bacteria. However, an inverse trend was observed with the ratio of biodegradation to adsorption as a function of log Kow, suggesting increasing mass transport limitation for the most hydrophobic chemicals used (log Kow > 4).

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Introduction Water pollution with polycyclic aromatic hydrocarbons (PAHs) is a ubiquitous problem.1 Many of these chemicals are known carcinogens, thus their removal from water is of great concern.2 Chemical treatments, adsorption, and biodegradation have been studied for removal of PAHs from water.3–5 Chemical treatments (e.g. oxidation, UV radiation, ultrasound) are effective ways to destroy PAHs, but the high energy demand and cost limit the use of these methods.6 Adsorption of PAHs to a highly porous and inexpensive adsorbent (e.g. granulated activated carbon) allows rapid removal, but requires renewal/regeneration and the concentrated levels of carcinogenic pollutants pose hazards for disposal.7–9 Therefore, biodegradation has been studied as an alternative low-cost water treatment option. Many naturally occurring bacteria can readily metabolize PAHs,10 but they are not ideal for integration into water treatment systems in their native form (either as a free cell suspension or as a biofilm). Containment, competition, and predation issues arise with cell suspensions, and biofilms do not have the desirable mechanical robustness and are prone to inadequate supplementation of nutrients due to limited porosity. Encapsulation of bacteria in an engineered, robust, and permeable structure facilitates their integration in water treatment systems.11 Silica gel bioencapsulation, in which biologicals (e.g. cells, enzymes, etc.) are physically confined within a 3D porous silica structure, has been studied extensively over the last few decades.12–16 The silica matrix is cytocompatible, keeps the cells protected from the environment, and retains the cells within the matrix. Some studies have focused on improving the cytocompatibility of silica encapsulation materials,17–21 but few have investigated whether the cells retain metabolic activity.22–26 We have previously shown that silica encapsulated Pseudomonas sp. NCIB 9816-4 remains metabolically active for biodegradation of PAHs.27–29 While our prior studies focused on the discovery of new substrates for biodegradation


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and silica matrix optimization, the rate and extent of PAH removal in a realistic scenario was not considered. Lastly, the mechanical properties of the silica matrix are an important factor for industrial-scale water treatment, since the required compressive strength in packed bed reactors range from 1-10 MPa.30,31 There are few studies of the mechanical properties in cytocompatible silica gels,21,29,32–34 but we have previously shown that the compressive strength and stiffness can be primarily controlled with silica content and there is a trade-off between high strength and cytocompatibility, due to the increased levels of alcohol produced during hydrolysis of the silica precursors.28 Functionalizing the encapsulation matrix to make it adsorbent allows rapid removal of pollutants from the water, while resolving the accumulation issue via biodegradation. While silica is an ideal material for encapsulation, it has very limited adsorption capacity for PAHs due its hydrophilic surface. However, organically modified silanes (ormosils) can be incorporated into the gel structure to synthesize adsorbent silica gels.35,36 Specifically, this is done by replacing one or more alkoxy (-O-R) groups with an organic group (e.g. methyl, ethyl, or phenyl). However, the common

use

of

organic

solvents,

desiccation,

and

high

temperature

treatment

(sintering/calcination) in these prior studies require adaptation to ensure the resulting encapsulation matrices are cytocompatible.37–39 A TMOS (tetramethoxysilane) and colloidal silica nanoparticle (SNP) gel was used as the basis for this work, since it was previously optimized for bioencapsulation of Pseudomonas sp. NCIB 9816-4.28 In this study, we engineered a silica gel bioencapsulation system for enhanced removal of PAHs from water via adsorption and biodegradation. In order to render the matrix hydrophobic, the ormosil MTMS (methyltrimethoxysilane) was incorporated into the matrix. Chemicals were selected for a model PAH mixture to span a range of molecular weights and hydrophobicities to


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gain insight into the role of biodegradation within an adsorbent matrix. The relative ratio of MTMS to TMOS was adjusted to maximize the adsorption of a model PAH mixture. We showed that the addition of NCIB 9816-4 encapsulated within the matrix increased the rate and capacity of removal. Furthermore, we investigated the effects of the matrix composition on its microstructure, mechanical properties, and hydrophobicity. The effectiveness of the developed silica biogel material with repeated use was also demonstrated. The quantity and composition of the PAH mixture which was removed by adsorption and biodegradation was also examined.

Materials and methods Materials Tetramethoxysilane (TMOS, 98% purity), methyltrimethoxysilane (MTMS, 98% purity), 22 nm diameter Ludox TM-40 colloidal silica nanoparticles (SNPs), and all other chemicals were purchased from Sigma-Aldrich (Sigma-Aldrich Corp., St. Louis, MO, USA) and used without further purification. Ultrapure water (UPW) was prepared by filtering distilled water using a MilliQ water purification system (Millipore, Billerica, MA, USA) to a final electrical resistance of >18.2 MΩ/cm.

Bacterial strains and growth conditions Cultures of Pseudomonas sp. NCIB 9816-4 were started on Luria Broth (LB), and later grown on naphthalene in minimal salts buffer (MSB), as previously reported.28 Cultures that reached a final OD600 of 1.5 to 2.5 were filtered through glass wool to remove any naphthalene crystals remaining in the solution prior to harvest. Cells were harvested by centrifugation at 5000 x g for 10 min followed by re-suspension at 0.5 g (wet weight)/mL in phosphate buffered saline (PBS) solution for encapsulation. The OD600 of the cell suspension was approximately 0.2 after a 1000-fold dilution.


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Silica biogel synthesis Silica and Ormosil gels were synthesized based on a previously developed method, with slight modification.28 TMOS and/or MTMS were added to 5 mM HCl solution, resulting in a final volumetric ratio of 1/1 (TMOS + MTMS)/HCl solution. The mixture was stirred at room temperature for 2 hours to allow for hydrolysis. The resulting alkoxide solution (the sol) was then mixed with the SNP and cell suspension or PBS at a ratio of 2.5/2.5/1 (v/v/v), respectively, and allowed to gel, for a final cell density of 0.083 g-cells/mL-gel. After gelation, which occurred within a few minutes, gels were washed twice for 30 seconds with 1 mL PBS to remove the methanol that formed during hydrolysis. Even before washing, the residual methanol in the subsequent solution would be at most ~1.5-3% (v/v), based on the volume of the gel and supernatant containing PAHs, and our previous measurements of methanol in a TMOS/nanoparticle gel.28 After washing twice, this is expected to decrease by two orders of magnitude. The MTMS content in all figures and text of this manuscript refers to the volumetric percentage of MTMS in the TMOS + MTMS mixture.

Hydrophobicity measurement Nile Red was used as a probe to determine gel hydrophobicity using microscopy and fluorescence spectroscopy. Cascade Blue was used in conjuction with Nile Red to illustrate the hydrophilic regions within the gel. Confocal laser scanning microscopy (CLSM) was performed with a Nikon A1si spectral confocal system mounted on a Nikon Ti2000E inverted fluorescence microscope equipped with DIC optics (Nikon Instruments Inc., Melville, NY, USA). NIS Elements imaging software was used for image acquisition and analysis was performed with ImageJ.40 Nile Red and/or Cascade Blue from stock solutions (1 mg/mL in EtOH) were added to the sol before gelation to reach a final concentration of 10 and 20 µg/mL, respectively. 50 µL of gel was prepared


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on glass microscopy slides for imaging. For Nile Red, samples were excited at 561 nm and emission was measured in the range of 570-620 nm. Cascade Blue was excited at 403 nm and emission was measured from 425-475 nm. GFP expressing E. coli were included in these samples in place of Pseudomonas sp. NCIB 9816-4 for visualization. GFP was excited at 488 nm and emission was measured from 500-550 nm. Additional samples were prepared for fluorescence spectroscopy with Nile Red using 300 µL of gel in a 96-well plate, and a SpectraMax M5 plate reader (Molecular Devices, LLC., Sunnyvale, CA, USA) was used to measure the fluorescence of the samples excited at 561 nm, using a cutoff filter at 590 nm while measuring emission in the range from 600-700 nm. Measurements were conducted in triplicate.

Mechanical testing The mechanical properties were measured as previously described.28 Cylindrical gel samples (12.5 mm x 12.5 mm) were tested by uniaxial compression using an MTS QT10 mechanical testing machine (MTS Systems, Eden Prairie, MN). Ten replicates were performed for each sample type.

Microstructural analysis Gel microstructure was examined with a Hitachi S-4700 cold field emission gun (CFEG) scanning electron microscope (SEM, Hitachi, Ltd., Tokyo, Japan). The samples were gradually dried in increasing ethanol concentrations of 50%, 75%, 90%, and 100% (twice) for 10 minutes per wash, left to air-dry overnight, and sputter-coated with 30 Å of platinum before examination with SEM.

Characterization of adsorption and biodegradation A mixture containing 400 ppb (each) of indole, tetralin, naphthalene, biphenyl, bibenzyl, dibenzothiophene, and phenanthrene in PBS was used for the adsorption and biodegradation measurement, for a total concentration of 2.8 ppm. Samples containing only the pollutant mixture


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in PBS were made for all experiments to control for variations in the respective concentrations of each chemical. Gel slabs for adsorption characterization were 200 µL in volume, with approximate dimensions of 10 mm in diameter x 2.5 mm in height (~69 mg dry gel mass, based on density of gel), contained PBS instead of the cell suspension, and were made at the bottom of 2 mL gas chromatography vials with PTFE-lined caps. The samples were left to gel for approximately 5 mins before washing twice with PBS. The vials were subsequently filled with 2 mL of the aromatic/heterocycle mixture described above and capped. Samples were incubated in this solution for 6 days to allow adsorption equilibration. Chemicals were extracted by pipetting a 1 mL aliquot from the supernatant and adding 400 µL of methyl tert-butyl ether (MTBE), vortexing the mixture for 10 seconds, and pipetting off the extract from the top of the phase-separated supernatant with a glass syringe. Solution composition was then determined by gas chromatography-mass spectrometry (GC-MS). Extracted samples were separated with an HP-1ms column, at a helium flow rate of 1.75 mL/min, and a temperature of 250°C at the injection port. The samples were split at the column outlet between a flame ionization detector (FID, 7890A, Agilent, Palo Alto, CA, USA) and a mass spectrometer (MS, 5975C, Agilent). An initial temperature of 80 °C was increased to 320 °C at 15 °C/minute and kept constant for 5 minutes. Electron impact mass spectra were collected at 70eV with positive polarity. Four replicates of each sample were tested. Kow values were either obtained from the published values by Sangster et al.41, if available, or calculated using Chem BioDraw Ultra software (Perkin Elmer, Waltham, MA, USA). Measurement of the combined adsorption and biodegradation was conducted using samples as described above, with the addition of bacteria. In these experiments, the sample size was approximately 15 mm in diameter x 2 mm in height, the volume of supernatant was 4 mL and the vials were 6 mL in volume, including 2 mL of headspace for better mixing to occur within the


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supernatant. The vials were filled with the aromatic/heterocycle mixture and incubated for 24 hours before each measurement. After the supernatant was sampled, the remaining solution was removed and replaced with a fresh solution. The aromatic/heterocycle mixture was prepared 24 hours prior to each experiment and sampled (as control) to account for variation in the mixture composition from batch to batch. Free cells (35 µL of cell suspension) were used as a positive control.

Metabolic Activity Metabolic activity of Pseudomonas sp. NCIB 9816-4 bacteria encapsulated in an 80% MTMS gel (silica biogel) was measured after exposure to a solution of saturated naphthalene (~250 µM) in PBS. Cylindrical samples 200 µL in volume, with approximate dimensions of 6 mm diameter and 7 mm height, were suspended in a sealed Oxygraph Oxytherm System chamber (Hansatech Instruments Ltd., United Kingdom). The samples were equilibrated, then measured from 2-5 mins and the slope of the oxygen concentration over that period was averaged to determine the oxygen consumption rate. Error bars indicate standard deviation, n=3.

Results and discussion Synthesis and characterization of organically-modified silica gel The organically-modified silica material was produced following the schematic shown in Figure 1.

First, a sol was prepared via acid-catalyzed hydrolysis of the silica precursors

tetramethoxysilane (TMOS) and methyltrimethoxysilane (MTMS), since these conditions are well known to produce a sol which is stable for several days when a strong acid is used.42 This allowed the pH induced gelation, from acidic to neutral, to be controlled by the addition of colloidal silica nanoparticles (SNPs) and cell suspension, forming the hydrophobic silica gel within minutes. For


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characterization of the silica gel matrix, phosphate buffered saline (PBS) was used in place of the cell suspension to maintain the same sample volume and pH as the biogel. In order to investigate the effect of MTMS content on the gel hydrophobicity, the relative ratio of MTMS to TMOS was adjusted. It was observed that hydrophobicity increased with MTMS content, as expected. Both fluorescence spectroscopy and contact angle measurements were used for hydrophobicity measurement, since there is no absolute measure of hydrophobicity. Nile Red, a compound that is only fluorescent in a hydrophobic environment, was used as a probe for fluorescence spectroscopy.43 Nile Red fluorescence intensity increased with increasing MTMS content, reaching a plateau at 80% MTMS content (Figure 2). The more than two-fold increase in Nile Red fluorescence from 60%-80% MTMS suggested significant changes in the way the material was structured. In addition, a blue shift in the emission peak location was noted from 655 nm at 0% MTMS (100% TMOS) to 640 nm at 80% MTMS (Figure S1), which was also consistent with increased hydrophobicity.44 Contact angle measurements were performed as an additional method to determine the effect of MTMS content on the hydrophobicity of the silica gel surface. It was observed that the water contact angle increased as the MTMS content increased, from 3.7 ± 0.5° (0% MTMS) to 90.2 ± 1.4° (100% MTMS) (Figure S2). However, the contact angle values plateaued near 90° in the 40-60% MTMS content range, which was attributed to structural heterogeneity in the samples. The maximum contact angle recorded in this study was similar to the lowest values reported in the literature for MTMS aerogels (95°)45. Due to the sensitivity of contact angle measurements to environmental conditions46 and surface roughness,47,48 as well as the tendency for hydrogels to absorb the liquid droplet over time, this method may not be a reliable metric for measuring hydrophobicity in silica hydrogels.


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After observing that the increase in hydrophobicity was non-linear, the gel microstructure was studied via scanning electron microscopy (SEM) to investigate structural changes which might explain this behavior. Generally, highly-networked, mesoporous microstructures were observed within the silica gel matrix, which was expected under water-rich synthesis conditions (moles H2O/moles -O-CH3 > 8).49 Little structural change from 0 to 60% MTMS content was observed (Figure 3a). In contrast, a drastic increase in the heterogeneity of the structure occurred with ≥ 80% MTMS, though most evident with 100% MTMS. In this range, spherical aggregates on the order of 1-10 µm were observed (Figure 3b), indicating phase separation between the hydrophobic phase, dominated by methyl groups, and the hydrophilic phase, containing surface silanol (Si-OH) groups and water molecules.50–52 The observed structural rearrangement helped to explain the sudden increase in hydrophobicity measured with Nile Red (Figure 2), as the more concentrated hydrophobic regions led to a drastic increase in fluorescence. The phase-separated regions also appeared to contain fewer visible pores, suggesting reduced specific surface area. Nitrogen adsorption measurements using the BET method53,54 for the 0% and 80% MTMS gels showed a drastic decrease in specific surface area from 290 to 176 m2/g, respectively, while the average pore diameter increased from 6 to 14 nm, which is similar to trends observed in the literature.45,55 These values approached the specific surface area of the 22 nm Ludox TM-40 silica nanoparticles, which made up nearly half of the initial gel mixture by volume, suggesting that MTMS alone provided little additional porosity. The effect of MTMS content on the gel mechanical properties was also characterized. Though not visible under SEM, the structural integrity of the gels declined with increasing MTMS content, as measured with uniaxial compression testing (Figure 4). The decrease in both modulus and stress at failure (Figure 4a/b) were a result of lower crosslinking density and to a lesser extent


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heterogeneity at high MTMS content (80-100%). The use of MTMS, which is trifunctional, instead of TMOS, which is tetrafunctional, is known to reduce crosslinking density due to the number of siloxane bonds (-Si-O-Si-) each precursor can form. The modulus and stress at failure were in the same range as, or greater than, commonly used encapsulation materials, such as, PVA (polyvinyl alcohol), alginate, and PEG (polyethylene glycol), which tend to be less stiff and more elastic materials.56–58 However, when compared with conventional or hybrid materials which do not contain biologicals, the silica biogel was orders of magnitude lower in stiffness and max stress.59 This is likely an unavoidable consequence of concessions made to ensure cytocompatibility and metabolic activity of the encapsulated cells, such as avoiding the use of heat treatment and organic solvents. The strain at failure increased with MTMS content, though still under 10% (Figure 4c). This is typical of silica gels, which resist deformation due to their highly-networked structure of siloxane (-Si-O-Si-) bonds, also making them prone to brittle failure. Further investigation of the gel structure was conducted to confirm that the drastic changes in hydrophobicity and microstructure were indeed a result of reorganization of MTMS molecules within the gel. Confocal laser scanning microscopy (CLSM) was used in conjunction with Cascade Blue and Nile Red as probes to identify localized hydrophilic/hydrophobic regions within the silica gel. Samples with ≤ 60% MTMS content exhibited uniform fluorescence (Figure 5a), indicating that the hydrophobic methyl groups were homogeneously distributed. At or above 80% MTMS content, the spatial distribution of fluorescence segregated into circular regions, similar to what was observed under SEM (Figure 3b). Furthermore, the hydrophobic aggregate size distribution observed in the CLSM images (Figure 5b) increased with MTMS content from 2.26 ± 1.51 µm to 3.51 ± 2.59 µm. These results suggested the presence of a metastable region, based on the synthesis conditions, in which phase separation was induced once a critical amount of MTMS was used.52 It


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should also be noted that TMOS and MTMS were co-hydrolyzed, which is known to promote covalent bonding between the organic and inorganic phases.60,61 The use of co-hydrolysis was intended to delay the onset of phase separation, due to greater bonding between the phases prior to gelation, in order to maintain a homogeneous structure. The Cascade Blue dye (Figure 5a) was used to highlight the distribution of hydrophilic/hydrophobic domains within the gel and also to confirm that encapsulated bacteria were co-localized with either of these domains. Green fluorescent protein (GFP) expressing E. coli were used to visualize the distribution of bacteria within the gel. In general, the bacteria were evenly distributed throughout the hydrophilic regions of the gel while they were generally excluded from the hydrophobic regions. We should also note that Cascade Blue is generally not cell permeable, which explains the lack of fluorescence in regions containing cells. Samples without cells were used to determine if there was any interaction between the cells and the matrix, and the omission of cells shifted the size distributions of hydrophobic aggregates, ranging from 1.89 ± 1.21 µm to 5.22 ± 2.91 µm (Figure S3, 80% and 100% MTMS, respectively), suggesting that there may be a slight interaction between the cells and the aggregates. See the supplementary materials for a video of the gelation process (Movie S1).

Adsorption characterization Aromatic hydrocarbons and heterocyclic chemicals (such as naphthalene and indole, respectively) are commonly found in the environment grouped with similar compounds.1,62,63 Therefore, a representative pollutant mixture was used in this study to simulate a typical water source contamination. Since the bacterium utilized in this study, Pseudomonas sp. NCIB 9816-4, was known to biodegrade a broad spectrum of aromatic chemicals and we have previously shown that it was possible to be used in complex mixtures,27 we were interested in designing a mixture


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such that the role of biodegradation in the presence of an adsorbent matrix could be better understood. Based on this premise, the mixture contained 7 chemicals which were all known substrates

for

NCIB

9816-4:

tetralin,64

naphthalene,65

indole,66

phenanthrene,64

dibenzothiophene,67 biphenyl,64 and bibenzyl.27 These chemicals span a range of molecular weights (MW) from 117.2 to 184.3 g/mol and hydrophobicity, as measured by the octanol-water partition coefficient (log Kow), ranging from 2.1 to 4.7. The silica gel composition was adjusted to maximize the adsorption of pollutants by tuning the MTMS to TMOS ratio. Equilibrium adsorption experiments were performed to determine the affinity and specificity of the silica gel to the chemicals used in this study and the role of gel hydrophobicity in adsorption of the mixture. A positive correlation was observed between MTMS content and adsorption, as was expected (Figure 6). When compared to the hydrophilic silica gel control (0% MTMS, 100% TMOS), a 7-fold increase in adsorption was observed with 80% MTMS (0.328 ± 0.037 ppm vs. 2.332 ± 0.242 ppm). This translated into a maximum specific adsorption capacity of 61 ± 6 µg/g (Figure S4). This data correlated well with the trends observed in the Nile Red experiments (Figure 2), suggesting that Nile Red fluorescence intensity may be used as a proxy for adsorption affinity. A positive correlation was observed between Langmuir adsorption coefficients (KL) measured with 80% MTMS and the Kow for each chemical in the mixture (Figure 6 inset), which showed higher adsorption affinity for more hydrophobic chemicals, consistent with reports in the literature.68–70 The only exception to this trend was bibenzyl, which had the highest Kow but a lower than expected KL of 8.3. This may be due to its large freedom of rotation and effective molecular volume, whereas the other chemicals were planar. Additionally, indole was poorly adsorbed by all compositions tested, potentially as a result of its comparatively low log Kow and slightly greater mobility (due to lower molecular weight). In general, this data indicates


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nonspecific interactions are responsible for the increased adsorption. However, the KL values measured in this study were much lower than comparable chemicals adsorbed on GAC,69 but KL,naphthalene determined with 80% MTMS was similar to zeolite71 and KL,phenanthrene was similar to certain types of limestone and sandstone.72 The adsorption capacities measured in this study were several orders of magnitude lower than other studies with naphthalene adsorbed onto carbon nanotubes, though the concentrations used in this study were much lower.73 While the intermediate gels (20-60% MTMS) were of interest for further study based on their mechanical properties, the silica gel composition with 80% MTMS had the highest adsorption affinity (KL,TOTAL=50.37 ± 8.17 L/g, Table 1) and specific adsorption capacity (61 ± 6 µg/g, Figure S4) of the gels tested, and thus it was used in subsequent biodegradation experiments.

Removal pollutant mixture from water with the silica biogel After adjusting the material composition to maximize adsorption, Pseudomonas sp. NCIB 9816-4 cells were encapsulated in the silica gel and their metabolic activity was verified. In order determine if the cells were indeed metabolically active after encapsulation, oxygen consumption was measured over a period of 4 weeks with a solution containing saturated naphthalene (Figure 7a). Oxygen consumption is directly related to naphthalene metabolism and the data showed slowly decreasing metabolic activity up to at least 4 weeks after encapsulation, similar to our previous results in a hydrophilic gel.28 It is important to note that the silica matrix rendered the cells incapable of dividing, due to physical confinement within the relatively stiff material (E = 5 MPa, Figure 4b), yet allowed them to be metabolically active.28,29 We have previously shown that Pseudomonas sp. NCIB 9816-4 can tolerate methanol concentrations of up to 30% (v/v) for 5 minutes, which is longer than the gelation time. Based on our previous work and the concentrations of MTMS and TMOS, we estimated the methanol concentration in this study to be 24% (v/v).


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After validating that the silica biogel was metabolically active, its ability to remove aromatic pollutants from water was evaluated over a short period (24 hrs). The silica biogel had increased removal (rate and capacity) of pollutants from the supernatant as compared to silica gel (Figure 7b). After 24 hours, the silica biogel removed most of the pollutants from the supernatant, leaving 10.3 ± 1.9% of the initial concentration, vs. 31.0 ± 2.5% with silica gel, which was attributed to biodegradation. While it was expected that the cells would easily biodegrade each individual chemical, consistent with previous reports,27,64 it was unknown whether this would be the case in a complex solution with competition from adsorption to the material. Since the enzyme of interest, naphthalene dioxygenase (NDO), binds to and reacts with different compounds with preference for smaller aromatics, a reduction in activity with log Kow was expected.74,75 Free (unencapsulated) NCIB 9816-4 cells were used as a positive control and after 24 hours of incubation, a total of 9.5 ± 1.3% of the initial pollutant concentration remained in the supernatant (Figure 7c). These results suggested that while the cells could oxidize each molecule in a complex solution, more time may be required for complete removal of the pollutants. Lastly, though the amount removed from solution by both free cells and the silica biogel were similar, the compositions were completely different. With the free cells, bibenzyl was the primary chemical remaining, whereas in the biogel both bibenzyl and biphenyl were the dominant remaining chemicals, though both present at less than half of the concentration of bibenzyl in the case of free cells. This indicated that bibenzyl was not well degraded by the free or encapsulated bacteria, which was consistent with our previous study,27 and also that adsorption likely helped to adsorb excess bibenzyl from solution. The silica biogel was also evaluated with repeated use to determine the quantity and composition of chemicals removed. Subsequent cycles showed that the silica biogel could be


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reused at least 4 times to remove the pollutants from the recharged supernatant, while the silica gel lost effectiveness after each use (Figure 8a). This illustrated that in the absence of bacteria to biodegrade pollutants and regenerate the material, the silica gel continued to gradually saturate with repeated use, which was expected. The cumulative removal of each pollutant in the mixture was also determined (Figure 8b). In contrast with the biogel, the silica gel showed an increasing trend with log Kow, which agreed with the previous results (Figure 6, inset). Together, these data suggest that when biodegrading bacteria are incorporated into the adsorbent silica matrix, biodegradation is the dominant removal mechanism for chemicals with log Kow ≤ 3.5 (biodegradation regime), whereas adsorption is dominant when log Kow > 4 (adsorption regime) (Figure 8c). Since the chemicals can all be biodegraded to some extent in the absence of adsorbent silica gel, this indicates that reduced mass transport due to adsorption hinders the rate of biodegradation when log Kow > 4. This effect was highlighted in the case of dibenzothiophene, phenanthrene, and bibenzyl, in which no statistically significant difference was observed between the silica gel and silica biogel (Figure 8b). We expected that PAHs are first adsorbed to the matrix since adsorption is nearly instantaneous, and the bacteria degrade the chemicals in their close proximity (in the pore liquid), allowing more chemicals to desorb from the hydrophobic regions and become bioavailable.76 Based on the data reported in Figure 8b, this suggested that chemicals with log Kow > 4 were slower to desorb from the matrix and therefore less bioavailable. Further testing of the material through wider concentration ranges and alteration of the synthesis techniques may be necessary for improved and more specific adsorption in the future. Two possible routes for improving the functionality of the material are the use of solely monomeric precursors in lieu of an alkoxide crosslinked SNP approach33 as well as exchanging the organic group used in the matrix (methyl) for another more hydrophobic group (e.g. ethyl, phenyl, butyl,


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etc.). The use of more hydrophobic organic groups in the precursors is likely to increase the adsorption capacity for aromatic pollutants, for example by stacking of the aromatic rings.77 Lastly, the procedure and material composition must be optimized to ensure that the material is mechanically robust to be useful in real-world applications requiring significant shear stress, such as packed bed reactors.

Conclusion In this study, a hydrophobic silica bioencapsulation method was developed with hydrocarbon-degrading Pseudomonas sp. NCIB 9816-4 to remove aromatic hydrocarbons from water via adsorption and biodegradation. This material has applications in water remediation from spill cleanup to continuous treatment of industrial waste. While this study focused on aromatic chemicals, the pollutant-degrading bacteria can be replaced for specific targeting of other chemicals of interest, making it a general approach for enhanced removal of hydrophobic chemicals.

Acknowledgements We thank Dr. Guillermo Marques for support with confocal microscopy and Ms. Sujin Yeom for assistance in growing cell cultures. This work was funded in part by a MnDRIVE seed grant, the Institute on the Environment, and an OVPR Transdisciplinary MnDRIVE grant from the University of Minnesota. Parts of this work were carried out in the Characterization Facility at the University of Minnesota, which receives partial support from NSF through the MRSEC program. Confocal microscopy was done with the help of the University Imaging Centers at the University of Minnesota.

References


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(1)

Page 18 of 36

Manoli, E.; Samara, C. Polycyclic Aromatic Hydrocarbons in Natural Waters: Sources, Occurrence and Analysis. TrAC - Trends Anal. Chem. 1999, 18 (6), 417–428.

(2)

Pashin, Y. V; Bakhitova, L. M. Mutagenic and Carcinogenic Properties of Polycyclic Aromatic Hydrocarbons. Environ. Health Perspect. 1979, 30 (June), 185–189.

(3)

Rubio-Clemente, A.; Torres-Palma, R. A.; Penuela, G. A. Removal of Polycyclic Aromatic Hydrocarbons in Aqueous Environment by Chemical Treatments: A Review. Sci. Total Environ. 2014, 478 (August), 201–225.

(4)

Yang, K.; Zhu, L. Z.; Xing, B. S. Adsorption of Polycyclic Aromatic Hydrocarbons by Carbon Nanomaterials. Environ. Sci. Technol. 2006, 40 (6), 1855–1861.

(5)

Bamforth, S. M.; Singleton, I. Bioremediation of Polycyclic Aromatic Hydrocarbons: Current Knowledge and Future Directions. J. Chem. Technol. Biotechnol. 2005, 80 (October 2004), 723–736.

(6)

Rivas, F. J. Polycyclic Aromatic Hydrocarbons Sorbed on Soils: A Short Review of Chemical Oxidation Based Treatments. J. Hazard. Mater. 2006, 138 (2), 234–251.

(7)

Mastral, A. M. Measurements of Polycyclic Aromatic Hydrocarbon Adsorption on Activated Carbons at Very Low Concentrations. Langmuir 2003, 42 (1), 155–161.

(8)

Brändli, R. C.; Hartnik, T.; Henriksen, T.; Cornelissen, G. Sorption of Native Polyaromatic Hydrocarbons (PAH) to Black Carbon and Amended Activated Carbon in Soil. Chemosphere 2008, 73 (11), 1805–1810.

(9)

Aktas, O.; Cecen, F. Bioregeneration of Activated Carbon: A Review. International Biodeterioration and Biodegradation. 2007, pp 257–272.

(10)

Cerniglia, C. E. Biodegradation of Polycyclic Aromatic Hydrocarbons. Curr. Opin.


18

Page 19 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Biotechnol. 1993, 4 (3), 331–338. (11)

Rodrigues, D.; Rocha-Santos, T. a P.; Freitas, A. C.; Gomes, A. M. P.; Duarte, A. C. Strategies Based on Silica Monoliths for Removing Pollutants from Wastewater Effluents: A Review. Sci. Total Environ. 2013, 461–462, 126–138.

(12)

Nassif, N.; Livage, J. From Diatoms to Silica-Based Biohybrids. Chem. Soc. Rev. 2011, 40 (2), 849–859.

(13)

Pannier, A.; Oehm, C.; Fischer, A. R.; Werner, P.; Soltmann, U.; Böttcher, H. Biodegradation of Fuel Oxygenates by Sol–gel Immobilized Bacteria Aquincola Tertiaricarbonis L108. Enzyme Microb. Technol. 2010, 47 (6), 291–296.

(14)

Blondeau, M.; Coradin, T. Living Materials from Sol–gel Chemistry: Current Challenges and Perspectives. J. Mater. Chem. 2012, 22 (42), 22335.

(15)

Wang, X.; Ahmed, N. Ben; Alvarez, G. S.; Tuttolomondo, M. V; Hélary, C.; Desimone, M. F.; Coradin, T. Sol-Gel Encapsulation of Biomolecules and Cells for Medicinal Applications. Curr. Top. Med. Chem. 2015, 15 (3), 223–244.

(16)

Ben-Yoav, H.; Melamed, S.; Freeman, A.; Shacham-Diamand, Y.; Belkin, S. Whole-Cell Biochips for Bio-Sensing: Integration of Live Cells and Inanimate Surfaces. Crit. Rev. Biotechnol. 2011, 31 (4), 337–353.

(17)

Coradin, T.; Livage, J. Aqueous Silicates in Biological Sol-Gel Applications: New Perspectives for Old Precursors. Acc. Chem. Res. 2007, 40 (9), 819–826.

(18)

Gill, I.; Ballesteros, A. Encapsulation of Biologicals within Silicate, Siloxane, and Hybrid Sol- Gel Polymers: An Efficient and Generic Approach. J. Am. Chem. Soc. 1998, 120 (34), 8587–8598.


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(19)

Page 20 of 36

Dickson, D. J.; Ely, R. L. Silica Sol-Gel Encapsulation of Cyanobacteria: Lessons for Academic and Applied Research. Appl. Microbiol. Biotechnol. 2013, 97 (5), 1809–1819.

(20)

Alvarez, G. S.; Foglia, M. L.; Copello, G. J.; Desimone, M. F.; Diaz, L. E. Effect of Various Parameters on Viability and Growth of Bacteria Immobilized in Sol-Gel-Derived Silica Matrices. Appl. Microbiol. Biotechnol. 2009, 82, 639–646.

(21)

Perullini, M.; Amoura, M.; Roux, C.; Coradin, T.; Livage, J.; Japas, M. L.; Jobbágy, M.; Bilmes, S. a. Improving Silica Matrices for Encapsulation of Escherichiacoli Using Osmoprotectors. J. Mater. Chem. 2011, 21 (12), 4546.

(22)

Perullini, M.; Jobbágy, M.; Mouso, N.; Forchiassin, F.; Bilmes, S. a. Silica-AlginateFungi Biocomposites for Remediation of Polluted Water. J. Mater. Chem. 2010, 20 (31), 6479.

(23)

Nassif, N.; Roux, C.; Coradin, T.; Bouvet, O. M. M.; Livage, J. Bacteria Quorum Sensing in Silica Matrices. J. Mater. Chem. 2004, 14 (14), 2264.

(24)

Nassif, N.; Roux, C.; Coradin, T.; Rager, M.-N.; Bouvet, O. M. M.; Livage, J. A Sol–gel Matrix to Preserve the Viability of Encapsulated Bacteria. J. Mater. Chem. 2003, 13 (2), 203–208.

(25)

Khongkhaem, P.; Intasiri, A.; Luepromchai, E. Silica-Immobilized Methylobacterium Sp. NP3 and Acinetobacter Sp. PK1 Degrade High Concentrations of Phenol. Lett. Appl. Microbiol. 2011, 52 (5), 448–455.

(26)

Branyik, T.; Kuncova, G.; Paca, J.; Demnerova, K. Encapsulation of Microbial Cells into Silica Gel. J. Sol-Gel Sci. Technol. 13 1998, 287, 283–287.

(27)

Aukema, K. G.; Kasinkas, L.; Aksan, A.; Wackett, L. P. Use of Silica-Encapsulated


20

Page 21 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Pseudomonas Sp. Strain NCIB 9816-4 in Biodegradation of Novel Hydrocarbon Ring Structures Found in Hydraulic Fracturing Waters. Appl. Environ. Microbiol. 2014, 80 (16), 4968–4976. (28)

Sakkos, J. K.; Kieffer, D. P.; Mutlu, B. R.; Wackett, L. P.; Aksan, A. Engineering of a Silica Encapsulation Platform for Hydrocarbon Degradation Using Pseudomonas Sp. NCIB 9816-4. Biotechnol. Bioeng. 2015, 113 (3), 513–521.

(29)

Mutlu, B. R.; Sakkos, J. K.; Yeom, S.; Wackett, L. P.; Aksan, A. Silica Ecosystem for Synergistic Biotransformation. Sci. Rep. 2016, 6, 27404.

(30)

Bartholomew, C. H. Mechanisms of Catalyst Deactivation. Appl. Catal. A Gen. 2001, 212 (1–2), 17–60.

(31)

Wu, D.; Zhou, J.; Li, Y. Mechanical Strength of Solid Catalysts: Recent Developments and Future Prospects. AIChE J. 2007, 53 (10), 2618–2629.

(32)

Krupa, I.; Nedelčev, T.; Račko, D.; Lacík, I. Mechanical Properties of Silica Hydrogels Prepared and Aged at Physiological Conditions: Testing in the Compression Mode. J. SolGel Sci. Technol. 2009, 53 (1), 107–114.

(33)

Mutlu, B. R.; Yeom, S.; Tong, H.-W.; Wackett, L. P.; Aksan, A. Silicon Alkoxide CrossLinked Silica Nanoparticle Gels for Encapsulation of Bacterial Biocatalysts. J. Mater. Chem. A 2013, 1 (36), 11051.

(34)

Mutlu, B. R.; Yeom, S.; Wackett, L. P.; Aksan, A. Modelling and Optimization of a Bioremediation System Utilizing Silica Gel Encapsulated Whole-Cell Biocatalyst. Chem. Eng. J. 2015, 259, 574–580.

(35)

Walcarius, A.; Mercier, L. Mesoporous Organosilica Adsorbents: Nanoengineered


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 36

Materials for Removal of Organic and Inorganic Pollutants. J. Mater. Chem. 2010, 20 (22), 4478. (36)

Pagliaro, M.; Ciriminna, R.; Man, M. W. C.; Campestrini, S. Better Chemistry through Ceramics: The Physical Bases of the Outstanding Chemistry of ORMOSIL. J. Phys. Chem. B 2006, 110 (5), 1976–1988.

(37)

Ciriminna, R.; Fidalgo, A.; Pandarus, V.; Béland, F.; Ilharco, L. M.; Pagliaro, M. The SolGel Route to Advanced Silica-Based Materials and Recent Applications. Chemical Reviews. June 19, 2013, pp 6592–6620.

(38)

Tripathi, V. S.; Kandimalla, V. B.; Ju, H. Preparation of Ormosil and Its Applications in the Immobilizing Biomolecules. Sensors Actuators, B Chem. 2006, 114 (2), 1071–1082.

(39)

Mackenzie, John, D.; Bescher, Eric, P. Structures , Properties and Potential Applications of Ormosils. J. Sol-Gel Sci. Technol. 1998, 377, 371–377.

(40)

Schneider, C. a; Rasband, W. S.; Eliceiri, K. W. NIH Image to ImageJ: 25 Years of Image Analysis. Nat. Methods 2012, 9 (7), 671–675.

(41)

Sangster, J. Octanol-Water Partition Coefficients of Simple Organic Compounds. J. Phys. Chem. Ref. Data 1989, 18 (3), 1111–1229.

(42)

Brinker, C. Hydrolysis and Condensation of Silicates: Effects on Structure. J. Non. Cryst. Solids 1988, 100, 31–50.

(43)

Fowler, S. D.; Greenspan, P. Application of Nile Red, a Fluorescent Hydrophobic Probe, for the Detection of Neutral Lipid Deposits in Tissue Sections: Comparison with Oil Red O. J. Histochem. Cytochem. 1985, 33 (8), 833–836.

(44)

Greenspan, P.; Mayer, E. P.; Fowler, S. D. Nile Red: A Selective Fluorescent Stain for


22

Page 23 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Intracellular Lipid Droplets. J. Cell Biol. 1985, 100 (3), 965–973. (45)

Standeker, S.; Novak, Z.; Knez, Z. Adsorption of Toxic Organic Compounds from Water with Hydrophobic Silica Aerogels. J. Colloid Interface Sci. 2007, 310 (2), 362–368.

(46)

Yasuda, T.; Okuno, T.; Yasuda, H. Contact Angle of Water on Polymer Surfaces. 1994, No. 12, 2435–2439.

(47)

Miwa, M.; Nakajima, A.; Fujishima, A.; Hashimoto, K.; Watanabe, T. Effects of the Surface Roughness on Sliding Angles of Water Droplets on Superhydrophobic Surfaces. Langmuir 2000, 16 (13), 5754–5760.

(48)

Wenzel, R. N. Surface Roughness and Contact Angle. J. Psychosom. Res. 1986, 30 (3), 387.

(49)

Brinker, C. J.; Scherer, G. W.; C. Brinker; Scherer, G. W. Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing; Academic press: San Diego, 1990.

(50)

Nakanishi, K.; Tanaka, N. Sol – Gel with Phase Separation . Hierarchically Porous Materials Optimized for High-Performance Liquid Chromatography Separations. Acc. Chem. Res. 2007, 40 (9), 863–873.

(51)

Kanamori, K.; Nakanishi, K.; Hirao, K.; Jinnai, H. Three-Dimensional Observation of Phase-Separated Silica-Based Gels Confined between Parallel Plates. Langmuir 2003, 19 (14), 5581–5585.

(52)

Nakanishi, K. Pore Structure Control of Silica Gels Based on Phase Separation. J. Porous Mater. 1997, 4 (2), 67–112.

(53)

Brunauer, S.; Emmett, P. H.; Teller, E. Adsorption of Gases in Multimolecular Layers. J. Am. Chem. Soc. 1938, 60 (1), 309–319.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(54)

Page 24 of 36

Barrett, E. P.; Joyner, L. G.; Halenda, P. P. The Determination of Pore Volume and Area Distributions in Porous Substances. I. Computations from Nitrogen Isotherms. J. Am. Chem. Soc. 1951, 73 (1), 373–380.

(55)

Reetz, M. T. Entrapment of Biocatalysts in Hydrophobic Sol-Gel Materials for Use in Organic Chemistry. Adv. Mater. 1997, 9 (12), 943–954.

(56)

Nemir, S.; Hayenga, H. N.; West, J. L. PEGDA Hydrogels with Patterned Elasticity: Novel Tools for the Study of Cell Response to Substrate Rigidity. Biotechnol. Bioeng. 2010, 105 (3), 636–644.

(57)

Liu, Y.; Geever, L. M.; Kennedy, J. E.; Higginbotham, C. L.; Cahill, P. A.; McGuinness, G. B. Thermal Behavior and Mechanical Properties of Physically Crosslinked PVA/Gelatin Hydrogels. J. Mech. Behav. Biomed. Mater. 2010, 3 (2), 203–209.

(58)

Jeon, O.; Bouhadir, K. H.; Mansour, J. M.; Alsberg, E. Photocrosslinked Alginate Hydrogels with Tunable Biodegradation Rates and Mechanical Properties. Biomaterials 2009, 30 (14), 2724–2734.

(59)

Mammeri, F.; Bourhis, E. Le; Rozes, L.; Sanchez, C. Mechanical Properties of Hybrid Organic-Inorganic Materials. J. Mater. Chem. 2005, 15 (35–36), 3787–3811.

(60)

Soler-Illia, G. J. A. A.; Innocenzi, P. Mesoporous Hybrid Thin Films: The Physics and Chemistry beneath. Chem. - A Eur. J. 2006, 12 (17), 4478–4494.

(61)

Novak, B. M. Hybrid Nanocomposite Materials—between Inorganic Glasses and Organic Polymers. Adv. Mater. 1993, 5 (6), 422–433.

(62)

Youngblood, W. W.; Blumer, M. Polycyclic Aromatic Hydrocarbons in the Environment: Homologous Series in Soils and Recent Marine Sediments. Geochim. Cosmochim. Acta


24

Page 25 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1975, 39 (9), 1303–1314. (63)

Giger, W.; Schaffner, C. Determination of Polycyclic Aromatic Hydrocarbons in the Environment by Glass Capillary Gas Chromatography. Anal. Chem. 1978, 50 (2), 243– 249.

(64)

Resnick, S.; Lee, K.; Gibson, D. Diverse Reactions Catalyzed by Naphthalene Dioxygenase from Pseudomonas Sp Strain NCIB 9816. J. Ind. Microbiol. 1996, 4, 438– 457.

(65)

Jeffrey, a M.; Yeh, H. J.; Jerina, D. M.; Patel, T. R.; Davey, J. F.; Gibson, D. T. Initial Reactions in the Oxidation of Naphthalene by Pseudomonas Putida. Biochemistry 1975, 14 (3), 575–584.

(66)

Ensley, B. D.; Ratzkin, B. J.; Osslund, T. D.; Simon, M. J.; Wackett, L. P.; Gibson, D. T. Expression of Naphthalene Oxidation Genes in Escherichia Coli Results in the Biosynthesis of Indigo. Sci. 1983, 222 (4620), 167–169.

(67)

Resnick, S. M.; Gibson, D. T. Regio- and Stereospecific Oxidation of Fluorene, Dibenzofuran, and Dibenzothiophene by Naphthalene Dioxygenase from Pseudomonas Sp. Strain NCIB 9816-4. Appl. Environ. Microbiol. 1996, 62 (11), 4073–4080.

(68)

Valderrama, C.; Gamisans, X.; de las Heras, X.; Farrán, A.; Cortina, J. L. Sorption Kinetics of Polycyclic Aromatic Hydrocarbons Removal Using Granular Activated Carbon: Intraparticle Diffusion Coefficients. J. Hazard. Mater. 2008, 157 (2–3), 386–396.

(69)

Hamdaoui, O.; Naffrechoux, E. Modeling of Adsorption Isotherms of Phenol and Chlorophenols onto Granular Activated Carbon. Part I. Two-Parameter Models and Equations Allowing Determination of Thermodynamic Parameters. J. Hazard. Mater.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 36

2007, 147 (1–2), 381–394. (70)

Karickhoff, S.; Brown, D.; Scott, T. Sorption of Hydrophobic Pollutants on Natural Sediments. Water Res. 1979, 13 (3), 241–248.

(71)

Chang, C. F.; Chang, C. Y.; Chen, K. H.; Tsai, W. T.; Shie, J. L.; Chen, Y. H. Adsorption of Naphthalene on Zeolite from Aqueous Solution. J. Colloid Interface Sci. 2004, 277 (1), 29–34.

(72)

Rügner, H.; Kleineidam, S.; Grathwohl, P. Long Term Sorption Kinetics of Phenanthrene in Aquifer Materials. Environ. Sci. Technol. 1999, 33 (10), 1645–1651.

(73)

Yang, K.; Wang, X.; Zhu, L.; Xing, B. Competitive Sorption of Pyrene, Phenanthrene, and Naphthalene on Multiwalled Carbon Nanotubes. Environ. Sci. Technol. 2006, 40 (18), 5804–5810.

(74)

Leblond, J. D.; Wayne Schultz, T.; Sayler, G. S. Observations on the Preferential Biodegradation of Selected Components of Polyaromatic Hydrocarbon Mixtures. Chemosphere 2001, 42 (4), 333–343.

(75)

Wammer, K. H.; Peters, C. a. Polycyclic Aromatic Hydrocarbon Biodegradation Rates: A Structure-Based Study. Environ. Sci. Technol. 2005, 39 (8), 2571–2578.

(76)

Li, N.; Jiang, J.; Chen, D.; Xu, Q.; Li, H.; Lu, J. A Reusable Immobilization Matrix for the Biodegradation of Phenol at 5000 mg/L. Sci. Rep. 2015, 5, 8628.

(77)

Martinez, C. R.; Iverson, B. L. Rethinking the Term “pi-Stacking.” Chem. Sci. 2012, 3 (7), 2191.


26

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Figure 1: Synthesis process of the silica biogel through hydrolysis of tetramethyl orthosilicate (TMOS) and methyltrimethoxysilane (MTMS), and condensation by mixing the hydrolyzed monomers with colloidal silica nanoparticles (SNP) and Pseudomonas sp. NCIB 9816-4. 75x32mm (300 x 300 DPI)



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Figure 2: Hydrophobicity characterization of the silica gel material as a function of methyltrimethoxysilane (MTMS) content in the gel. Nile Red fluorescence intensity measurements were conducted in triplicate. Error bars indicate standard deviation. 66x52mm (300 x 300 DPI)


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Figure 3: Microstructure characterization of the silica gel by scanning electron microscopy (SEM) with varying MTMS content. a) High magnification (40kX) images of the gel surface show little structural change with up to 60% MTMS, but at 80% MTMS or higher, phase separation occurred. Scale bars are the same across panels (500 nm). b) Spherical aggregates induced by phase separation in 80% and 100% MTMS gels.

94x50mm (300 x 300 DPI)



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Figure 4: Mechanical properties of the silica gel as a function of MTMS content. a) Stress at failure. b) elastic modulus. c) strain at failure. d) toughness. The samples were cylinders 12 mm in height and 12 mm in diameter and were tested in uniaxial compression until failure. Error bars indicate standard deviation, n=10. 136x105mm (300 x 300 DPI)


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Figure 5: Characterization of hydrophobic silica gels with confocal laser scanning fluorescence microscopy. a) Fluorescence images illustrating the distribution of bacteria and hydrophobic regions in the gel with the onset of phase separation. Cascade Blue was used to stain the hydrophilic regions, while Nile Red highlighted the hydrophobic regions. GFP-expressing E. coli were used in place of Pseudomonas sp. NCIB 9816-4 for visualization. Scale bars are the same across panels. Images have been adjusted for brightness to better illustrate the distribution of the hydrophobic methyl groups. b) Size distribution of hydrophobic aggregates in gels with encapsulated E. coli showing that the size of phase-separated hydrophobic domains increased with MTMS content (n > 3,000). 221x275mm (300 x 300 DPI)



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Figure 6: Increasing the methyltrimethoxysilane (MTMS) content of the silica gel to maximize the adsorption of the aromatic chemicals. Adsorption was measured by the change in the supernatant concentrations of chemicals after 6 days of exposure to an initial solution concentration of 400 ppb for each chemical (2.8 ppm total). Inset shows the linear relationship between Log (Kow) vs Log (KL) for the silica gel. Error bars indicate standard deviation, n=4. 130x95mm (300 x 300 DPI)


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Figure 7: Removal of chemicals through adsorption and biodegradation by the Silica Biogel. a) Long-term metabolic activity of Pseudomonas sp. NCIB 9816-4 encapsulated in an 80% MTMS gel measured by naphthalene degradation. b) Time course of chemical removal by the Silica Gel and Silca Biogel showing faster removal and higher removal capacity in gels that contained encapsulated cells (n=2). c) Initial use of the Silica Biogel after 24 hours. The right panel shows the composition of chemicals remaining in the supernatant. (a-b) Error bars indicate standard deviation. 136x104mm (300 x 300 DPI)



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Figure 8: Repeated use of the Silica Biogel. a) Overall chemical concentration in the supernatant after repeated use of Silica Gel and Silica Biogel with 24 hours per reuse cycle with fresh chemical solution (400 ppb per chemical, 2.8 ppm total). b) Cumulative amount of each chemical removed from solution v after 4 reuses. Chemicals are sorted from lowest to highest based on their log Kow. c) Ratio of biodegradation to adsorption calculated after 4 reuse cycles. Error bars indicate standard deviation, n=4. * p

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