1 2 AN ABSTRACT OF THE THESIS OF Jonathan R. Giska for the degree of Master of Science in Environmental Engineering presented on May 28, 2013 Title: The Effects of Silver Ions and Nanoparticles on Biofilms and Planktonic Cultures of Nitrosomonas europaea Abstract approved: Lewis Semprini Due to the effective antimicrobial properties of silver nanoparticles they represent a significant proportion of all consumer-­‐related nanomaterials. The introduction of silver nanoparticles from these products into domestic and industrial wastewater streams poses potential complications for biological treatment systems. A critical component of biological treatment that is extremely sensitive to perturbation is the nitrification process that converts ammonia to nitrate. The purpose of this study was to examine the effects of silver ions and silver nanoparticles on the model nitrifying bacteria, Nitrosomonas europaea, in both suspended cell batch and continuous biofilm cultures. These cultures were exposed to varying concentrations of either silver ions or nanoparticles and their nitrification activity, the associated silver masses of both the media and biomass, protein content of the N.europaea cells, and silver nanoparticle size and aggregation state were monitored during the experiments. The major findings of this study are that silver ions inhibit nitrification activity in both suspended batch and continuous biofilm cultures to a greater extent than silver nanoparticles and that biofilms are more tolerant to inhibition by silver ions and nanoparticles than suspended cells. These results indicate that silver nanoparticles may not pose a significant risk to nitrification processes in biological wastewater treatment systems. 3 © Copyright by Jonathan R. Giska May 28, 2013 All Rights Reserved 4 The Effects of Silver Ions and Nanoparticles on Biofilms and Planktonic Cultures of Nitrosomonas europaea by Jonathan R. Giska A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented May 28, 2013 Commencement June 2013 5 Master of Science thesis of Jonathan R. Giska presented on May 28, 2013 APPROVED: Major Professor, representing Environmental Engineering Head of the School of Chemical, Biological, and Environmental Engineering Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Jonathan R. Giska, Author 6 ACKNOWLEDGEMENTS I would like to express sincere appreciation to my colleagues Jamie Hughes and Mohammad Azizian whom I could not have gotten through many lab days. I would also like to thank fellow CBEE graduate students Daniel Vogler, Anna Herring, Adam Lambert, and Sassan Ostvars for countless inspirational, spirited discussions that helped maintain focus and motivation to complete this work. I am eternally indebted to my family – Rich, Mary Lou, Nicole, Tyras, and Hugh – for always providing positivity and encouragement when it was needed. Finally, I must thank my dog, Yanpa Fred Giska, for keeping me grounded with all of our walks and your brilliant insights. 7 TABLE OF CONTENTS Page 1 INTRODUCTION ..................................................................................................................................... 1 2 LITERATURE REVIEW ......................................................................................................................... 3 SECTION 2.1 SILVER NANOPARTICLE PROPERTIES ................................................................................ 3 SECTION 2.2 FATE AND TRANSPORT OF SILVER NANOPARTICLES ....................................................... 6 SECTION 2.3 THE NITROGEN CYCLE AND NITROSOMONAS EUROPAEA AS A MODEL AOB .................... 8 SECTION 2.4 PROPERTIES OF BIOFILMS ................................................................................................... 9 SECTION 2.5 SILVER ION AND NANOPARTICLE TOXICITY STUDIES ..................................................... 10 3 MATERIALS AND METHODS ........................................................................................................... 14 SECTION 3.1 N.EUROPAEA CULTIVATION ............................................................................................... 14 SECTION 3.2 SUSPENDED CELL BATCH EXPERIMENTS ......................................................................... 14 SECTION 3.3 CONTINUOUS DRIP FLOW REACTOR BIOFILM EXPERIMENTS ........................................ 16 SECTION 3.4 ANALYTICAL METHODS ..................................................................................................... 18 8 TABLE OF CONTENTS (Continued) Page 4 RESULTS AND DISCUSSION ............................................................................................................ 19 SECTION 4.1 RESPONSES TO SILVER IONS .............................................................................................. 21 Subsection 4.1.1 suspended cell batch experiments .............................................................. 21 Subsection 4.1.2 drip flow reactor biofilm ............................................................................... 24 Subsection 4.1.3 summary .............................................................................................................. 30 SECTION 4.2 RESPONSES TO SILVER NANOPARTICLES ......................................................................... 31 Subsection 4.2.1 suspended cell batch experiments .............................................................. 31 Subsection 4.2.2 drip flow reactor biofilm ............................................................................... 36 Subsection 4.2.3 summary .............................................................................................................. 43 5 CONCLUSION ......................................................................................................................................... 65 6 BIBLIOGRAPHY ..................................................................................................................................... 67 9 LIST OF FIGURES Figure Page 1 NITRITE PRODUCTION RATES OF N.EUROPAEA SUSPENDED CELL BATCH SILVER ION EXPERIMENTS DURING 3-­‐HOUR EXPOSURE PERIODS ................................................ 45 2 PERCENT NITRIFICATION ACTIVITY FROM 3-­‐HOUR BATCH EXPOSURES AND 3-­‐
HOUR RECOVERY DATA FROM THIS STUDY AND RADNIECKI ET AL. (2011) ............ 46 3 BIOMASS-­‐ASSOCIATED SILVER VERSUS PERCENT NITRIFICATION ACTIVITY FROM 3-­‐HOUR SILVER ION EXPOSURE BATCH EXPERIMENTS. ........................................ 47 4 NITRITE PRODUCTION RATES (A) AND PERCENT INHIBITION (B) OF N.EUROPAEA DFR BIOFILM EXPERIMENTS DURING 3-­‐HOUR SILVER ION EXPOSURE PERIODS ...................................................................................................................................................... 48 5 SILVER CONCENTRATION IN THE DFR EFFLUENT FROM 3-­‐HOUR SILVER ION EXPOSURES ................................................................................................................................................ 49 6 BIOMASS-­‐ASSOCIATED SILVER VERSUS PERCENT NITRIFICATION ACTIVITY FROM 3-­‐HOUR SILVER ION EXPOSURE BATCH AND DFR EXPERIMENTS .................... 50 7 NITRITE PRODUCTION RATES FOR BATCH EXPERIMENTS IN DFR (A) AND LOW MG2+ (B) MEDIA AS WELL AS PERCENT INHIBITION OF NITRIFICATION ACTIVITY (C) FROM N.EUROPAEA SILVER NANOPARTICLE EXPERIMENTS DURING 3-­‐HOUR EXPOSURE PERIODS. ............................................................................................................................. 51 10 LIST OF FIGURES (Continued) Figure Page 8 BIOMASS-­‐ASSOCIATED SILVER VERSUS PERCENT NITRIFICATION ACTIVITY FROM 3-­‐HOUR EXPOSURE BATCH SILVER NANOPARTICLE EXPERIMENTS. ............. 52 9 UV-­‐VIS SPECTROPHOTOMETER DATA FROM SILVER NANOPARTICLES IN HEPES BUFFER (PH 7.8) ...................................................................................................................................... 53 10 UV-­‐VIS SPECTROPHOTOMETER DATA FROM BATCH SILVER NANOPARTICLE EXPERIMENTS IN DFR MEDIA AND 1.0 PPM AGNP FOR CONTROLS CONTAINING NO CELLS (A) AND 3-­‐HOUR EXPOSURES (B). ............................................................................. 54 11 UV-­‐VIS SPECTROPHOTOMETER DATA FROM BATCH SILVER NANOPARTICLE EXPERIMENTS IN LOW MG2+ MEDIA AND 20 PPM AGNP FOR CONTROLS CONTAINING NO CELLS (A) AND 3-­‐HOUR EXPOSURES (B). ................................................ 55 12 N.EUROPAEA BIOFILMS WITH NO AG+ OR SILVER NANOPARTICLE EXPOSURE (A) AND WITH 3-­‐HOUR 20 PPM SILVER NANOPARTICLE EXPOSURE AND HEPES ONLY SYRINGE SOLUTION (B). ......................................................................................................... 56 13 NITRITE PRODUCTION RATES (A) AND PERCENT INHIBITION (B) OF NITRIFICATION ACTIVITY OF N.EUROPAEA DFR BIOFILM SILVER NANOPARTICLE EXPERIMENTS DURING 3-­‐HOUR EXPOSURE PERIODS. ......................................................... 57 14 SILVER EFFLUENT CONCENTRATIONS FOR 20 PPM SILVER NANOPARTICLE DFR BIOFILM 3-­‐HOUR EXPOSURES. ............................................................................................... 58 11 LIST OF FIGURES (Continued) Figure Page 15 BIOMASS-­‐ASSOCIATED SILVER VERSUS PERCENT NITRIFICATION ACTIVITY FROM 3-­‐HOUR EXPOSURE BATCH AND DFR SILVER NANOPARTICLE EXPERIMENTS. ......................................................................................................................................... 59 16 UV-­‐VIS SPECTROPHOTOMETER DATA FROM DFR BIOFILM SILVER NANOPARTICLE EXPERIMENTS. ...................................................................................................... 60 17 SILVER NANOPARTICLE CONCENTRATIONS FROM UV-­‐VIS DATA FOR DFR BIOFILM EFFLUENT COLLECTED DURING THE 3-­‐HOUR 20 PPM SILVER NANOPARTICLES EXPOSURES WITH HEPES ONLY AS THE SYRINGE SOLUTION .... 61 12 LIST OF TABLES Table Page 1 AVERAGE TOTAL PROTEIN MASS FROM BOTH DFR BIOFILM AND BATCH SUSPENDED CELL EXPERIMENTS ................................................................................................... 62 2 SILVER MASS DATA FROM ALL SILVER ION EXPERIMENTS PERFORMED UNDER ALL CONDITIONS ..................................................................................................................................... 63 3 SILVER MASS DATA FROM ALL SILVER NANOPARTICLE EXPERIMENTS PERFORMED UNDER ALL CONDITIONS ....................................................................................... 64 1 1. Introduction Nanotechnology is a rapidly growing industry concerned with the fabrication of materials and products at the nanoscale (1 – 100 nm). The increased surface area to volume ratio of nanomaterials relative to larger particles or bulk materials significantly alters the physico-­‐chemical properties of these materials and remains an active area of research and product development. (Moore 2006; Nel et al. 2006; Buzea et al. 2007) Nanomaterials can be found in a wide range of consumer products: medicine, cosmetics, environmental remediation, and electronic devices. (Fabrega et al. 2011) A public database is available from the Woodrow Wilson Institute that, although not comprehensive, contains an actively updated inventory of consumer products containing nanomaterials. (http://www.nanotechproject.org) Currently this database contains 1,317 products, and of these products 313 contain silver nanoparticles (AgNP). Silver has long been used for its antimicrobial properties as its toxicity to microorganisms is greater than many other metals while maintaining low toxicity to mammalian cells. (Zhao and Stevens 1998). It has been shown that AgNP are more efficient in mediating antimicrobial activity (Lok et al. 2006; Rai et al. 2009) than silver ions and as a result have been incorporated into wound dressings, medical devices, water purification systems, linings of washing machines, dishwashers, refrigerators, toilet seats, and clothing. (Li et al. 2011) Benn and Westerhoff (2008) have demonstrated that after several rounds of washing AgNP originally embedded within clothing such as socks leach from the fabrics. These AgNP can then enter both domestic and industrial wastewater streams, eventually arriving at treatment facilities. (Blaser et al. 2008; Geranio et al. 2009; Hagendorfer et al. 2010) The effects of these AgNP on biological wastewater treatment are relatively unknown and may present significant concerns for meeting discharge requirements. (Sheng and Liu 2011) 2 Numerous studies have explored the effects of AgNP on bacteria growing in suspension as well as biofilms, but few have focused specifically on bacteria associated with biological treatment. Sheng and Liu (2011) examined AgNP effects on wastewater biofilms, while Radniecki et al. (2010) and Yuan et al. (2012) studied AgNP effects on nitrifying bacteria in batch cultures, relating toxicity to a specific process of biological treatment. However there has been little research on AgNP effects of attached cell/biofilm systems in regards to biological treatment, specifically nitrification. The process of nitrification is a two-­‐stage process used for the treatment of ammonia in wastewaters and requires two distinct classes of bacteria. The first stage oxidizes ammonia to nitrite while the second oxidizes nitrite to nitrate, which are carried out by ammonia-­‐oxidizing bacteria (AOB) and nitrite-­‐oxidizing bacteria (NOB), respectively. These bacterial groups are typically the most sensitive to perturbation in biological treatment systems and serve as an excellent indicator species. This study addresses attached growth treatment systems with a process-­‐
oriented experimental design by assessing the toxicity of AgNP relative to silver ions (Ag+) using the model AOB, Nitrosomonas europaea, in both batch and continuous biofilm cultures. The objectives of this study are: •
Compare nitrification activity of N.europaea suspended cells and biofilms when exposed to Ag+ and AgNP •
Determine the suspended and cell-­‐associated silver mass fractions after exposure to Ag+ and AgNP •
Examine the aggregation and dissolution behavior of AgNP during the exposure tests 3 2. Literature Review 2.1 Silver Nanoparticle Properties AgNP were first synthesized in 1951; Turkevich et al. (1951) reported a wet chemistry synthesis using silver nitrate as a Ag+ source and sodium citrate as a reducing agent. Since then material scientists and chemists have discovered ways to manipulate the size, shape, and surface chemistry to further develop unique physico-­‐chemical properties including high electrical and thermal conductivity, surface-­‐enhanced Raman scattering, chemical stability, catalytic activity, and non-­‐
linear optical behavior. (Capek 2004; Frattini et al. 2005) The Woodrow Wilson Institute (http://www.nanotechproject.org) provides an extensive database of consumer products containing nanoparticles, which currently contains listings for 1,317 products and of these 313 contain AgNP. However, it is the broad-­‐spectrum antibacterial activity of silver (Luoma et al. 2008; Ratte et al. 1999; Silver 2003; Silver et al. 2006) and low manufacturing cost of AgNP that has driven their use in a diverse number of consumer products such as wound dressings, medical devices, water purification systems, and the linings of washing machines, dishwashers, refrigerators, toilet seats, and clothing. (Li et al. 2011) Several studies have reported enhanced antimicrobial activity of AgNP relative to bulk silver due to the large surface area to volume ratio, providing better contact with microorganisms (Morones 2005); thus the antimicrobial activity of AgNP is responsible for driving their use in consumer products. The antimicrobial activity of AgNP can be characterized based on suspended Ag+ or nanoparticle-­‐specific effects. Liu and Hurt (2010) studied the kinetics of Ag+ release from AgNP and proposed a two-­‐stage mechanism for dissolution consisting of a first stage where O2 and H+ react slowly with AgNP to form Ag+ and peroxide intermediates, leading to the second stage where the peroxide intermediates rapidly dissolve Ag+ from the AgNP forming water in the process. They further proposed a 4 rate law dependent on both pH and natural organic matter (NOM). As previously stated the H+ facilitates diffusion of Ag+ from the AgNP, whereas the NOM acts to reduce dissolution rates, increasing the stability of AgNP. Further controlling the stability of AgNP are the capping agents used to provide specified functionality (e.g. polar or non-­‐polar) to the AgNP for downstream usage in consumer products. Arnaout and Gunsch (2012) studied the stability of AgNP with different capping agents and found citrate-­‐coated AgNP were the most stable particles of the study. Thus Ag+ dissolution is primarily controlled by environmental conditions but is also influenced by AgNP capping agents. Suspended Ag+ have been shown to interact with cytoplasmic components and nucleic acids, to react with thiol groups, to inhibit respiratory chain enzymes, and to interfere with membrane permeability. (Russell and Hugo 1994) For example, Dibrov et al. (2002) demonstrated low levels of Ag+ induced proton leakage in Vibrio cholerae causing a collapse of the proton motive force. (Lok et al. 2007) Holt and Bard (2005) have shown that Ag+ inhibits at a low potential point of the respiratory chain, possibly the NADH dehydrogenase of complex I, in Escherichia coli cells. (Lok et al. 2007) The nanoparticle-­‐specific antimicrobial activities are only partly understood and have been difficult to distinguish from the effects of Ag+ as similar mechanisms have been observed in experimental treatments using both materials. Previous studies have shown AgNP localized to severely damaged cell membranes via transmission electron microscopy (Dror-­‐Ehre et al. 2009; Li et al. 2010; Sondi and Salopek-­‐Sondi 2004), and have been predicted to disrupt the permeability of the cell membranes leading to the efflux of reducing sugars, proteins, and adenosine triphosphate as well as the collapse of the membrane potential and proton motive force. (Li et al. 2010; Lok et al. 2006) In addition to these effects a proteomic study by Lok et al. (2006) revealed accumulation of envelope protein precursors; they found these mechanisms to be consistent with those of Ag+ but nano-­‐ versus micromolar 5 concentrations respectively. When AgNP enter cells Shrivastava et al. (2007) showed they not only interrupt the electron transport chain by binding to thiol groups but also dephosphorylate peptides on tyrosine residue, interfering with cell signaling mechanisms. Again, the aforementioned AgNP-­‐specific antimicrobial mechanisms are difficult to distinguish from Ag+, but a study by Xu et al. (2012) established AgNP generate reactive oxygen species (ROS), possibly leading to the enhanced activity relative to Ag+. Their results indicated the production rate of ROS was dependent on temperature and oxygen concentration, consistent with the rate law for AgNP dissolution developed by Liu and Hurt (2010). Therefore it is evident that dissolution of Ag+ from AgNP is responsible for the majority of antimicrobial activity but that ROS generated upon dissolution provide a nanoparticle-­‐specific toxicity mechanism. Aside from the antimicrobial activity of AgNP, they also exhibit optical properties not observed in bulk Ag or suspended Ag+ -­‐-­‐ specifically localized surface plasmon resonance (LSPR), which is a direct effect of the size of the nanoparticles. The free electrons (d-­‐orbital) of silver are able to travel through the material with a mean free path of ~50 nm; therefore no scattering is expected from the bulk with particles smaller than this, thus all interactions are expected to be with the surface. (Eustis and El-­‐Sayed 2006) Surface plasmons are coherent electron oscillations between the particle surface and the surrounding medium, and when the wavelength of incident light is much greater than the nanoparticle size photons can induce a standing resonance, which couples to the surface plasmon oscillation. As the incident light wave front passes and is absorbed by the surface the electron density of the particle is polarized to one surface and oscillates in resonance with the light’s frequency (Eustis and El-­‐Sayed 2006) and this is referred to as the LSPR. The oscillation frequency of the surface electrons is dependent on the size and shape of the particles, the dielectric constant of the surrounding medium, and the capping material. (Eustis and El-­‐Sayed 2006) 6 MacCuspie et al. (2011) found that LSPR could provide qualitative information about the size and size distribution of AgNP, as well as quantitative information about their concentration. AgNP roughly 10 nm in diameter exhibit a LSPR absorbance peak (i.e. λmax) at 390 nm (Link and El-­‐Sayed 1999), and as they increase in size the peak begins to broaden and λmax begins to red-­‐shift (Evanoff and Chumanov 2004 and 2005). AgNP shape asymmetry or coupling of the LSPR across multiple particles via agglomeration can cause similar effects, as well as broad, multiple or asymmetric peaks. (Duan et al. 2009; Zook et al. 2011; Elghanian et al. 1997) Exploiting LSPR via UV-­‐Vis spectroscopy provides a rapid means to monitor AgNP through time and has been used previously to characterize their behavior under environmentally relevant conditions. (MacCuspie et al. 2011) 2.2 Fate and Transport of Silver Nanoparticles Despite silver being relatively innocuous to humans, suspended Ag+ are persistent and highly toxic to prokaryotes and many freshwater and marine invertebrates and fish (Fabrega et al. 2011; Bianchini et al. 2002; Erickson et al. 1998; Fisher and Wang 1998; Hogstrand and Wood 1998). Ag+ tend to bioaccumulate in organisms due to the similar chemical properties with Na+ and Cu+, enabling cellular uptake via cell membrane ion transporters. (Luoma et al. 2008) The potential for bioaccumulation led the Environmental Protection Agency (EPA) and the European Economic Community (EEC) to begin regulating silver discharges in the late 1970s. (Fabrega et al. 2011) There is no current evidence to suggest that humans are being negatively impacted by AgNP incorporation into consumer goods, but studies do indicate that AgNP-­‐
containing products have the potential to release both AgNP and Ag+ into the environment. (Benn and Westerhoff 2008; Geranio et al. 2009; Gottschalk et al. 2009) The environmental fate of Ag+ in freshwater and marine ecosystems is largely controlled by ligands such as organic matter and sulfide, as well as complexation with chloride ions, respectively. (Adams and Kramer, 1998; Erickson et al. 1998; 7 Luoma et al. 1995) These interactions control the speciation and bioavailability rates in their respective environments. The dissolution of Ag+ from AgNP varies greatly with particle shape, size, and coating (Arnaout and Gunsch 2012) and is controlled in large part by aggregation of AgNP. NOM have been shown to interact with AgNP by increasing their stability and causing aggregation in solution, which decreases their rate of dissolution. (Liu and Hurt 2010; Wirth et al. 2012) Aside from the impact AgNP have on natural ecosystems, the fate and transport of AgNP in engineered systems, specifically wastewater treatment systems containing biological treatment, are of serious concern. Benn and Westerhoff (2008) demonstrated the ability of AgNP to be released from commercially available socks and be transported to wastewater treatment plants (WWTP). In another study Kim et al. (2010) detected Ag-­‐sulfide complexes in WWTP and determined sludge is a probable sink for Ag+ and AgNP given the high thiol content of sludge. In biological wastewater treatment the effects of AgNP and Ag+ are a serious concern for the nitrifying bacteria. These bacteria consist of two classes, the first metabolize ammonia to nitrite and the second further oxidize nitrite to nitrate – these are the ammonia-­‐oxidizing bacteria (AOB) and the nitrite-­‐oxidizing bacteria (NOB) respectively. These microbes are of particular concern because they are inefficient growers that are extremely sensitive to minor perturbations (e.g. pH, temperature change) in the treatment system and are easily outcompeted by the heterotrophic bacteria. (Hooper et al. 1997) The nitrifying bacteria have also demonstrated a tenfold increase in toxicity from organic contaminants relative to aerobic heterotrophs. (Blum and Speece 1992) The extreme sensitivity of these organisms coupled to their distinct biochemical transformations of nitrogen species make them an ideal indicator species useful for studying the effect of AgNP fate and transport to WWTP. 8 2.3 The Nitrogen Cycle and Nitrosomonas europaea as a Model Ammonia-­‐Oxidizing Bacteria The National Academy of Engineering (NAE) of the National Academy has listed managing the nitrogen cycle as one of the Grand Challenges for engineering. (www.nae.edu) This cycle follows nitrogen through many compounds and oxidation states: initially dinitrogen gas is fixed by microbes into ammonia, which is then incorporated into organic nitrogen with carbon to form amines. This organic nitrogen may then be consumed and released as ammonia from animals and humans; in the latter case the ammonia is transported to a WWTP where nitrifying bacteria oxidize the ammonia to nitrate in a two-­‐step process requiring two classes of bacteria – AOB oxidize ammonia to nitrite and the NOB oxidize nitrite to nitrate. In some WWTP the nitrate is treated further in anaerobic conditions to induce nitrite reduction back to dinitrogen gas, via intermediate species, thus completing the cycle. (Grady et al. 1999) The last two steps of nitrification and denitrification occur in nature along with a dissimilatory nitrate reduction pathway; however it is the part of the nitrogen cycle that engineers have influence over in WWTP that are critical for addressing the NAE Grand Challenge. Understanding how AgNP will effect nitrification in WWTP will be critical given the rapid growth of AgNP in consumer products. As previously mentioned the nitrifying bacteria make an excellent indicator species for biological treatment systems in WWTP. Because AOB carry out the first step in the nitrification process they are a natural candidate for study. Nitrosomonas europaea is a model AOB that has been studied for many years. Engel and Alexander (1958) first described the growth and autotrophic metabolism of N.europaea in the late 50s. Today the entire genome of N.europaea has been sequenced and physiological and molecular data have been combined to determine the functionality of many genes responsible for nitrogen-­‐
related biochemical pathways. (Chain et al. 2003; Bergmann et al. 1994; Sayavedra et al. 1998) The pathway of ammonia oxidation is now well understood and occurs in two stages: (i) transfer of an oxygen from bimolecular oxygen to ammonia via the enzyme ammonia monooxygenase (AMO) creating hydroxylamine, with a concomitant reduction of the other oxygen molecule and two hydrogen ions to water; (ii) oxidation of hydroxylamine with a water molecule via hydroxylamine monooxygenase resulting in the production of nitrite and five hydrogen ions, along with four electrons (two net positive given the first step). (Arp et al. 2002) N.europaea is also relatively easy to culture in batch and continuous systems as either suspended cells or biofilms. (Radniecki et al. 2008; Lauchnor et al. 2011) A rapid colorimetric assay has also been established for measuring the nitrification activity of N.europaea that consists of adding a small sample of the culture media into a solution of 1% w/v sulphanilamide in 1 M HCl, then adding 0.2% w/v N(1-­‐
naphthyl) ethylenediamine dihydrochloride, and measuring the absorbance at 540 nm. (Hageman and Hucklesby 1971) Slightly more complicated methods have also been developed to measure the specific oxygen uptake rates (SOURs) from both the AMO and HAO enzymes individually (Ely et al. 1995), allowing for elucidation of enzyme-­‐specific inhibition. The large amount of data and established methods relating to the culturing and experimentation of N.europaea make it an ideal AOB to study the effects of AgNP on nitrifying bacteria. 2.4 Properties of Biofilms Biological wastewater treatment systems exist in a variety of configurations, but two popular designs in use today are continuous stirred tank reactors (CSTR) that have a known volume and continuous flow rate, and trickling filter reactors that flow wastewater over a fixed substrate, typically rocks. (Grady et al. 1999) These two designs contain similar microbes but in significantly different cultures – in a CSTR the bacteria exist as suspended cells in flocs (i.e. activated sludge), whereas in a trickling filter reactor the bacteria adhere to the substrate and form biofilms. Biofilms differ greatly from suspended cells, as they are multilayer coatings of bacterial cells that accumulate at a living or inert surface and are surrounded by a 9 10 matrix of extracellular polymeric substances (EPS). EPS usually contributes ~85% of the mass of biofilms and is critical for the formation and integrity of biofilm structure as well as facilitating surface attachment (Smirnova et al. 2010; Hall-­‐
Stoodley et al. 2004) The composition of EPS can vary greatly for different bacterial species and growth conditions (Smirnova et al. 2010) but typically is composed of proteins, polysaccharides, and humic substances. (Wilen et al. 2003) EPS also provides a physical barrier between active cells and the environment that generates chemical gradients of important parameters such as oxygen, pH, nutrients, and contaminants (Flemming and Wingender 2001), where diffusion is the dominant transport mechanism for these particles in a biofilm. (Stewart 1998) Peulen and Wilkinson (2011) found the density of EPS to be an important factor in controlling the diffusion coefficients of nanoparticles in biofilms, as well as the charge on the particles – they observed a higher than expected coefficient for negatively charged AgNP. This physical barrier also provides increased protection relative to suspended cells via sorption to and subsequent sloughing of EPS from the biofilm (Fabrega et al. 2009). Kahn et al. (2011) found that under environmentally relevant conditions AgNP preferentially adsorb to EPS. Given the ubiquity of biofilms in engineered treatment systems and their complex physical and chemical characteristics, determining how AgNP interact with biofilms is necessary for understanding the potential risks of AgNP fate and transport. 2.5 Silver Ion and Nanoparticle Toxicity Studies Numerous studies have focused on the effects of AgNP on bacteria for a variety of species cultivated in different formats (e.g., batch, continuous stirred tank reactors (CSTR), drip flow reactors (DFR), and microplates) using AgNP of various sizes capping agents, as well as different experimental designs and analytical techniques; many of these studies utilize a variety of metrics for determining the magnitude of these effects, either qualitatively or quantitatively. (Radniecki et al. 2010; Choi et al. 2008; Fabrega et al. 2009; Lauchnor et al. 2011; Wirth et al. 2012) Despite significant variation in these studies, they tend to follow the same general format: (i) 11 qualitative and quantitative characterization of the AgNP employed in the study; (ii) description of the bacterial species, culturing methods, and analytical findings related to AgNP toxicity – most studies also perform the same methods using Ag+ for comparison; (iii) an analysis of AgNP stability/dissolution and subsequent Ag+ release. A major challenge in discerning the implications and potential risks of AgNP in the environment derived from these studies remains in the interpretation of multiple datasets with minimal overlap. For example, Fabrega et al. (2009) examined the effects of ~65 nm AgNP on Pseudomonas fluorescens in batch cultures while varying AgNP concentration, pH, and NOM. They used transmission electron microscopy (TEM), dynamic light scattering (DLS), and electron dispersive X-­‐ray spectrometry (EDX) to characterize the AgNP before, during, and after the experiments, but used only optical density (OD) readings to interpret the effects of exposure on the microbes. This last method is prone to interferences and is not a robust measure of toxicity to the overall metabolism of the bacteria, as it does not distinguish active from inactive cells. Choi et al. (2010) studied the how differently 20 nm AgNP capped with polyvinyl alcohol affect Escherichia coli cells grown in batch and cells grown as biofilms in microplates. They characterized the AgNP using TEM and DLS, and used microrespirometry to assay the toxicity effect on the E.coli cells. This bioassay is dependent on a calibration curve fitted from oxygen uptake versus cell density via OD measurements, which in turn is based on a fitted curve. This assay, while providing more information regarding the productivity of the bacteria, still represents an indirect and general measurement. However this work did provide insight into AgNP aggregation on biofilm surfaces. Fabrega et al. (2009b) also studied AgNP toxicity on biofilms of Pseudomonas putida grown in a flow cell reactor. Again, TEM, EDX, and DLS were used to characterize the 65 nm spherical AgNP; however they included a fluorescent staining method to 12 distinguish active and dead cells, analysis for estimating the amount of EPS that sloughed off the biofilms during AgNP exposure, and metals analysis to determine the biomass-­‐associated silver mass. This work advanced the understanding of adsorption and aggregation of AgNP to biofilms and the subsequent sloughing off of EPS with AgNP bound. Wirth et al. (2012) exposed P.fluorescens biofilms grown in microplates to PVP-­‐coated AgNP and Ag+, with and without humic acids (HA) and demonstrated: stable AgNP in solution are more toxic to biofilms than Ag+; HA induced AgNP aggregates that were unable to penetrate the EPS layers of the biofilms resulting in significantly reduced toxicity; and that even at extremely high levels of either Ag+ or AgNP, a viability loss of 100% was never observed. All of these studies were significant in their findings but lacked a specific metabolic pathway to target for AgNP toxicity investigations. Nitrifying bacteria such as the model AOB N.europaea offer the ability to study the effects of AgNP exposure on a process-­‐related metabolic pathway – nitrite production. Several studies have looked at dose-­‐dependent toxicity effects of AgNP on N.europaea in batch cultures. The functionality of these studies emerges from the colorimetric assay described previously to monitor nitrite levels, which can then be used to generate a percent inhibition of treatments versus controls. Radniecki et al. (2011) used 20 nm citrate-­‐coated AgNP, Yuan et al. (2012) used 7 and 40 nm AgNP coated with polyvinyl alcohol or adenosine triphosphate, and Arnaout and Gunsch (2012) used particles from 20 – 30 nm coated with either citrate, gum Arabic, or polyvinyl pyrrolidone (PVP). These works demonstrated just how significantly surface coating affected nitrification inhibition in N.europaea, and Arnaout and Gunsch (2012) determined that citrate-­‐coated AgNP were the most toxic. Given the body of work on AgNP exposures on suspended cell batch cultures of N.europaea, this current work seeks to contribute by filling the gap in AgNP exposures on N.europaea biofilms. Nitrification activity data from AgNP exposure 13 tests on N.europaea biofilms is critical for understanding how the fate and transport of AgNP can affect biologically mediated treatment processes in engineered systems. Further, understanding the aggregation and dissolution behavior of the AgNP in these systems may aid in mitigating any adverse affects they have on treatment. 14 3. Materials and Methods 3.1 N.europaea Cultivation N.europaea (ATCC 19718) cells, provided by Dan Arp (Oregon State University), were cultured in batch at 30°C in minimal growth media, as previously described (Radniecki et al. 2008). For suspended cell batch tests these cells were harvested by centrifugation at ~10,000 g for 30 minutes, washed with 30 mM HEPES (pH 7.8) and used to inoculate the batch test bottles. For biofilm experiments no centrifugation/washing was performed before DFR inoculations, as these cells required nutrients for their initial incubations and attachment to the glass slides. For both batch and biofilm experiments media was prepared as previously described (Lauchnor et al. 2011) and will be referred to as DFR media: 20 mM HEPES; 2.5 mM (NH4)2SO4; 10 µM KH2PO4; 3.77 mM Na2CO3; 730 µM MgSO4; 200 µM CaCl2; 9.9 µM FeSO4; 16.5 µM EDTA free acid; and 0.65 µM CuSO4. Additional experiments were conducted in a reduced Mg2+ media containing 200 µM MgSO4 and this preparation shall be referred to as low Mg2+ (LM) media. For tests involving AgNP media was prepared by mixing AgNP in nanopure water and shaking at 250 rpm for 20 minutes, then adding 10X concentrated media (either DFR or LM) and shaking for another 15 min, and finally inoculated with cells. This method was developed in Radniecki et al. (2011) to reduce AgNP aggregation prior to the addition of cells. For biofilm tests using AgNP a similar protocol was used for the syringe solution rather than the experimental media. 3.2 Suspended Cell Batch Experiments Suspended cell batch tests were performed on cells harvested during their exponential growth phase. Batch tests were run in two modes: inhibition and 15 inhibition/recovery. For the inhibition tests 35 ml of DFR or LM media was inoculated to optical density readings at 600 nm (OD600) of ~0.072 using harvested cells from the previously mentioned laboratory cultures. Treatments containing either free Ag+ or citrate-­‐stabilized 20 nm BioPure AgNP, purchased from Nanocomposix (San Diego, USA), were run alongside controls containing only experimental media for three hours and sampled to monitor nitrite levels. Again, batch tests involving AgNP were run using both DFR and LM media. Percent inhibition was calculated relative to the controls as: # # treatment nitrite concentration/protein concentration & &
% Inhibition =100%! % 1 " %
$ $ control nitrite concentration/protein concentration (' ('
For the recovery tests a larger volume of test media (100 ml) was prepared then inoculated and the inhibition portion was conducted for three hours as above. A larger volume was used to ensure minimal cell loss during washing steps between the exposure and recovery periods. At the end of the exposure period the entire volume was centrifuged at 12,000g for 20 minutes and the pelleted cells were washed with 30 mM HEPES (pH 7.8) to remove nitrate and any unbound silver. After three washes with 30 mM HEPES (pH 7.8), the cells were re-­‐suspended in 100 ml of fresh media with no inhibitors. OD600 measurements were taken prior to beginning the recovery portion to verify minimal cell loss. The recovery portion was run until the treatment experiments NO2-­‐ levels met or exceeded that of the controls. Nitrite levels were measured during the experiment by collecting 10 µL from each bottle and analyzed colorimetrically as described below. At the end of the experiments the cells were centrifuged at 12,000g for 20 minutes and the supernatant was collected for ICP-­‐OES analysis. In order to determine the total amount of silver associated with the cells, the cells were washed and centrifuged three times in 30 mM HEPES solution and then resuspended in 1 mL nanopure water for digestion and subsequent ICP-­‐OES analysis as described below. 16 3.3 Continuous Drip Flow Reactor Biofilm Experiments N. europaea biofilms were cultured in four channel DFR (BioSurface Technologies Inc., Bozeman, USA) as previously described (Lauchnor et al. 2011). For drip flow experiments, in a laminar flow hood aseptic techniques were used to add 10 ml of laboratory culture to each channel of an autoclaved DFR containing frosted glass slides. In order to promote cell attachment to the glass slides, the DFR were then incubated in batch mode at 30°C for 3-­‐4 days. After the initial incubation the influent and effluent tubing were attached to the DFR and growth media (either DFR or LM media) was then continuously pumped into the DFR at a rate of ~ 0.2 mL h-­‐1 using a peristaltic pump. DFR effluent was sampled aseptically as previously described in (Lauchnor et al. 2011) and nitrite levels were monitored until steady state productivity values (0.02 – 0.03 mmol h-­‐1) were achieved, after approximately 4-­‐6 weeks – these are considered standard biofilms. Additionally experiments were run on mature biofilms; these biofilms were cultured for an extra month after reaching steady state nitrite production rates. Inhibition experiments were then performed by injecting Ag+ or AgNP via syringe pump at ~ 1/10 the DFR flow rate to final concentrations ranging from 0.05 – 0.75 and 20 ppm, respectively, for a duration of three hours. Percent inhibition for each biofilm experiment was calculated independently relative to the initial nitrite production rates as: ' ' #(sample nitrite concentration ) ( flow rate )%& * *
% Inhibition = 100%! ) 1 " ) $
,,
( ( #$( initial nitrite concentration ) ( flow rate )%& + + Percent inhibition of the biofilms receiving only control media was then subtracted as background to establish the final, adjusted percent inhibition of the treatment biofilms. Each DFR consists of four channels – two control channels and two 17 treatment channels. The two control channels are further divided as one received media input solely from the peristaltic pump (i.e. growth media) and the other received growth media along with additional input from a syringe pump during the experiment to deliver control syringe solution (i.e. solution containing no silver). The two treatment channels received inputs from the syringe pump with solutions containing silver and either LM media or HEPES (pH 7.8) only. Flow from the peristaltic and syringe pumps to the DFR channels was controlled using a three-­‐way valve that allowed mixing of the solutions prior to entering the DFR. During the experiment, channel effluent was collected in 30 minute intervals from each channel, with flow rates determined by weighing the effluent in pre-­‐weighed glass vials, and subsequently analyzed for nitrite concentrations. Effluents from experiments involving AgNP were further analyzed by UV-­‐Vis spectroscopy to identify AgNP aggregation or dissolution. Finally after the three-­‐hour exposures, the frosted glass slides were removed and the biofilms were harvested by rinsing repeatedly with 2 mL of nanopure water and scraping with a sterile razor blade. The biofilm suspension was then vortexed for one minute, placed in a sonication bath for two minutes, and vortexed for an additional minute in order to disperse the biological material for subsequent protein and ICP-­‐OES analyses for silver content. Ag+ recovery experiments were also performed on the biofilms, but only on mature biofilms. The exposure portion of these experiments was conducted as previously mentioned, but after exposure the biofilms were cultured until nitrite production rates returned to pre-­‐test levels. An additional exposure was performed on the same biofilms as just described for a total of two exposures on the same biofilms. 18 3.4 Analytical Methods Nitrite levels for samples from the batch and DFR experiments were determined using a colorimetric assay with absorbance at 540nm as described previously (Hyman 1995). Protein content of biofilms was determined via the Biuret assay with absorbance readings at 540 nm on 1 ml samples digested in 3 M NaOH for 30 minutes (Hyman 1995). All spectrophotometer measurements for nitrite and protein levels were made using Beckman Coulter DU530 Life Science UV-­‐Vis spectrophotometer. (Brea, CA USA) DFR effluent and supernatant from batch experiments was collected (~ 6 ml) and acidified for ICP-­‐OES analysis with concentrated sulfuric acid (3%) to maintain Ag in solution and diminish interference from HEPES. Cell pellets from batch experiments and biofilm suspensions (1 mL) were digested overnight at 60°C in 2 mL of concentrated phosphoric acid and 3 mL of concentrated nitric acid. Silver measurements were made using a Teledyne Leeman Labs Prodigy ICP-­‐OES (Hudson, NH USA) set in axial mode with a detection limit of ~ 25 ppb for Ag, using the standard silver element line 328.068 nm. Triplicate peak integrations were made with averages reported for final values and standard curves from 50 ppb to 5.0 ppm were performed for quantification. AgNP aggregation and dissolution data from batch supernatant and DFR effluent was monitored using a Hewlitt-­‐Packard 8453 UV-­‐VIS spectrophotometer (Palo Alto, CA USA) with a wavelength scan from 300 – 700 nm. Nanoparticle controls were suspended in 30 mM HEPES buffer (pH 7.8) and used to generate a standard curve (2 – 20 ppm AgNP) from a maximum absorbance value (λmax) of 400 nm for quantification of samples. Batch supernatant and DFR effluent (~1 ml) was centrifuged at ~17,000 g for one minute to remove cells while maintaining AgNP in suspension. Samples were then scanned from 300 – 700 nm and AgNP concentration was determined by absorbance at λmax and aggregation was inferred by examining the entire spectra as demonstrated in Zook et al. (2011) 19 4. Results and Discussion The results and discussion of this thesis focus on the interactions of Ag+ and AgNP with N.europaea in both suspended cell batch and DFR biofilm experimental systems. These findings are separated into physiological data and silver analyses: physiological data is given by rates of nitrite production coupled to protein content measurements that are used to estimate the extent of inhibition; silver concentrations of both suspended and biomass-­‐associated Ag species are presented along with data on the stability and aggregation of AgNP. The results and discussion section is divided into three sub-­‐sections: responses of N.europaea cells to silver ions; responses of N.europaea cells to silver nanoparticles; and comparison of these responses to both the Ag+ and AgNP. These sub-­‐sections are further divided into results for suspended cell batch experiments and DFR biofilm experiments. The physiological data is first presented for each experimental system, followed by the Ag metal analysis, then the spectrophotometer data for AgNP experiments, and finally a summary of the findings. The suspended cell batch experiments closely parallel recently published work (Radniecki et al. 2011) done previously by this group that examined the effects of Ag+ and AgNP on the nitrification activity of N.europaea in batch systems. The fundamental difference between the batch experimental systems in the two studies is the media used during the exposure and recovery tests – in the previous study cells were tested in a HEPES buffer (pH 7.8) and 2.5 mM (NH4)2SO4 only; whereas in this study the fully defined growth media (DFR media) described in the materials and methods section was used. The use of the DFR media and its implications is discussed in the following sub-­‐sections as previous studies have demonstrated divalent cations and chloride ions interact with both Ag+ and AgNP in solution. (Choi et al. 2008; Li et al. 2010) The results from Radniecki et al. are used primarily for comparison of physiological responses of the batch systems and Ag analyses, as well as the effects of using different media. Due to the novelty of cultivating N.europaea in biofilms using a DFR scheme there is limited data to compare the results of the Ag+ and AgNP exposures in this study. Lauchnor et al. (2011) used the same DFR system to determine inhibition of nitrification activity of N.europaea biofilms by phenol and toluene exposure. Their results showed that biofilms were less inhibited than suspended cells, but can only be used for corroborating biofilm nitrite production rates and protein content regarding this study. Previous work with Ag+ and AgNP exposures to biofilms has been performed (Fabrega et al. 2009; Wirth et al. 2012; Choi et al. 2010; Xiao and Weisner 2013) and despite significant differences to this work, many concepts from these studies, focused on Ag+ and AgNP sorption and diffusion into biofilms as well as AgNP aggregation by biofilm EPS and NOM, are applicable to the experimental system in this study. Therefore the results of DFR biofilm experiments from this study are compared only within this study between Ag+ and AgNP results, while some of the broader concepts of Ag+ and AgNP interactions with biofilms from previous studies are integrated into interpreting the results. The general overview of the findings from this study indicates the biofilm systems were less sensitive to Ag+ and AgNP than suspended cells as observed by nitrification inhibition data, but that suspended cells recovered at a faster rate for similar inhibition levels. The physiological results also show that per mg of protein the suspended cells were more productive than biofilms; however, biofilms maintained higher percent nitrification activity at similar μg Ag bound per mg protein. Finally a major finding is that the aggregation and stability of the AgNP were significantly affected by the media used in the experimental systems, which greatly influenced the degree of nitrification inhibition observed. 20 21 4.1 Responses to Silver Ions 4.1.1 Suspended cell batch experiments Results from Ag+ suspended cell batch experiments are presented in (Figure 1). Nitrite production normalized to protein content (Figure 1a) for both the control and Ag+ treatments were extremely consistent among replicates, nearly identical in some cases, as demonstrated by their associated error bars. Linear production rates were observed for the controls as well as the 0.05 and 0.10 ppm Ag+ treatments across the 3-­‐hour exposure. At 0.25 ppm Ag+ treatments the production rates decreased from 0.75 – 2.25 hours with a slight increase from 2.25 – 3.0 hours. Treatments at 0.50 and 0.75 ppm Ag+ show no significant difference in their nitrite production rates, which remained nearly constant after 0.75 hours indicating no significant nitrification occurred from 0.75 – 3.0 hours. The percent inhibition of nitrification activity for the treatments relative to the controls is shown in (Figure 1b). In these experiments percent inhibition reached a maximum of ~95% at Ag+ concentrations ≥0.50 ppm with no significant difference in the observed inhibition at ≥0.25 ppm Ag+. The inhibition observed at 0.05 and 0.10 ppm Ag+ parallels the results observed for 0.05 and 0.09 ppm Ag+ treatments in Radniecki et al. (2011), shown in (Figure 2a), while the 0.25 ppm Ag+ inhibition data agrees with Arnaout and Gunsch (2012). Cell biomass was also monitored before and after the three-­‐hour exposure periods with average values for all batch experiments shown in [Table 1]. For all batch experiments no detectable biomass loss occurred during the exposure period, and only minimal losses occurred during the wash steps prior to the recovery period. The corresponding percent recovery data for the batch Ag+ experiments is shown in (Figure 1c). These data were normalized to the nitrite production rate after a three-­‐
hour recovery period of the non-­‐exposed controls. The results from (Figure 1c) illustrate a significant increase in recovery time for cells exposed to >0.25 ppm Ag+ 22 and no recovery after six days for cells exposed to 0.75 ppm Ag+. The increased recovery times are due to significant reductions in the viable cell mass in each experiment; this is reflected in the nitrite production rates (Figure 1a) where 0.25 ppm Ag+ treatments maintain an increasing production rate and 0.50 ppm Ag+ treatments remain relatively constant after 0.75 hours. The large variance observed by the 95% confidence interval for the 0.50 ppm Ag+ recovery data at just over five and half days was likely due to variability in cell loss during the wash steps prior to the recovery portion of the experiments. Because cell recovery is related to cell growth, an exponential process, during the longer recovery periods the variability in the initial cell number significantly affected the recovery rates of the individual experiments. Comparing the recovery results for 0.05 and 0.10 ppm Ag+ of this study at three hours to those at 0.05 and 0.09 ppm Ag+ presented in Radniecki et al. (2011) demonstrate a significant increase in recovery of nitrification activity at 0.05 ppm Ag+ in this study, with no significant difference in recovery at 0.10 and 0.09 ppm Ag+ (Figure 2b). Metal analysis was performed to determine the initial silver mass present in the batch tests prior to the addition of cells; upon completion of the exposures the amount of silver present in the supernatant and cell digests was also analyzed and all these data are summarized in [Table 2]. The ‘measured’ mass added to the bottles was recorded as the concentration of Ag in the experimental media prior to adding cells, and ranged from ~50 – 75% of the ‘nominal’ value for the associated experimental concentration (i.e., 0.05, 0.10, 0.25, 0.50, or 0.75 ppm Ag+). This loss of mass was likely due to sorption of the Ag+ onto the glass bottle during the initial dispersion into the media; however subsequent acid rinsing of the bottles yielded non-­‐detectable Ag concentrations. The ‘suspended’ Ag mass, collected as the supernatant after pelleting the biomass via centrifugation, approximately doubled with increasing concentrations of initial Ag+ mass for all treatments. [Table 2] The suspended Ag mass fraction recovered from the solutions ranged from 7-­‐15% of the measured Ag mass added with no 23 trend associated to the initial Ag mass. The biomass associated or ‘bound’ Ag was measured from the digest of the biomass pellets that were collected after the three-­‐
hour exposure following centrifugation of the batch experiments. At all Ag+ treatment concentrations the majority of the Ag mass recovered existed as biomass-­‐
associated Ag and increased up to 0.25 ppm then decreased slightly at 0.5 ppm. Interestingly the bound Ag mass showed a decreasing trend in its mass fraction of the total with increasing initial Ag mass. It is unclear if saturation of the sorption sites were reached at 0.25 ppm given these observations and more rigorous experimentation would need to be performed to develop an accurate sorption model. Data for biomass-­‐associated μg Ag/mg protein versus percent nitrification activity is shown in (Figure 3) for both the batch Ag+ experiments, plotted along with their associated linear regression model fit through 100 percent nitrification activity. In comparing the data from this study with previous work of Radniecki et al. (2011) the current work found a reduced percent nitrification at similar bound μg Ag/mg protein values. The results also indicate a steeper slope for the results of this study, suggesting a greater sensitivity to increases in biomass-­‐associated silver. These differences are likely due to the increased cell concentrations and volumes employed specifically to investigate the biomass-­‐associated silver mass by the previous study (Radniecki 2011); this is further supported by no significant difference in nitrification activity observed in the exposure tests between the two studies. The results of this work as compared with the previous study (Radniecki 2011) conducting similar Ag+ suspended cell batch experiments indicates similar inhibition of nitrification inhibition by Ag was achieved in the DFR media used in this study and 30 mM HEPES and 2.5 mM (NH4)2SO4 used in Radniecki et al. (2011) This finding was unanticipated due to the presence of Mg2+ and Cl-­‐ in the DFR media, which have been shown to interfere with Ag+ cellular uptake and form AgCl colloids, respectively (Fabrega 2011 and Wirth 2012). However, in another study by Choi et 24 al. (2008) researchers performed similar exposures using Ag+ as well as AgCl colloids on mixed autotrophic, nitrifying cell suspensions from a wastewater treatment plant. Their results demonstrated that at Ag+ concentrations ≤0.50 ppm no significant difference in nitrification inhibition between exposures with Ag+ or AgCl colloids was observed – these data are relevant given the Ag+ concentrations used in this current study. These previous observations may help to account for the nearly identical results between the current work and that of Radniecki et al. (2011) given the significant difference in ionic composition between the two experimental media – i.e. AgCl colloid formation has little effect on nitrification inhibition. 4.1.2 Drip Flow Reactor biofilm experiments Results from the Ag+ DFR biofilm experiments are with nitrite production rates and corresponding percent nitrification inhibition for the three-­‐hour exposure periods. (Figure 4a,b) These results also include experiments performed on ‘mature’ biofilms, which contained approximately four times the protein content of the standard biofilms used in all other DFR experiments [Table 1]. As mentioned in the materials and methods section, ‘standard’ biofilms reached a steady state of nitrite production (3 – 4 weeks) prior to experimentation; whereas the ‘mature’ biofilms were maintained at steady state for an extra month prior to experimentation. The initial average nitrite production rates (Figure 4a) for the standard biofilms ranged from 0.02 – 0.03 mmol NO2-­‐/hour while the initial average production rate of the mature biofilms was > 0.04 mmol NO2-­‐/hour – initial production rates were those at which the biofilms reached steady state and remained constant for 2 -­‐ 3 days prior to experimentation. The observed range of initial nitrite production rates for the standard biofilms is in agreement with those observed previously from the same DFR system (Lauchnor 2011 et al.). For the DFR experiments the nitrite production was normalized to flow rate (mmol NO2-­‐/hour) rather than protein (mmol NO2-­‐/mg protein) as was done for the batch experimental results (figures 4a and 1a, respectively). This convention was adopted due to the inability to measure protein content of the biofilms during the exposure. Comparing production rates for both suspended cells and biofilms normalized to protein content and time, the final production values for the biofilms are on average less than the rates of the suspended cell experiments – i.e., 0.005 – 0.032 mmol NO2-­‐/mg protein/hr for the biofilms versus 0.124 – 0.372 mmol NO2-­‐/mg protein/hr for suspended cells. These observations suggest planktonic cells have an increased metabolic rate relative to biofilm cells. It is also evident from (Figure 4a) that the mature biofilms maintained significantly greater production levels at 2.0 ppm Ag+ exposures. As protein content was the only variable between the biofilm exposures at 2.0 ppm (i.e., four times greater in the mature biofilms) this suggests protein content is a critical factor influencing dose response in biofilms. The percent inhibition results (Figure 4b) show no significant response difference between the standard biofilm exposures at Ag+ concentrations of 0.5 and 1.0 ppm and 2.0 and 7.5 ppm, with inhibition levels of ~50 and 75% respectively. It is interesting to note that similar inhibition at 2.0 and 7.5 ppm Ag+ were observed during the 3-­‐hour exposure, as 7.5 ppm is higher than any previous study on biofilms. This result suggests two phenomena: i.) at 2.0 ppm Ag+ the toxicity to cells at or near the surface is saturated; and ii.) diffusion limitations caused by the complex porous EPS structure and adsorption to the biofilm (Puelen and Wilkinson 2011) provide protection for active cells embedded deeper within the film’s structure. These two phenomena are likely responsible for a sustained basal metabolism of ~25% of the initial nitrification activity for biofilms; this phenomena was not observed in the planktonic cells at < 1/10 the Ag+ dosage. Figure 4b does indicate that biofilm maturity level (i.e. protein content) correlated to a significant decrease in nitrification inhibition at 2.0 ppm Ag+. At nearly four times the protein content the percent inhibition of mature biofilms was 15% -­‐-­‐ a decrease of ~80% relative to the percent inhibition of the standard biofilms at 75%. Recovery data for Ag+ DFR experiments on mature biofilms only are shown in (Figure 4c). These recovery tests were performed sequentially on individual 25 26 biofilms exposed to 2.0 ppm Ag+. The results indicate a faster recovery rate for the second exposure relative to the first exposure, from 7 to 4 days to reach 60% recovery. Given the similar conditions of both experiments, it is likely the difference in recovery rates may be due to a molecular mechanism within the active cells –e.g. transcriptional control of specific metabolic pathways. One possible explanation of this is the upregulation of the merA gene after the first exposure. This gene codes for the mercuric reductase enzyme and is responsible for reducing metals using NADPH thus reducing the oxidative stress to the organism. (Rosen 1996; Su et al. 2009) Arnaout and Gunsch (2012) showed a four-­‐fold increase in merA transcription in N.europaea exposed to Ag+ and AgNP. Therefore it is possible the N.europaea biofilms from this study increased their mercuric reductase enzyme after the first exposure and maintained its activity through the second exposure, leading to a faster recovery rate. Comparing the recovery of the biofilms and planktonic cells it is clear the biofilms recovered at a much slower rate. It should be noted that the planktonic cells were rinsed three times to remove any excess Ag+, whereas the biofilms were not. The rate of recovery for the 0.05 ppm Ag+ planktonic cell tests at a similar percent inhibition from ~ 20% was nearly two orders of magnitude faster than that of the mature biofilms. Possible explanations for these observations include: i.) growth rates of planktonic cells are much higher than biofilm cells (i.e., planktonic cells typically reach stationary phase in 3 – 4 days, while biofilms do not reach a steady state for 3 -­‐4 weeks), which implies higher global metabolic rates in planktonic cells; ii.) Ag+ associated with planktonic cells/biomass after exposure is no longer available to interact with newly synthesized cells during the recovery period; iii.) Ag+ remains associated with (i.e. sorbed and/or slowly diffusing through) the biofilms continuing to cause cellular damage until sufficient growth/EPS synthesis has occurred to slough off Ag-­‐associated biomass. Comparing the recovery rates from this study with previous work using phenol to inhibit N.europaea biofilms demonstrates a significantly increased recovery time for 27 the biofilms inhibited by Ag+ relative to phenol. (Lauchnor and Semprini 2013, in press) The previous study found that biofilms at ~ 60% inhibition by phenol reached nearly complete recovery of nitrification activity by 50 hours as compared to 7 days for the faster recovery from Ag+ exposure. These results suggest the Ag+ persists longer in the biofilms than the phenol and continues to hinder recovery. Possible explanations for this persistence are that Ag+ does not degrade over time (either biotically or abiotically) as phenol does and the ability of Ag+ to sorb to the EPS may increase its retention time in the biofilms. Metal analysis results for the DFR effluent collected during the Ag+ exposures to the biofilms are shown in (Figure 5) and the Ag mass balance data is summarized in [Table 2]. Control experiments containing no biofilms at 2.0 ppm Ag+ are also shown in (Figure 5). Examining the breakthrough curves for the effluent values from the control, 0.5, and 2.0 ppm Ag+ biofilm exposures (Fig. 5) reveals they do not differ significantly from one another after the first hour; however the 7.5 ppm Ag+ biofilm exposure effluent is an order of magnitude greater than the other treatments and as such was plotted on the secondary y-­‐axis on the right of the plot. The breakthrough curve Ag concentrations for the Ag+ treatments remained relatively low for the first hour then reached an apparent steady state for the remainder of the test. These results indicate significant sorption occurred in both the control and treatment DFR experiments. Interestingly, biofilms at all exposures of Ag+ sloughed visible biomass during the first 30 minutes of every exposure test, which may explain the similar breakthrough responses of all exposure tests. The sloughing off of biomass has been observed previously upon exposure to Ag+ and AgNP (Fabrega et al. 2009 and 2011) and this biomass is able to sorb Ag, thus reducing the amount available during the ICP-­‐OES measurements. The control data showed the opposite behavior in its breakthrough response, reaching its highest effluent concentration at one hour and then declining for the remainder of the exposure. This behavior is likely due to sorption of Ag+ to components of the system, especially the glass syringe, increasing with time. 28 Examining [Table 2] shows the 0.5 ppm exposures retained 95% of the nominal Ag+ mass in the injection syringes; whereas the 2.0 and 7.5 ppm Ag+ exposures retained only 45-­‐50% of their nominal Ag+ mass. These actual Ag mass values lead to expected effluent concentrations from these two exposure levels of approximately ~ 1.0 and 3.5 ppm respectively. The breakthrough curve in (Figure 5) indicates the standard and mature biofilm 2.0 ppm effluents reach averages of ~ 0.05 and 0.025 ppm respectively, while the 7.5 ppm exposures maintain an average of ~ 2.5 ppm after an hour. Again, as observed in the suspended cell batch tests, the discrepancy between the nominal and actual Ag+ mass added to the systems was likely due to sorption of Ag+ to the glass syringes and/or the DFR apparatus itself; this effect is diminished at higher concentrations likely due to saturation of sorption sites. The ‘suspended’ Ag mass [Table 2] from the DFR tests differs from that of the batch tests; in the biofilm tests this value represents the cumulative total of the effluent samples for the duration of the 3-­‐hour exposure. The ‘biomass’ Ag mass [Table 2] is similar to that of the batch tests and was taken from digests prepared from the entire biofilm present on the glass slides after exposure rather than the cell pellets as in the batch tests. For the 0.5 ppm tests the suspended and bound Ag masses were nearly equal, while for both the standard and mature biofilm 2.0 ppm tests the majority of the Ag mass (75 and 85% respectively) retained was present as bound Ag, and for the 7.5 ppm test the majority (90%) was present in the suspended samples. In the 0.5 and 2.0 ppm Ag+ tests less than 20% of the actual Ag mass was recovered, while 72% Ag mass was recovered for the 7.5 ppm Ag+ experiments with ~ 90% in the suspended fraction. The bound Ag mass from the 7.5 ppm exposures at ~ 9 μg was only twice that of the 2.0 ppm exposures; this, combined with the significant suspended Ag mass recovered in the effluent, suggests that sorption of Ag+ played a less significant role at the higher exposures and may indicate that sorption sites associated with the DFR system itself were saturated at 7.5 ppm. 29 [Table 2] also indicates that the 0.5 ppm exposures bound a greater Ag mass per mg of protein than either of the 2.0 ppm exposures. The standard biofilm exposure at 2.0 ppm bound more Ag per mg of protein than the mature biofilms at 2.0 ppm. This trend is a direct result of the protein content of the individual biofilms at each exposure – i.e., the protein levels increased from biofilms at the 0.5 ppm exposures to the 2.0 ppm standard and then to the mature biofilm exposures at 2.0 ppm from ~ 0.25 to 1.0 to 4.5 mg protein, respectively. The assumption that the Ag sorption to the biofilms reached equilibrium may not be valid, as the 7.5 ppm exposure biofilms had a significantly lower protein content than the mature biofilms (0.19 mg compared to 4.5 mg, respectively) yet were able to bind twice as much Ag mass. Further, the only treatments held constant Ag+ were the standard and mature exposures at 2.0 ppm, and these data are insufficient to develop a sorption model. Work is needed to establish a rigorous sorption model for Ag binding to N.europaea biofilms. The biomass-­‐associated μg Ag/mg protein versus percent nitrification data for DFR biofilm Ag+ individual experiments is plotted in (Figure 6) along with the batch Ag+ data from this study. The biofilm data has two associated regression models plotted: the first is a linear fit through 100% activity based on data points with biomass-­‐
associated Ag per protein values < 10 μg/mg; and the second is a power law fit using all of the biofilm data points. It is clear from (Figure 6) that biofilms had increased nitrification activity relative to suspended cells at similar biomass-­‐associated μg Ag/mg protein levels when exposed to Ag+ at bound Ag per mg protein values > 10 μg/mg. At values < 10 μg Ag/mg protein the slopes of the linear regression models for both the batch and DFR exposures show similar trends. However, the importance of this latter trend is unclear when interpreting the effects of biomass-­‐
associated Ag per protein on nitrification activity as the biofilm exposures were at such elevated concentrations. One possibility is that during the biofilm exposures biomass with bound Ag may have continually sloughed off, leading to lower values at the end of the experiment when the films were sampled. Finally, a significant finding of this work is also presented in (Figure 6) -­‐-­‐ the power law regression model for the entire biofilm data set approaches an asymptote, suggesting a saturation of nitrification inhibition relative to biomass-­‐associated Ag exists. This observation, coupled with the percent inhibition data from the 7.5 ppm exposure (i.e. similar to 2.0 ppm exposure), further supports the postulate that the biofilms offer protection from Ag+ toxicity via EPS, enabling them to maintain a basal metabolism at significantly higher Ag+ exposures than planktonic cells. 4.1.3 Summary The physiological results of the Ag+ exposures for the batch and DFR experiments (Figures 1 and 4) demonstrate that biofilms tolerated Ag+ exposure concentrations up to ten times that of the suspended cell batch experiments with similar percent inhibition levels. These results also show that suspended cell nitrite production rates on a per mg protein per hour basis ranged from three to ten times higher than biofilms, leading to significantly greater recovery rates at similar inhibition levels than biofilms. The Ag mass balance data for the ‘actual’ mass added [Table 2] shows that despite significant loss of Ag mass prior to cell inoculation, the biofilms were exposed to ≥ 150 times the Ag mass of suspended cells across the 3-­‐hour exposures. However the planktonic cells consistently bound a greater mass fraction of the total Ag than biofilms and demonstrated greater biomass-­‐associated Ag mass per mg protein than the biofilms at similar exposures (i.e. 0.50 ppm Ag+). On average the total percent Ag mass recovered was greater for the batch systems (except for the 7.5 ppm Ag+ DFR exposure). Finally, the bound μg Ag/mg protein data from (Figure 6) illustrates a linear regression model for the suspended cell experimental data that predicts loss of all nitrification activity at ~7.7 bound μg Ag/mg protein; whereas the power law regression model for the biofilm data suggests the percent nitrification activity tends towards some minimum asymptotic value, suggesting that even at high Ag+ some nitrification activity may be retained by the biofilms. 30 31 Wirth et al. (2012) also observed a basal metabolic rate of N.europaea biofilms at AgNP exposures up to 100 ppm, suggesting protection of the active cells by the EPS of the biofilms. Peulen and Wilkinson (2011) have also indicated the EPS of biofilms is a complex, heterogeneous matrix able to sorb charged particles as well as act as a diffusion barrier from the bulk solution. Further supporting the idea of a basal nitrification activity of the biofilms from this study is that mature biofilms were less affected at similar exposure levels than standard biofilms with less protein content, which is a proxy for biomass/EPS. 4.2 Responses to Silver Nanoparticles 4.2.1 Suspended cell batch experiments AgNP batch experiments were conducted using both DFR media as well as LM media containing reduced MgSO4. It should be mentioned that all previous experiments with Ag+ were conducted with DFR media and the LM media was only introduced to the AgNP batch experiments to reduce agglomeration of AgNP by divalent cations as shown by Chinnapongse et al. (2011). In preliminary work using DFR media large aggregates were visible as suspended black particles that formed after the addition of AgNP into the media and prior to the addition of N.europaea cells, corroborating the effects of divalent cations observed in Chinnapongse et al. (2011). Given the composition of the media (i.e., inclusion of Cl-­‐ salts as well as divalent cations), these particulates are likely composed of various AgCl compounds, Ag+, and sequestered AgNP. (Chinnapongse et al. 2011; Carlson et al., 2008; Choi and Hu 2008) In a similar study by Choi et al. (2010) comparing the toxicity of planktonic cells and biofilms of E.coli in nutrient media using microrespirometry when exposed to 20 nm AgNP, a 15X increase in particle size via AgNP aggregation due to media constituents was observed. In our study these large aggregates formed to a much greater size and extent in the DFR media but were still visible in the LM media. When AgNP agglomerate/aggregate their toxicity can decrease by a significant 32 reduction in available surface area for Ag+ dissolution as well as reaching sufficient density to precipitate out of solution (Carlson et al., 2008; Choi and Hu 2008). Results for AgNP suspended cell batch experiment nitrite production rates and corresponding percent nitrification inhibition are shown in (Figure 7). Nitrite production rates for both the DFR and LM media controls display no significant difference in their average values from one another nor from the Ag+ batch experiment controls (Figures. 7a,b and 1a), indicating consistency regardless of media used for tests. It is evident that AgNP treatments in the LM media decreased nitrite production to a greater extent than in the DFR media. However nitrite production rates for all batch experiments, except the 20 ppm AgNP treatment, increased approximately linearly across the 3-­‐hour inhibition. Also shown in (Figure 7b) is the significant reduction of nitrite production at 20 ppm AgNP in the LM media, similar to the effect of 0.5 ppm Ag+ on suspended cells (Figure 1a). These results suggest a threshold dosage for the LM media experimental conditions at AgNP concentrations >10 ppm. The percent inhibition values (Figure 7c) for both the 1.0 and 4.0 ppm AgNP treatments in DFR media are ≤1.0 ppm AgNP treatment in LM media, indicating an increased inhibition in LM media. This slightly increased inhibition is likely due to a slight reduction in aggregation of AgNP as a result of reduced Mg2+ in the LM media. The final percent inhibition among the LM media experiments follows a nearly exponential trend reaching a value of approximately 85% at 20 ppm AgNP. These findings deviate significantly from the percent inhibition results in Radniecki et al. (2011) as they demonstrated approximately 90% inhibition at 0.7 ppm AgNP compared with 85% at 20 ppm AgNP in this study. The AgNP batch experiments in Radniecki et al. (2011) used experimental media containing only HEPES (pH 7.8) and (NH4)2SO42-­‐, indicating a significant effect of media composition, specifically divalent cations and chloride, on AgNP nitrification inhibition in batch experiments involving N.europaea. Again, this is due to aggregation of the AgNP caused by the divalent cations and AgCl formation that has been shown to sequester AgNP into 33 particulates – this is supported by findings of previous studies using similar media to the LM media used in this study. (Chinnapongse et al. 2011; Carlson et al., 2008; Choi and Hu 2008) Metal analysis of Ag for the AgNP batch experiments is shown in [Table 3] for both the DFR and LM media at 1.0 and 20.0 ppm AgNP, respectively. For the DFR media batch tests the initial Ag mass measured, prior to the addition of cells, was 8.94 μg, at ~30% of the nominal concentration. The suspended and biomass associated Ag were approximately equal and added to a total mass of 4.79 μg – i.e. 54% of the initial measured Ag mass. For the LM media batch tests an initial Ag mass of 172 μg was observed at ~30% of the nominal Ag mass. The majority of the Ag recovered was present as suspended Ag at 186 μg, with 14 μg as biomass-­‐associated Ag. The total Ag mass recovered for the LM media tests was 275 μg at 116% of the initial measured Ag mass. This increase in the recovered mass from the initial Ag mass is likely due to the formation of large aggregates leading to sampling bias when pipetting volumes for ICP-­‐OES analysis, which is consistent with the observed black particulate formation. Examining the biomass-­‐associated Ag mass per mg protein [Table 3], there was roughly a six-­‐fold increase at the 20 ppm exposure in the LM media relative to the 1 ppm exposure in DFR media. These data from the 20 ppm AgNP exposure may over-­‐predict the actual silver associated with biomass as it is likely that larger, more dense silver particulates co-­‐pelleted with the biomass during centrifugation. The percent nitrification activity versus the mass of Ag associated per mg of protein is shown in (Figure 8). Two distinct clusters for the 1.0 and 20.0 ppm AgNP treatments from this study are shown along with data from the previous study (Radniecki et al. 2011). The regression model fit to the data from Radniecki et al. (2011) implies that at ~14 μg Ag/mg protein nitrification activity should be completely inhibited. However, the results from this study demonstrate levels of nitrification activity inhibition of ~80 – 95% begin to occur at levels >60 μg Ag/mg protein. Also to be noted for both the DFR and LM media results from this study in 34 (Figure 8) is the variance of μg Ag/mg protein among the 1.0 and 20.0 ppm clusters. This phenomenon is likely due to the wide range of larger aggregates, which would account for the variable Ag masses associated with the biomass. Finally, the absence of complete inhibition of nitrification activity of the 20 ppm treatments in this study relative to Radniecki (2011) suggests reduced exposure of the cells to Ag+ from a possible reduction in AgNP surface area for dissolution, which is related to the different media used in the two studies – i.e., the media in this study contained divalent cations and Cl-­‐; whereas that in Radniecki et al. (2011) contained only HEPES buffer and ammonium sulfate. UV-­‐Vis spectroscopy was used to characterize the extent of agglomeration and dissolution of AgNP by exploiting the localized surface plasmon resonance (LSPR) as promulgated by MacCuspie et al. (2011). Control UV-­‐Vis data for AgNP suspended in HEPES (pH 7.8) from 2.0 – 20.0 ppm with no cells are presented in (Figure 9a) as absorbance intensity versus wavelength. A maximum absorbance (i.e., λmax) was identified at 400 nm, consistent with previous studies (Petit 1993; Kong and Jang 2006; Zook 2011). A standard curve was generated of AgNP concentration versus absorbance intensity at λmax to quantify the experimental results (Figure 9b), showing an excellent fit was achieved. UV-­‐Vis data from batch experiments at 1.0 ppm AgNP in DFR media are shown in (Figure 10). Control data containing only media and AgNP without cells are shown in (Figure 10a) and are scaled to observe the behavior of the spectra in the range of the intensity equal to 1.0 ppm AgNP (i.e., ~0.10 absorbance intensity from Figure 9b). These spectra show a significant absorbance peak at ~300 nm that has not been previously described in the literature. The spectra in (Figure 10a) indicate that at t = 0 hour (i.e. after AgNP were added to the media but prior to inoculation with N.europaea cells) there exists a very broad absorbance at longer wavelengths and small but distinct peaks at ~360 and 610 nm. The broad absorbance at longer wavelengths suggests much larger aggregates (Chinnapongse et al. 2011), which agrees well with the observed black particulate formation in the test bottles prior to 35 the addition of cells. The peak at 360 nm is likely due to nitrite, which absorbs at 280 and 360 nm. The remaining spectra from 1 -­‐ 3 hours follow no consistent trend with λmax absorbance dropping from hour 1 to 2 and then increasing again from 2 to 3. These results illustrate potential sampling bias when pipetting the sample volume from the test bottles, which would be a direct result of larger aggregate formation and precipitation from solution. The UV-­‐Vis data for the DFR media AgNP batch experiments at 1.0 ppm AgNP (Figure 10b) shows nearly identical results at t = 0 hours relative to the control data, but significantly different spectra for the 3-­‐hour exposure after the cells were added. Here the intensity axis is scaled to capture the behavior of the spectra during the 3-­‐
hour exposure. The addition of the cells introduced a significant interference peak not observed in the controls that experiences a red-­‐shift (i.e. the peak migrates to longer wavelengths) during the exposure period and ranges from ~320 – 330 nm. It has been shown that AgNP in media containing no interfering materials tend to red-­‐
shift as they increase in size. However, the peaks observed in (Figure 10b) have not been previously described in the literature and were only present in solutions containing media, cells, and AgNP; beyond concluding they are a direct result of interactions with the N.europaea cells or their associated biomass, further investigation is needed to determine the exact phenomena responsible. These spectra also include the nitrite peak at 360 nm and show that it increased during the exposure, consistent with the nitrification activity observed in (Figure 7a). Also, the broad absorbance from the exposure spectra reveal intensities at λmax well above those correlated to 1.0 ppm (i.e., ~0.10) due to the significant intensities observed at 320 – 330 nm and subsequent peak tailing. The UV-­‐Vis data for the LM media batch experiments at 20.0 ppm AgNP is shown in (Figure 11). The control data (Figure 11a) shows similar small peaks at ~300, 360, and 620 nm to the DFR media controls (Figure 10a), but little to no broad absorbance at higher wavelengths suggesting minimal formation of larger particles. The LM media spectra were also more consistent across time with absorbance 36 intensity values remaining near 0.10 indicating greater stability of AgNP, but at significantly lower than expected values ranging from ~0.9 -­‐1.3 ppm, due to dissolution or removal by flocculation. The experimental results for the LM media tests (Figure 11b) across the 3-­‐hour exposure are nearly identical to the DFR media results (Figure 10b). Again, there are large interference peaks ranging from ~300 – 316 nm that experience a red-­‐shift through time and broad absorbances at higher wavelengths. The spectra indicate AgNP concentrations >1.0 ppm at λmax, but may be an artifact of the tailing from the peaks at 300 – 316 nm. These experimental results in (Figure 10b and 11b) indicate the presence of larger aggregates in both the 1.0 ppm DFR media and 20.0 ppm LM media treatments, but are currently unable to neither accurately describe the agglomeration state of the AgNP nor quantitatively predict AgNP concentration during the 3-­‐hour exposures. The presence of divalent cations and chloride in the media used in this study clearly affected the aggregation state of the AgNP as observed by black particulate formation and broad absorbance at higher wavelengths in the UV-­‐Vis spectra. This range of large aggregate formation potentially causing sampling bias that affected both UV-­‐Vis and ICP-­‐OES measurements. The aggregation and particulate formation is also responsible for the reduced inhibition observed in this study by reducing the available suspended Ag+ from the AgNP. The findings related to nitrification activity from the batch AgNP experiments of this study differed significantly from those of the previous study by Radniecki et al. (2011), indicating that media composition plays a critical role in determining the affects of AgNP on nitrification by N.europaea. 4.2.2 Drip Flow Reactor biofilm experiments Biofilms were inoculated and grown using LM media for the AgNP experiments and the 3-­‐hour AgNP exposures were conducted using either LM media or HEPES (pH 7.8) only as the injection syringe solution containing the AgNP. As in the batch experiments, these solutions were chosen to minimize aggregation by reducing the amount of divalent cations in the media. Further complicating these experiments was the necessity for the syringe injection solutions to contain 10X the desired exposure AgNP concentration due to dilution by the media feed stream – approximately 200 ppm depending on flow rates. At these concentrations immediate formation of black precipitates were observed when the AgNP were dispersed into the LM media syringe solution but not in the HEPES-­‐only solution. However, after both experiments the treated biofilms were covered in black precipitates, suggesting the biofilms increased aggregation and precipitation of AgNP (Figure 12). These results are consistent with observations in Choi et al. (2010) that demonstrated a 40X increase in particle size after exposure of E.coli biofilms in nutrient broth to 20 nm AgNP. Nitrification production rates and percent inhibition for the AgNP exposures are shown in (Figure 13). The production rates for both the controls and exposures using either the LM media or HEPES-­‐only syringe solutions are presented in (Figure 13a). The results indicate a slight reduction in the HEPES-­‐only controls due to the ~10% dilution of the growth media to the biofilms by the injection volume into the feed stream. The treatments using the LM media as syringe solution demonstrated slightly more consistent production rates relative to the HEPES-­‐only syringe solution treatments. Again this is likely an artifact from the dilution of the growth media confounding any effects solely from the AgNP. The corresponding percent inhibition data (Figure 13b) illustrates no significant difference between the treatments, with both at ~5% inhibition after the 3-­‐hour exposures. Clearly aggregation of the AgNP from mixing with the LM media after injection into the feed stream, along with the increased protection of the active cells in the biofilm by the EPS, contributed to significantly reducing Ag+ dissolution and access to active cells; thus accounting for the decrease in percent inhibition at 20 ppm AgNP from 80% in the batch tests to 5% in the biofilm tests for the same test media. It is difficult to compare the results of this study with previous work on AgNP toxicity and biofilms as multiple culturing, exposure, and monitoring methods have been employed. Thus there is no consistent metric that can be used to assess 37 38 physiological effects. Fabrega et al. (2009 and 2011) used both pure culture (Pseudomonas putida) and mixed culture (from marine sediments) biofilms grown in flow cell reactors and found significant sloughing of biofilm material during the first four hours of exposure. Lacking any physiological assays they used the ratio of biofilm volume to surface area to determine the effects of AgNP exposure. In this study sloughing of biofilm material was observed during the first 30 minutes of the exposures but clearly had little effect on nitrite production, indicating that sloughing is a poor measure of biofilm productivity. Wirth et al. (2012) exposed Pseudomonas fluorescens grown in microplates in 200 μL of Minimal Davis Media (containing ~400 μm Mg2+) to ~20 nm AgNP and measured toxicity as a function of viable cell loss using a BacLight Live/Dead staining kit. They demonstrated an ~60% decrease in cellular viability at 20 ppm AgNP. These results are surprising given that Wirth et al. (2012) observed significant aggregation (from ~20 to ~300 nm) and sedimentation of nanoparticles. This discrepancy may be due to the significantly decreased biomass of the biofilm cultures relative to this current study. Choi et al. (2010) cultured E.coli biofilms in microplates in 200 μL of nutrient broth, exposed them to ~20 nm AgNP, and measured an active cellular fraction using microrespirometry. Their results showed only ~10% active cell fraction at 20 ppm AgNP exposure. Despite the increased accuracy of biofilm productivity utilizing a physiological assay, the significantly reduced biomass relative to the current study may have been responsible for the considerable disparity observed in toxicity. Therefore findings of this study in comparison with previous works demonstrate the importance of selecting culturing methodology, media composition, total biomass, and assay selection in placing the results of biofilm inhibition upon exposure to AgNP. Ag mass data collected from the AgNP biofilm experiments is shown in [Table 3] and Ag breakthrough curves for both control (no biofilm) and the 20 ppm AgNP treatment with HEPES only syringe solution are shown in (Figure 14). As mentioned previously the immediate formation of black precipitates in the syringe solution comprised of the low ionic strength DFR media is responsible for the significant 39 reduction (~77%) in Ag mass added from the calculated target concentration as measured from the initial concentration of the syringe solution. [Table 3] This corresponds well with the lack of observed precipitates in the HEPES-­‐only syringe solution that led to only a 26% reduction in the initial measured mass of Ag added from nominal concentration. The abundance of black precipitates in the LM media syringe solution treatments also likely caused variations in the effluent Ag mass that possibly accounts for the suspended Ag mass found to be higher than the Ag mass input into the system. For the HEPES-­‐only syringe solution treatments the majority of Ag mass recovered (~88%) was found suspended and collected in the effluent. [Table 3] As mentioned previously the biofilms from both treatments contained a significant amount of associated black precipitates (Figure 12) and this is reflected in reasonably close values of biomass-­‐associated Ag mass for both treatments. [Table 3] The biomass-­‐associated Ag for both the batch and biofilm AgNP exposures is greater at all levels than the Ag+ exposures, which also corresponds well with the black particulate formation increasing the Ag mass in this fraction. The aggregation and precipitation of AgNP in the LM media syringe solution resulted in the Ag mass recovery of 116% and must be taken into account when interpreting the results and utility of these experiments. The HEPES-­‐only syringe solution treatments offered an experimental setup that did not allow aggregation to occur in the injection syringe. With this method of addition we could account for nearly half of the Ag mass added to the system. [Table 3] The breakthrough curves in (Figure 14) show the control (i.e., containing no biofilm but a frosted glass slide) and treatments; the average value of the HEPES-­‐only syringe initial concentration (dashed orange line at ~15 ppm Ag) is also indicated. The control breakthrough curve shows concentration values reached ~13 ppm Ag after the first hour and remained relatively constant until the end of the 3-­‐hour exposure, demonstrating minimal sorption or aggregation/precipitation from solution in the absence of a biofilm. The HEPES-­‐only syringe solution treatments 40 with a biofilm reached a steady concentration of ~7 ppm Ag after the first hour with minimal variation, indicating less breakthrough of Ag compared to the control. The LM media syringe solution treatments reached a maximum effluent concentration of ~5 ppm Ag and declined to a final value of ~2.5 ppm and was more variable than the either the control or HEPES-­‐only treatment, indicating AgNP aggregation and precipitation from solution over the course of the exposure. These observations re-­‐
iterate the variability and subsequent lack of utility of the LM media syringe solution due to the significant black particulate formation. They also demonstrate that in the treatments using HEPEs-­‐only syringe solution ~50% of the Ag in the control effluent was lost during the exposures. The percent activity versus biomass-­‐associated Ag per mg protein for the biofilm AgNP experiments is shown, along with the batch AgNP results from this study, in (Figure 15). The biomass is represented by a digest of the entire biofilms. The small number of samples is unable to capture any significant trends between treatments of the biofilms aside from an increase in μg biomass-­‐associated Ag per mg protein for the HEPES-­‐only treatments at similar percent activity levels relative to the LM media treatments; this effect must be related to the reduced Ag mass in the effluent across the exposure period in the LM media treatments. The reduced values of biomass-­‐associated Ag per protein for the biofilm exposures at 20 ppm AgNP versus the batch exposures at 20 ppm is likely due to co-­‐pelleted silver containing black particulates present in the batch cell digests, leading to a significant over-­‐prediction of the values in [Table 3] and data points on (Figure 15). UV-­‐Vis absorbance data for the DFR effluent collected during the 3-­‐hour exposure AgNP periods is shown in (Figure 16). Due to the significant particulate formation and precipitation in the LM media syringe solution treatments, only the effluent from the controls and HEPES-­‐only syringe solution treatments were analyzed by UV-­‐Vis spectroscopy (Figure 16). Spectra from four data sets are shown in (Figure 16a,b): Pre-­‐20X are the pre-­‐exposure syringe solutions diluted 20X; Uncentrifuged are the effluent samples; Centrifuged are the effluent samples after centrifugation 41 for 1 minute to remove cell-­‐associated interference; and Post-­‐20X are the post-­‐
exposure syringe solutions diluted 20X. The spectra for the pre-­‐ and post-­‐exposures for both the control (i.e. containing no biofilms) and treatments show peaks at 400 nm with no tailing or elevated background at higher wavelengths, similar to the HEPES controls (Figure 9a). Interestingly the post-­‐exposure absorbance was slightly greater than the pre-­‐
exposure values, and this was consistent for both the controls and treatments. One possible explanation is that a small amount of AgNP settled from solution in the syringes during the exposures leading to a slight AgNP concentration increase in the post-­‐exposure samples. Also in both the control and treatment data the absorbance of the pre-­‐ and post-­‐exposures, taken as 20X diluted samples from the injection syringes, ranged from 1.4 – 1.6, indicating pre-­‐ and post-­‐exposure syringe solution values of ~26 and 29 ppm, respectively (via the regression model from Figure 9b. These UV-­‐Vis values for the pre-­‐exposure syringe AgNP concentrations are nearly twice that measured by ICP-­‐OES (i.e., approximately 15 ppm Ag as seen in (Figure 14)). Due to the sensitivity of the ICP-­‐OES method and minimal interferences with the corresponding Ag analyses, these results should be used rather than the UV-­‐Vis data as the measured pre-­‐exposure syringe Ag mass. However the UV-­‐Vis data is useful in illustrating that the pre-­‐ and post-­‐exposure syringe Ag mass was composed primarily of 20 nm AgNP; thus demonstrating consistency in nanoparticle size mixing with the growth media and entering the DFR throughout the 3-­‐hour exposure during the 20 ppm AgNP treatments. Control data is shown in (Figure 16a) and reveal a decrease in λmax absorbance across the 3-­‐hour exposures, as well as broad absorbance at higher wavelengths. These results suggest loss of 20 nm AgNP with time along with the formation of aggregates (Chinnapongse et al. 2011), respectively. Because LM media was used as growth media for these experiments, it is likely the divalent cations in solution were responsible for the observed aggregation. However it is unclear from this data if the 42 reduction in 20 nm AgNP is due entirely to the formation of the larger aggregates, or if particle dissolution also plays a role. The HEPES-­‐only syringe solution treatment data (Figure 16b) contain spectra from both the uncentrifuged and centrifuged effluent samples. It is clear from the uncentrifuged samples that significant reduction in 20 nm AgNP occurred and larger precipitates formed during the 3-­‐hour exposures given the broad absorbance at higher wavelengths. This reduction of absorbance intensities at λmax is likely to due aggregation and dissolution induced by the biofilms, as can be inferred from comparison with the control data. The spectra from (Figure 16b) also indicate minimal variation in absorbance intensities among both the centrifuged and uncentrifuged samples. It is also observed that centrifugation of the samples removed all particles with an absorbance range 400 – 700 nm, further demonstrating the large size of the particulates formed during the exposure. Utilizing the λmax absorbance intensities (Figure 16) and the regression model from (Figure 9b), AgNP concentrations for the control and HEPES treatments, as well as the pre-­‐exposure syringe HEPES solution, were calculated and are presented in (Figure 17). The dashed orange line represents the average pre-­‐exposure syringe solution AgNP concentration and, as mentioned previously, suggests all Ag mass present in the syringe solution was present as 20 nm AgNP. The results for the control and treatments show that ~75-­‐80% of the pre-­‐exposure syringe 20 nm AgNP concentration was lost during the control experiments, and ~93% was lost during the 20 ppm treatment experiments, as observed by the uncentrifuged data. This suggests a small, but significant effect of the biofilms in reducing the 20 nm AgNP in solution. Again, these data only represent the AgNP concentration in the effluent not the entire Ag mass as was represented in the breakthrough curve in (Figure 14). A comparison of the effluent values between these figures demonstrates roughly a third of both the control and treatment effluent was present as 20 nm AgNP. It should be noted, as previously discussed, the UV-­‐Vis regression model significantly over-­‐predicted (nearly double) the Ag concentration in the syringe solution as compared with the ICP-­‐OES data so caution must be taken in assuming the values shown in (Figure 17). However despite the possible inconsistency with the 20 nm AgNP mass, the uncentrifuged data (Figure 17) does suggest that a fraction (i.e., < 30%) of 20 nm AgNP were able to make it through the DFR unaltered, but the centrifuged data shows nearly all of the 20 nm AgNP have been removed or agglomerated during their transport through the DFR. In comparing the control and uncentrifuged treatments in (Figure 17), nearly two thirds of the 20 nm AgNP present in the controls were lost in the treatment, suggesting a significant effect of the biofilms in either precipitating/adsorbing or inducing aggregation of the AgNP. Reconciling these results in regards to the fate and transport of unaltered AgNP in engineered systems is difficult and highly dependent on flow regimes and mixing phenomena. 4.2.3 Summary The results from these experiments echo the critical dependence on media selection as shown in the AgNP batch experiments. The immediate particle formation and precipitation observed when the AgNP were dispersed into the LM media syringe solution and the subsequent inconsistencies in measuring suspended Ag mass demonstrated the requirement for the HEPES-­‐only syringe solution to maintain consistency of original particle size and reliably run repeatable experiments. Furthermore, the growth media constraint of the biofilms, requiring at least the LM media, ensured sufficient levels of cations to induce aggregation of larger particles in the control samples that lead to a reduction in 20 nm AgNP as observed from the UV-­‐Vis spectra (Figures 13a and 14). However, these samples maintained suspended Ag mass levels just below the pre-­‐exposure syringe values from (Figure 11) and [Table 3]. Despite the significant loss of 20 nm AgNP from the system observed in the control experiments, the HEPES-­‐only treatments illustrated significant removal of the 20 nm AgNP relative to the controls with increased larger 43 44 particles, but failed to account for nearly 50% of the Ag mass. Given the previously described diffusion limitations within biofilms from Puelen and Wilkinson (2011), as well as their finding that virtually no spherical nanoparticles >50 nm were able to penetrate a P.fluorescens biofilm, it is expected that little to none of the AgNP penetrated into the biofilm. This is reasonable given the UV-­‐Vis data and visual confirmation of black precipitates coupled with the observations that under similar media conditions Choi et al. (2010) found AgNP size increased from 20 to 800 nm after 4 hours. Thus, AgNP toxicity is highly dependent on particle size and in this study that aggregation and low rates of Ag-­‐NP dissolution were likely responsible for the high nitrification activity being maintained mmol NO2-­‐/mg protein 45 0.4 Control 0.05 ppm 0.10 ppm 0.25 ppm 0.50 ppm 0.75 ppm 0.3 0.2 0.1 0 0 1 Time (hr) 2 3 (a) % Inhibition 100 80 0.05 ppm 60 0.10 ppm 40 0.25 ppm 0.50 ppm 20 0.75 ppm 0 0 1 Time (hr) 2 3 (b) 0.05 ppm 80 0.10 ppm % Activity 100 0.25 ppm 60 0.50 ppm 40 0.75 ppm 20 0 0 1 2 3 4 Time (days) 5 6 (c) Figure 1: Nitrite production rates (a) of N.europaea suspended cell batch silver ion experiments during 3-­‐hour exposure periods. Percent inhibition of nitrification (b) for the exposure period and percent nitrification activity (c) for the recovery period is shown. Error bars represent 95% confidence intervals. 46 100 This study % Activity 80 Radniecki 2011 60 40 20 0 0.05 0.10 Ag+ (ppm) (a) 100 This study % Activity 80 Radniecki 2011 60 40 20 0 0.05 0.10 Ag+ (ppm) (b) Figure 2: Percent nitrification activity from 3-­‐hour batch exposures (a) and 3-­‐hour recovery data (b) from this study and Radniecki et al. (2011). Data from Radniecki et al. is from experiments run at 0.05 and 0.09 ppm. Error bars represent 95% confidence intervals. 47 100 Batch (this study) % Nitri-ication Activity 80 Batch (Radniecki 2011) 60 y = -­‐5.4455x + 100 R² = 0.88189 40 y = -­‐12.832x + 100 R² = 0.97294 20 0 0 2 4 6 8 10 12 14 µg Ag/mg protein Figure 3: Biomass-­‐associated silver versus percent nitrification activity from 3-­‐hour silver ion exposure batch experiments. Data from this study are presented along with data from the previous Radniecki et al. (2011) study. Regression models forced through 100 percent activity are shown with their corresponding R2 value. 48 0.06 0.05 mmol NO2-­‐/hr Control 0.04 0.5 ppm 0.03 1.0 ppm 0.02 2.0 ppm 0.01 7.5 ppm 0.00 2.0 ppm** 0 1 2 Time (hr) 3 (a) 0.5 ppm 80 1.0 ppm 60 2.0 ppm % Inhibi(on 100 7.5 ppm 40 2.0 ppm** 20 0 0 1 Time (hr) 2 3 (b) 100 1st Exposure % Activity 80 2nd Exposure 60 40 20 0 0 2 4 6 Time (days) 8 10 (c) Figure 4: Nitrite production rates (a) of N.europaea DFR biofilm experiments during 3-­‐hour silver ion exposure periods. Percent inhibition of nitrification (b) for the exposure period that includes results from biofilms at 2.0 ppm is shown. Recovery data were collected for a mature biofilm with multiple exposures at 2.0 ppm Ag+ with subsequent recovery periods and percent nitrification activity (c) for these recovery periods is shown. Error bars represent 95% confidence intervals. 49 1.0 3 2 Control -­‐ 2.0 ppm 0.6 0.5 ppm 2.0 ppm 0.4 2.0 ppm** 1 7.5 ppm Ag (ppm) Ag (ppm) 0.8 0.2 0.0 0 0 1 2 3 Time (hr) Figure 5: Silver concentration in the DFR effluent from 3-­‐hour silver ion exposures. Control data (no biofilm) and 0.5 and 2.0 ppm experimental results correspond to the primary Ag concentration axis on the left, while 7.5 ppm exposures correspond to the secondary Ag secondary axis on the right (see arrows). The (**) represents exposures performed on mature biofilms at 2.0 ppm Ag+. Error bars represent 95% confidence intervals. 50 100 y = -­‐10.146x + 100 R² = 0.56622 % Nitri-ication Activity 80 Batch (this study) DFR 60 y = 81.958x-­‐0.305 R² = 0.9363 40 20 y = -­‐12.832x + 100 R² = 0.97294 0 0 10 20 30 40 50 60 µg Ag/mg protein Figure 6: Biomass-­‐associated silver versus percent nitrification activity from 3-­‐hour silver ion exposure batch and DFR experiments. Regression models fit through 100 percent activity are shown with their corresponding R2 values – two models are shown for the DFR data: a linear regression (dashed line) for data with < 10 μg/mg protein and a power law regression (solid line) for all DFR data points. 51 mmol NO2-­‐/mg protein 0.5 Control -­‐ DFR 0.4 1.0 ppm DFR 0.3 4.0 ppm DFR 0.2 0.1 0 0 1 2 3 Time (hr) 2 3 Time (hr) (a) mmol NO2-­‐/mg protein 0.5 Control -­‐ Low Mg 0.4 1.0 ppm Low Mg 10 ppm Low Mg 0.3 20 ppm Low Mg 0.2 0.1 0 0 1 (b) 100 % Inhibition 80 1.0 ppm -­‐ DFR 4.0 ppm -­‐ DFR 60 1.0 ppm -­‐ Low Mg 10 ppm -­‐ Low Mg 40 20 ppm -­‐ Low Mg 20 0 0 1 Time (hr) 2 3 (c) Figure 7: Nitrite production rates for batch experiments in DFR (a) and low Mg2+ (b) media as well as percent inhibition of nitrification activity (c) from N.europaea silver nanoparticle experiments during 3-­‐hour exposure periods. Error bars represent 95% confidence intervals. 52 100 Batch (this study) % Nitri-ication Activity 80 Batch (Radniecki 2011) 60 y = -­‐5.7803x + 100 R² = 0.78636 20 ppm AgNP Low Mg 40 20 0 0 10 20 30 40 50 60 70 80 90 100 µg Ag/mg protein Figure 8: Biomass-­‐associated silver versus percent nitrification activity from 3-­‐hour exposure batch silver nanoparticle experiments. Data from batch experiments from this study using DFR media and low Mg2+ media are presented along with data from the previous Radniecki et al. (2011) study. Regression model trendline fit through 100 percent activity is shown for data from Radniecki et al. (2011) 53 2.5 20 ppm Intensity 2 10 ppm 5 ppm 1.5 2 ppm 1 0.5 0 300 400 500 600 700 Wavelength (nm) (a) AgNP Concentration (ppm) 25 y = 9.2061x R² = 0.98572 20 15 10 5 0 0 0.5 1 1.5 2 2.5 Intensity (b) Figure 9: UV-­‐Vis spectrophotometer data (a) from silver nanoparticles in HEPES buffer (pH 7.8). The standard curve for absorbance at λmax (400 nm) is shown (b) along with the best fit regression fit through the origin. 54 0.2 0 hr 1 hr Intensity 0.15 2 hr 3 hr 0.1 0.05 0 300 400 500 600 700 Wavelength (nm) (a) 2.5 0 hr Intensity 2 0.75 hr 1.5 hr 1.5 2.25 hr 3 hr 1 0.5 0 300 400 500 600 700 Wavelength (nm) (b) Figure 10: UV-­‐Vis spectrophotometer data from batch silver nanoparticle experiments in DFR media and 1.0 ppm AgNP for controls containing no cells (a) and 3-­‐hour exposures (b). 55 0.2 0 hr 1 hr Intensity 0.15 2 hr 3 hr 0.1 0.05 0 300 400 500 600 700 Wavelength (nm) (a) 2.5 0 hr 0.75 hr 2 Intensity 1.5 hr 1.5 2.25 hr 3 hr 1 0.5 0 300 400 500 600 700 Wavelength (nm) (b) Figure 11: UV-­‐Vis spectrophotometer data from batch silver nanoparticle experiments in low Mg2+ media and 20 ppm AgNP for controls containing no cells (a) and 3-­‐hour exposures (b). 56 (a)
(b) Figure 12: N.europaea biofilms with no Ag+ or silver nanoparticle exposure (a) and with 3-­‐hour 20 ppm silver nanoparticle exposure and HEPES only as the syringe solution (b). The dimensions of the slides are 75 mm by 25 mm. 57 0.045 mmol NO2-­‐/hr 0.035 0.025 Control -­‐ Low Mg 20 ppm Low Mg 0.015 Control -­‐ HEPES 20 ppm HEPES 0.005 0 1 2 3 Time (hr) (a) 100 20 ppm -­‐ Low Mg 80 % Inhibition 20 ppm -­‐ HEPES 60 40 20 0 0 1 2 3 Time (hr) (b) Figure 13: Nitrite production rates (a) and percent inhibition (b) of nitrification activity of N.europaea DFR biofilm silver nanoparticle experiments during 3-­‐hour exposure periods. Both low Mg2+ media and HEPES (pH 7.8) were used as the syringe AgNP injection solution. Error bars indicate 95% confidence intervals. 58 20 Ag (ppm) 15 Control -­‐ HEPES 10 Bio]ilm -­‐ Low Mg Bio]ilm -­‐ HEPES 5 Syringe -­‐ Initial 0 0 1 2 3 Time (hr) Figure 14: Silver effluent concentrations for 20 ppm silver nanoparticle DFR biofilm 3-­‐hour exposures. Results are shown for HEPES only AgNP syringe solution controls containing no biofilms, low Mg2+ media AgNP syringe solution, and HEPES only AgNP syringe solution. The initial Ag concentration of the injection syringe is also shown. Error bars represent 95% confidence intervals 59 100 80 % Nitri.ication Activity Batch (this study) HEPES only DFR Low Mg 60 20 ppm AgNP Low Mg 40 20 0 0 10 20 30 40 50 60 70 80 90 100 µg Ag/mg protein Figure 15: Biomass-­‐associated silver versus percent nitrification activity from 3-­‐hour exposure batch and DFR silver nanoparticle experiments. Data from batch experiments from this study using DFR media and low Mg2+ media are presented along with data from DFR experiments using both low Mg2+ media or HEPES (pH 7.8) only syringe solutions. 60 2 Pre-­‐20X 0.5 hr 1.6 Intensity 1.0 hr 1.2 1.5 hr 2.0 hr 0.8 3.0 hr Post-­‐20X 0.4 0 300 400 500 600 700 Wavelength (nm) (a) 2 Pre -­‐ 20X Intensity 1.6 Uncentrifuged Centrifuged 1.2 Post -­‐ 20X 0.8 0.4 0 300 400 500 600 700 Wavelength (nm) (b) Figure 16: UV-­‐Vis spectrophotometer data from DFR biofilm silver nanoparticle experiments. Control data containing no cells with only HEPES (pH 7.8) in the injection syringe solution and low Mg2+ growth media is shown in (a). The pre-­‐ and post-­‐experiment syringe data at 20X dilutions are shown along with effluent samples collected during the 3-­‐hour exposure period. UV-­‐Vis data from (b) the HEPES only syringe solution experiments with average effluent data shown for both centrifuged and uncentrifuged samples collected at 1, 2, and 3 hours. 61 30 AgNP (ppm) 25 Control Uncentrifuged 20 Centrifuged 15 10 5 0 1 2 3 Time (hr) Figure 17: Silver nanoparticle concentrations from UV-­‐Vis data for DFR biofilm effluent collected during the 3-­‐hour 20 ppm silver nanoparticles exposures with HEPES only as the syringe solution. The dashed orange line indicates the average initial AgNP concentration in the injection syringes. Bar graphs are shown for the controls (no biofilms) and the treatment effluent samples, both centrifuged and uncentrifuged. Error bars represent 95% confidence intervals. 62 Table 1: Average total protein mass from both DFR biofilm and batch suspended cell experiments – 95% confidence intervals in parentheses. (a) Mature biofilms. Protein (mg) DFR Batch 1.1 (±0.545) 4.8a (±0.850) 0.18 (±0.016) 63 Table 2: Silver mass data from all silver ion experiments performed under all conditions. Mass added is given as the nominal (a) mass of silver given the target concentration and the volume of the batch experiments or injected during the DFR experiments, which is 3.8 ml for all tests; whereas the actual (b) mass was measured at the end of the experiment in either control bottles for batch tests or with the remaining volume present in the syringes after exposure periods for the biofilm tests. The final mass is divided into suspended (c) and biomass (d) components, which is the mass present in either batch solution/cumulative DFR effluent or that associated with the digested cellular material, respectively. Ag/protein (e) is the Ag mass associated with cells normalized to the protein content and is given in ug of Ag per mg of protein. Data from mature biofilm experiments (f) at 2.0 ppm are also shown. Ag+ (ppm) Nominala Actualb Suspendedc Boundd (ug/mg) (ug) Recovered 0.05 1.5 0.89 0.10 0.66 3.5 0.76 85 0.10 3.0 2.40 0.24 1.20 6.4 1.44 60 0.25 7.5 6.64 0.48 1.71 7.4 2.19 33 0.50 15.0 8.21 1.24 1.32 7.0 2.56 31 0.5 19 18 1.47 1.30 5.3 2.77 15 2.0 76 35 1.57 4.33 3.6 5.90 17 30f 0.84 4.74 1.0 5.58 16 7.5 138 90.13 8.98 48.6 99.11 72 Batch DFR Mass Added (ug) 285 Final Mass (ug) Ag/proteine Total Mass % Mass 64 Table 3: Silver mass data from all silver nanoparticle experiments performed under all conditions. Mass added is given as the nominal (a) mass of silver given the target concentration and the volume of the batch experiments or injected during the DFR experiments, which is 3.8 ml for all tests; whereas the actual (b) mass was measured at the end of the experiment in either control bottles for batch tests or with the remaining volume present in the syringes after exposure periods for the biofilm tests. The final mass is divided into suspended (c) and biomass (d) components, which is the mass present in either batch solution/cumulative DFR effluent or that associated with the digested cellular material, respectively. Ag/protein (e) is the Ag mass associated with cells normalized to the protein content and is given in ug of Ag per mg of protein. Here data from the batch experiments with DFR (f) and low Mg2+ media (g), as well as data from DFR biofilm experiments in low ionic strength media with both low Mg2+ media (h) and HEPES only (i) syringe solution are shown. AgNP (ppm) Nominala Measuredb Suspendedc Biomassd (ug/mg) (ug) Recovered 1f 30 8.94 2.51 2.28 12.0 4.79 54 20g 600 172 186 14.00 74.0 275 160 DFR 20 760 175h 180 22.85 10.4 203 116 564i 270 31.55 17.8 302 54 Batch Mass Added (ug) Final Mass (ug) Ag/proteine Total Mass % Mass 65 5. Conclusion The significant findings of this work are: silver ions inhibit nitrification activity in both N.europaea suspended cells and biofilms to a greater extent than silver nanoparticles, with the latter inhibition highly dependent on media selection; and N.europaea biofilms are more tolerant to inhibition by either silver ions or nanoparticles than suspended cells. Previous work has shown silver ion dissolution from nanoparticles to be responsible for their toxicity to bacteria and the results from this work support those observations. (Morones et al. 2005) Nanoparticle dissolution depends on particle stability and aggregation and this study has shown the critical role media plays in controlling these processes. The divalent cations required in the test media caused significant aggregation and precipitation in both the batch and drip flow reactor experiments, leading to reduced silver ion dissolution. The reduced availability of silver ions led to substantially decreased nitrification inhibition relative to the silver ion exposures. In batch experiments this translated to an increase in nanoparticle concentration of nearly two orders of magnitude to reach similar nitrification inhibition as observed in the silver ion exposures. For biofilm exposures, not only was the growth media important for reducing the toxicity of the nanoparticles, further protection was conferred by the structure of the biofilm itself. The complex extracellular polymeric matrix surrounding the active cells in the biofilm served to adsorb silver ions and nanoparticles and likely provided resistance to particle diffusion to the active cells. The combined effects of the media and biofilm structure were responsible for the dramatic increase in toxicological resistance to silver nanoparticles relative to suspended cells, while the latter likely provided the basis for the increased resistance to silver ions. 66 The findings of this study suggest that nanoparticle toxicity depends critically on the aqueous chemistry of the system and, given the increased ionic strength and organic matter content of municipal wastewater streams, would likely pose little threat to biological water treatment processes However, the observed adsorption of black, silver-­‐containing particulates to the biofilms and increased biomass-­‐associated silver mass of the AgNP exposures, suggests significant amounts of silver would likely need to be accounted for when disposing of or applying sludge to agricultural systems. 67 6. BIBLIOGRAPHY Adams, N. and Kramer, J. Reactivity of Ag+ ion with thiol ligands in the presence of iron sulfide. Environ. Toxicol. Chem. 1998, 17, 625-­‐629. Arnaout, C. and Gunsch, C. Impacts of silver nanoparticle coating on the nitrification potential of Nitrosomonas europaea. Environ. Sci. Technol. 2012, 46 (10), 5387-­‐5395. Arp, D.; Sayavedra-­‐Soto, L.; Hommes, N. Molcular biology and biochemistry of ammonia oxidation by Nitrosomonas europaea. Arch. Microbiol. 2002, 178 (4), 250-­‐
255. Benn, T. and Westerhoff, P. Nanoparticle silver released into water from commercially available sock fabrics. Environ. Sci. Technol. 2008, 42 (18), 7025-­‐7026. Bergmann, D.; Arciero, D.; Hooper, A. Organization of the hao gene cluster of Nitrosomonas europaea: genes for two tetraheme C cytochromes. J. Bacteriol. 2003, 176 (11), 3148-­‐3153. Bianchini, A.; Bowles, K.; Brauner, C.; Gorsuch, J.; Kramer, J.; Wood, C. Evaluation of the effects of reactive sulfide on the acute toxicity of silver (i) to Daphnia magna. Part 2: toxic results. Environ. Toxicol. Chem. 2002, 21, 1294-­‐1300. Blaser, S.; Scheringer, M.; MacLeod, M.; Hungerbuhler, K. Estimation of cumulative aquatic exposure and risk due to silver: contribution of nano-­‐functionalized plastics and textiles. Sci. Total Environ. 2008, 390 (2-­‐3), 396-­‐409. Blum, D. and Speece, R. The toxicity of organic chemicals to treatment processes. Water Sci. and Technol. 1992, 25 (3), 23-­‐31. Buzea, C.; Pacheco, I.; Robbie, K. Nanomaterials and nanoparticles: sources and toxicity. Biointerphases. 2007, 2 (4), MR17-­‐MR71. Capek, I. Preparation of metal nanoparticles in water-­‐in-­‐oil (w/o) microemulsions. Adv. Colloid Interface Sci. 2004, 110, 49-­‐74. Carlson, C.; Hussain, S.; Schrand, A.; Braydich-­‐Stolle, L.; Hess, K.; Jones, R.; Schlager, J. Unique cellular interaction of silver nanoparticles: size-­‐dependent generation of reactive oxygen species. J. Phys. Chem. 2008, 112, 13608-­‐13619. Chain, P.; Lamerdin, J.; Larimer, F.; Regala, W.; Lao, V.; Hauser, L.; Hooper, A.; Klotz, M.; Norton, J.; Sayavedra-­‐Soto, L.; Arciero, D.; Hommes, N.; Whittaker, M.; Arp, D. 68 Complete genome sequence of the ammonia-­‐oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea. J. Bacteriol. 2003, 185 (9), 2759-­‐
2773. Chinnapongse, S.; MacCuspie, R.; Hackley, V. Persistence of singly dispersed silver nanoparticles in natural freshwaters, synthetic seawater, and simulated estuarine waters. Sci. Tot. Environ. 2011, 409 (12) 2443-­‐2450. Choi, O.; Deng, K.; Kim, N.; Ross, L.; Hu., Z. The inhibitory effects of silver nanoparticles, silver ions, and silver chloride colloids on microbial growth. Water Res. 2008, 42, 3066-­‐3074. Choi, O. and Hu, Z. Size dependent and reactive oxygen species related nanosilver toxicity to nitrifying bacteria. Environ. Sci. Technol. 2008, 42, 4583-­‐4588. Choi, O.; Yu, C.; Esteban Fernandez, G., Hu., Z. Interactions of nanosilver with Escherichia coli cells in planktonic and biofilm cultures. Water Res. 2010, 44, 6095-­‐
6103. Dibrov, P.; Dzioba, J.; Gosink, K.; Hase, C. Chemiosmotic mechanism of antimicrobial activity of Ag+ in Vibrio cholerae. Antimicrob. Agents Chemother. 2002, 46 (8), 2668-­‐
2670. Dror-­‐Ehre, A.; Mamane, H.; Belekova, T.; Markovich, G.; Adin, A. Silver nanoparticle – E.coli colloidal interaction in water and effect on E.coli survival. J. Colloid Interface Sci. 2009, 339 (2), 521-­‐526. Duan, J.; Park, K.; MacCuspie, I.; Vaia, R.; Pachter, R.Optical properties of rodlike metallic nanostructures: insight from theory and experiment. J. Phys. Chem. C. 2009, 113, 15524-­‐15532. Elghanian, R.; Storhoff, J.; Mucic, R.; Letsinger, R.; Mirkin, C. Selective colorimetric detection of polynucleotides based on the distance-­‐dependent optical properties of gold nanoparticles. Science. 1997, 277, 1078-­‐1081. Ely, R.; Williamson, K.; Guenther, R.; Hyman, M.; Arp, D. A co-­‐metabolic kinetics model incorporating enzyme inhibition, inactivation, and recovery. 1. Model development and testing. Biotechnol. Bioeng. 1995, 46 (3), 218-­‐231. Engel, M. and Alexander, M. Growth and autotrophic metabolism of Nitrosomonas europaea. J. Bacteriol. 1958, 76 (2), 217. 69 Erickson, R.; Brooke, L.; Kahl, M.; Vende Venter, F.; Harting, S.; Markee, T. Effects of laboratory test conditions on the toxicity of silver to aquatic organisms. Environ. Toxicol. Chem. 1998, 17, 572-­‐578. Eustis, S. and El-­‐Sayed, M. Why gold nanoparticles are more precious than pretty gold: Noble metal surface plasmon resonance and its enhancement of the radiative and nonradiative properties of nanocrystals of different shapes. Chem. Soc. Rev. 2006, 35, 209-­‐217. Evanoff, D. and Chumanov, G. Size-­‐controlled synthesis of nanoparticles. J. Phys. Chem. B. 2004, 108, 13948-­‐13956. Evanoff, D. and Chumanov, G. Synthesis and optical properties of silver nanoparticles and arrays. ChemPhysChem. 2005, 6, 1221-­‐1231. Fabrega, J.; Renshaw, J.; Lead, J. Interactions of silver nanoparticles with Pseudomonas putida biofilms. Environ. Sci. Technol. 2009, 43, 9004-­‐9009. Fabrega, J.; Fawcett, S.; Renshaw, J.; Lead, J. Silver nanoparticle impact on bacterial growth: effect of pH, concentration, and organic matter. Environ. Sci. Technol. 2009(b), 43 (19), 7285-­‐7290. Fabrega, J.; Zhang, R.; Renshaw, J.; Liu, W.; Lead, J. Impact of silver nanoparticles on natural marine biofilm bacteria. Chemosphere. 2011, 85 (6), 961-­‐966. Fisher, N. and Wang, W. Trophic transfer of silver to marine herbivores: a review of recent studies. Environ. Toxicol. Chem. 1998, 17, 562-­‐571. Flemming, H. and Wingender, J. Relevance of microbial extra-­‐cellular polymeric substances (EPSs) – Part I: Structural and ecological aspects. Water Sci. Technol. 2001, 43 (6), 1-­‐8. Frattini, A.; Pellegri, N.; Nicastro, D.; Sanctis, O. Mater. Chem. Phys. 2005, 94, 148-­‐
152. Geranio, L.; Heubberger, M.; Nowack, B. The behavior of silver nanotextiles during washing. Environ. Sci. Technol. 2009, 43, 8113-­‐8118. Gottschalk, F.; Sonderer, T.; Scholz, R.; Nowack, B. Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, fullerenes) for different regions. Environ. Sci. Technol. 2009, 43 (24), 9216-­‐9222. 70 Grady, C.; Daigger, G.; Lim, H. Biological wastewater treatment, 2nd ed. CRC Press: 1999. Hageman, R. and Hucklesby, D. Nitrate reductase in higher plants. Methods Enzymol. 1971, 23, 491-­‐503. Hagendorfer, H.; Lorenz, C.; Kaegi, R.; Sinnet, B.; Gehrig, R.; Goetz, N.; Scheringer, M.; Ludwig, C.; Ulrich, A. Size-­‐fractionated characterization and quantification of nanoparticle release rates from a consumer spray product containing engineered nanoparticles. J. Nanopart. Res. 2010, 12 (7), 2481-­‐2494. Hogstrand, C. and Wood, C. Toward a better understanding of the bioavailability, physiology, and toxicity of silver in fish: implications for water quality criteria. Environ. Toxicol. Chem. 1998, 17, 547-­‐561. Holt, K. and Bard, A. Interaction of silver (I) ions with the respiratory chain of Escherichia coli: an electrochemical and scanning electrochemical microscopy study of the antimicrobial mechanism of micromolar Ag+. Biochemistry. 2005, 44, 13214-­‐
13223. Hooper, A.; Vannelli, T.; Bergmann, D.; Arciero, D. In Enzymologyof the oxidation of ammonia to nitrite by bacteria. 1997, 59-­‐67. Hyman, M.; Russell, S.; Ely, R.; Williamson, K.; Arp; D. Inhibition, inactivation, and recovery of ammonia-­‐oxidizing activity in cometabolism of trichloroethylene and Nitrosomonas europaea. Appl. Environ. Microbiol. 1995, 61 (4), 1480-­‐1487. Kahn, S.; Mukherjee, N.; Chandrasekaran. Impact of exopolysaccharides on the stability of silver nanoparticles in water. Water Res. 2011, 45, 5184-­‐5190. Kim, B; Park, C.; Murayama, M.; Hochella, M. Discovery and characterization of silver sulfide nanoparticles in final sewage sludge products. Environ. Sci. Technol. 2010, 44 (19), 7509-­‐7514. Kong, H. and Jang, J. One-­‐step fabrication of silver nanoparticle embedded polymer nanofibers by radical mediated dispersion polymerization. Chem. Commun. 2006, 3101-­‐3012. Lauchnor, E.; Radniecki, T.; Semprini, L. Inhibition and gene expression of Nitrosomonas europaea biofilms exposed to phenol and toluene. Biotechnol. Bioeng. 2011, 108 (4), 750-­‐757. 71 Lauchnor, E. and Semprini, L. Inhibition of phenol on the rates of ammonia oxidation by Nitrosomonas europaea grown under batch, continuous fed, and biofilm conditions. Water Research. 2013, http://dx.doi.org/ 10.1016/j.watres.2013.04.052. Li, W.; Xie, X.; Shi, Q.; Duan, S.; Ouyang, Y.; Chen, Y. Antibacterial effect of silver nanoparticles on Staphylococcus aureus. Biometals. 2011, 24, 135-­‐141. Li, X.; Lenhart, J.; Walker, H. Dissolution-­‐accompanied aggregation kinetics of silver nanoparticles. Langmuir. 2010, 26 (22), 16690-­‐16698. Link, S. and El-­‐Sayed, M. Size and temperature dependence of the plasmon absorption of colloidal gold nanoparticles. J. Phys. Chem. B. 1999, 103 (21), 4212-­‐
4217. Liu, J. and Hurt, R. Ion release kinetics and particle persistence in aqueous nano-­‐
silver colloids. Environ. Sci. Technol. 2010, 44, 2169-­‐2175. Lok, C.; Ho, C.; Chen, R.; He, Q.; Yu, W.; Sun, H.; Tam, P.; Chiu, J.; Che, C. Proteomic analysis of the mode of antibacterial action of silver nanoparticles. J. Proteome. Res. 2006, 5, 916-­‐924. Lok, C.; Ho, C.; Chen, R.; He, Q.; Yu, W.; Sun, H.; Tam, P.; Chiu, J.; Che, C. Silver nanoparticles: partial oxidation and antibacterial activities. J. Biol. Inorg. Chem. 2007, 12, 527-­‐534. Luoma, S.; Ho, Y.; Bryan, G. Fate, bioavailability and toxicity of silver in estuarine environments. Mar. Pollut. Bull. 1995, 31, 44-­‐54. Luomo, S. and Rainbow, P. Metal contamination in aquatic environments: science and lateral management. Cambridge: Cambridge University Press: 2008. MacCuspie, R. Colloidal stability of silver nanoparticles in biologically relevant conditions. J. Nanopart. Res. 2011, 13, 2893-­‐2908. Moore, M. Do nanoparticles present ecotoxicological risks for the health of the aquatic environment? Environ. Int. 2006, 32, 967-­‐976. Morones, J.; Elechiguerra, J.; Camacho, A.; Holt, K.; Kouri, J.; Ramirez, J.; Yacaman, M. The bactericidal effect of silver nanoparticles. Nanotechnology. 2005, 16, 2346-­‐
2353. Nel, A.; Xia, T.; Madler, L.; Li, N. Toxic potential of materials at the nanolevel. Science. 2006, 311, 622-­‐627. 72 Petit, C.; Lixon, P.; Pileni, M. In situ synthesis of silver nanocluster in AOT reverse micelles. J. Phys. Chem. 1993, 97 (49), 12974-­‐12983. Puelen, T. and Wilkinson, K. Diffusion of nanoparticles in a biofilm. Environ. Sci. Technol. 2011, 45, 3367-­‐3373. Radniecki, T.; Dolan, M.; Semprini, L. Physiological and transcriptional responses of Nitrosomonas europaea to toluene and benzene inhibition. Appl. Environ. Microbiol. 2008, 74 (17), 5475-­‐5482. Radniecki, T.; Stankus, D.; Neigh, A.; Nason, J.; Semprini, L. Influence of liberated silver from silver nanoparticles on nitrification inhibition of Nitrosomonas europaea. Chemosphere. 2011, 85, 43-­‐49. Rai, M.; Yadav, A.; Gade, A. Silver nanoparticles as a new generation of antimicrobials. Biotechnol. Adv. 2009, 27, 76-­‐83. Ratte, H. Bioaccumulation and toxicity of silver compounds: a review. Environ. Toxicol. Chem. 1999, 18, 89-­‐108. Rosen, B. Bacterial resistance to heavy metals and metalloids. J. Biolog. Inorg. Chem. 1996, 1 (4), 273-­‐277. Russell, A. and Hugo, W. Antimicrobial activity and action of silver. Prog. Med. Chem. 1994, 31, 351-­‐370. Sayavedra-­‐Soto, L.; Hommes, N.; Alzerreca, J.; Arp, D.; Norton, J.; Klotz, M. Transcription of the amoC, amoA, and amoB genes in Nitrosomonas europaea and Nitrsospira sp. NpAV. FEMS Microbiol. Let. 1998, 167 (1), 81-­‐88. Sheng, Z. and Liu, Y. Effects of silver nanoparticles on wastewater biofilms. Water Res. 2011, 45 (18), 6039-­‐6050. Shrivastava, S., Bera, T., Roy, A.; Singh, G.; Ramachandrarao, P.; Dash, D. Characterization of enhanced antibacterial effects of novel silver nanoparticles. Nanotechnology. 2007, 18:225103. Silver, S. Bacterial silver resistance: molecular biology and uses and misuses of silver compounds. FEMS Microbiol. Rev. 2003, 27, 341-­‐353. 73 Silver, S.; Phung, L.; Silver, G. Silver as biocides in burn and wound dressings and bacterial resistance to silver compounds. J. Ind. Microbiol. Biotechnol. 2006, 33, 627-­‐
634. Smirnova, T.; Didenko, L.; Azizbekyan, R.; Romanova, Y. Structural and functional characteristics of bacterial biofilms. Microbiology. 2010, 79 (4), 413-­‐423. Sondi, I. and Salopek-­‐Sondi, B. Silver nanoparticles as antimicrobial agent: a case study on E.coli as a model for gram-­‐negative bacteria. J. Colloid Interface Sci. 2004, 275 (1), 177-­‐182. Stewart, P. A review of experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms. Biotechnol. Bioeng. 1998, 59 (3), 261-­‐272. Stoodley, P.; Sauer, K.; Davies, D.; Costerson, J. Biofilms as complex differentiated communities. Annu. Rev. Microbiol. 2004, Su, H.; Chou, C.; Hung, D.; Lin, S.; Pao, I.; Lin, J.; Huang, F.; Dong, R.; Lin, J. The disruption of bacterial membrane integrity through ROS generation induced by nanohybrids of silver and clay. Biomaterials. 2009, 30 (30), 5979-­‐5987. Turkevich, J.; Stevenson, P.; Jillier, J. A study of the nucleation and growth of processes in the synthesis of colloidal gold. Discuss. Faraday Soc. 1951, 55-­‐75. Wilen, B.; Jin, B.; Lant, P. Relationship between flocculation of activated sludge and composition of extracellular polymeric substances. Water Sci. Technol. 2003, 47 (12), 95-­‐103. Wirth, S.; Lowry, G.; Tilton, R. Natural organic matter alters biofilm tolerance to silver nanoparticles and dissolved silver. Environ. Sci. Technol. 2012, 46, 12687-­‐
12696. Xiao, Y. and Weisner, M. Transport and retention of selected engineered nanoparticles by porous media in the presence of a biofilm. Environ. Sci. Technol. 2013, 47, 2246-­‐2253. Xu, H.; Qu, F.; Xu, H.; Lai, W.; Wang, Y.; Aguilar, Z.; Wei, H. Role of reactive oxygen species in the antibacterial mechanism of silver nanoparticles on Escherichia coli O157:H7. Biometals. 2012, 25, 45-­‐53. 74 Yuan, Z.; Li, J.; Xu, B.; Zhang, H.; Yu, C. Interaction of silver nanoparticles with pure nitrifying bacteria. Chemosphere. 2012, http://dx.doi.org/ 10.1016/j.chemosphere.2012.08.032. Zhao and Stevens. Multiple parameters for the comprehensive evaluation of the susceptibility of Escherichia coli to the silver ion. Biometals. 1998, 11, 27-­‐32. Zook, J.; MacCuspie, R.; Locascio, L.; Halter, M.; Elliott, J. Stable nanoparticle aggregates/agglomerates of different sizes and the effect of their size on hemolytic toxicity. Nanotoxicology. 2011, 5 (4), 517-­‐530.