Investigating the Toxicity of Silver Ions to Chronically Exposed Nitrifying Bacteria

Transcription

1 Investigating the Toxicity of Silver Ions to Chronically Exposed Nitrifying Bacteria Issa El Haddad San Diego State University Summer 2012-Fall 2012 Dr. Tyler Radniecki Department of Civil, Construction, and Environmental Engineering San Diego State University February 11, 2013

2 Table of Contents Acknowledgements... 3 Executive Summary... 3 Project Objectives... 3 Project Approach... 4 Project Outcomes... 7 Conclusions... 10

3 Acknowledgements This project was supported by Agriculture and Food Research Initiative Competitive Grant no from the USDA National Institute of Food and Agriculture. I would also like to acknowledge Dr. Tyler Radniecki for his guidance and support over the course of this project. In addition, I want to thank my coworkers in the lab for all their help in conducting experiments and running samples. Executive Summary Silver (Ag) has been known to inhibit the growth of bacteria and fungi. Due to these anti-bacterial and anti-fungal properties, the use of Ag in consumer-based products has skyrocketed over the past few years (Figure 1). Silver nanoparticles are used in applications such as clothing, electronics, information technology, healthcare, biotechnology, food, and agriculture. Through the process of integrating Ag-NPs into clothing and other commercial products, the resulting effects on the environment due to the loss of Ag from these products remain unknown. In particular, environmental engineers are concerned with the implications of Ag-NP ecotoxicity to wastewater Figure 1: Major Nanoparticles bacteria, specifically ammonia oxidizing bacteria (AOB), which is present in the activated sludge process of the wastewater treatment plant (WWTP). As nanoparticles exhibit a high surface area to mass ratio, Ag-NPs are highly reactive and dissolve into silver ions (Ag + ) over a short period of time. AOB are important in wastewater treatment because they play a key role in the removal of nitrogen from municipal wastewater, in a process called nitrification, which is the biological oxidization of ammonia (NH 3 ) to nitrite (NO 2 - ). The nitrification activity of the AOB is highly sensitive to disruption, which is why the ecotoxicity of Ag-NPs is being studied. Nitrosomonas europaea is used as the model AOB in this study. This project will examine the toxicity of Ag +, an important end-product of Ag-NP dissolution and chief source of Ag-NP toxicity, to chronically exposed N. europaea cells. Project Objectives The central hypothesis of this project is that in a reactor in which the hydraulic retention time is shorter than the cell retention time (e.g. WWTPs), sub-inhibitory concentrations of Ag + will become lethal to N. europaea cells chronically exposed to these low concentrations, due to an accumulation of Ag + onto the N. europaea cells. El Haddad Page 3

4 To test this hypothesis, N. europaea cells were cultured in sequencing batch reactors (SBRs) which had a hydraulic retention time of 1 day and a cell retention time of 21 days. This allowed the cells to be washed over by a large volume of growth media containing low concentrations of Ag +. The cell growth, ph, and nitrification activity was measured daily. The Ag + concentrations accumulating on the cells was measured using an inductively coupled plasma mass spectrometry (ICP-MS). Once steady-state had been achieved in the SBRs, as indicated by a constant ph, cell density, and NO 2 - concentration, Ag + were introduced to the SBRs through the daily dosing of fresh growth media. As a potential USDA employee, this project presents critical thinking and analysis as well as practical and theoretical knowledge of raw data and experimental methods generated and employed towards understanding the ecotoxicity of Ag + and their effect on WWTPs, and eventually on the receiving watersheds. This project is crucial in identifying at what Ag + concentrations will WWTP nitrification mechanisms fail, thus releasing contaminated water into the subsequent watersheds. Project Approach This project utilized a variety of procedures, which are detailed below. These include the optimization of the growth media, the daily fill-and-draw procedures and measurements, preparing samples for use in the ICP-MS, and other side experiments that further supports the stated hypothesis. Optimization of Growth Media The SBRs are bioreactors where fresh media is continuously added to the reactor while old media is continuously removed, to keep the total volume constant. By changing the rate at which new media is added into the reactor, the growth rate of the bacteria can be controlled. This relationship is shown mathematically through the following formulas: Where HRT and SRT are the hydraulic retention time and sludge (cell) retention time, respectively, V is the total volume in the reactor, and Q is the flow rate. These formulas will be used in the following set of procedures. One of the most challenging tasks in this project was the preparation of the optimal growth media in the SBRs. The ideal growth environment for the N. europaea cells was determined through a process of trial and error, especially with the buffers. The buffers prevent the bacteria from quickly acidifying the growth environment through their nitrification activity. If the ph, which is normally around 7.8, decreases, the equilibrium between NH 3 + NH 4 + shifts heavily to NH 4 +. N. europaea can only oxidize NH 3, thus a shift to NH 4 + will slow down N. europaea's metabolism and cause a decrease in the overall cell density. The following tables will present which compounds were added to make the growth media for the bacteria. El Haddad Page 4

5 Figure 2: AOB Growth Media NOTE: 0.5 ml of mm Phenol Red (a ph indicator) stock solution was added to Part I, to make the final concentration of the ph indicator at µm. Each part of the growth media is prepared separately, then autoclaved, to prevent the precipitation of the trace metals. The ph of Part II is adjusted to 8 with 10N NaOH before autoclaving. After the flasks cool to room temperature, usually the following day, the growth media is combined inside a sterile environment (e.g. in a laminar flow hood). All of Part II and 16 ml of Part III (3.8 mm final concentration) are combined with Part I. Because phenol red, a ph indicator, was added, changes in color can be noticed. Part I was initially yellow, and then became reddish-brown after adding Part II, and finally reddish-pink when Part III was added. The SBRs were inoculated with N. europaea cells and allowed to grow in batch mode until the bacteria reaches early stationary phase. Growth media was then be removed and replaced at a rate that achieves a hydraulic retention time of 1 day and a cell retention time of 21 days, in a "fill-and-draw" process. The ph, cell density, and NO 2 - concentrations reached a steady-state in the SBRs, varying slightly with each fill-and-draw event (Radniecki et al. 2011). Once this El Haddad Page 5

6 steady-state has been reached, Ag + doses were infused into the SBRs at sub-inhibitory concentrations. UV-vis spectrophotometry was used to measure cell densities (or OD 600, optimal density at 600 nm absorbance) and NO 2 - concentrations (absorbance at 352 and 400 nm). Based on these measurements, the growth rate and nitrification activity of the N. europaea cells was calculated. Furthermore, these measurements will be used to determine inhibition of N. europaea by Ag +. The NO 2 - concentrations produced by the N. europaea were calculated using the following formula: The SBRs were inoculated with 30 ml of previously grown N. europaea cells that were in the mid-exponential growth phase (OD 600 ~ 0.050). The SBRs were shaken at 110 rpm at 30 o C in the dark. Once the cells reached early stationary phase (OD 600 ~ 0.070), cells and growth media were removed from the SBRs and replaced with fresh growth media as outlined below (Daily Fill-and- Draw Procedure) to achieve a 21-day hydraulic retention time. Daily Fill-and-Draw Procedure 1. Prepare six 50 ml falcon tubes and six 15 ml falcon tubes, label them (1-6 each), and place them on a tube-rack. Also bring a dry 2000 ml beaker (you can use the same beaker as above). Place the tube rack and the beaker in the laminar flow hood. 2. Take the six flasks off the shaker and place them in the laminar flow hood. 3. Place three serological pipets (two 35 ml and one 7.5 ml) in the laminar flow hood. 4. Put on gloves, and spray them with 70% ethanol (i.e. wash your hands with ethanol) before inserting your hands into the hood. 5. Spray the tweezers with 70% ethanol, and proceed to take off the aluminum foil off of the SBRs (you can do this using your hands). Using the tweezers, take off the foam stopper and place it inside the foil. 6. Take the caps off of the centrifuge tubes. Using one of the 35 ml serological pipets, take out 117 mls from each SBR. 30 ml will go inside the 50 ml centrifuge tubes, while the rest will be dumped in the beaker. Close the 50 ml centrifuge tubes. 7. Using the second 35 ml serological pipet, add 120 ml of fresh AOB Growth Media to each SBR. 8. Using autoclaved glass pipettes, add drops of 10N NaOH to each SBR until the color become pink (ph around 7.8). Hand shake SBRs slowly to mix. 9. After adjusting the ph, use the 7.5 ml serological pipet to take out 3 ml of each SBR to place them in the corresponding 15 ml centrifuge tubes. 10. Dose SBRs #4-6 with the appropriate amount of Ag + (in the form of AgNO 3 ). 11. Spray the tweezers with 70% ethanol, and then use it to close off the flasks with the foam stoppers. Place aluminum foil at the flask opening to seal them. 12. Place the flasks back on the shaker at 110 rpm, in the dark at 30 o C. El Haddad Page 6

7 13. Take the measurements using the UV-Vis using the following steps: a. Assuming you are on the last page, select "General Tests" b. Select "Smart Start" c. Select "OD-NO3" which measures absorbances at 600 nm, 352 nm, and 400 nm. d. Select "Run Test" e. Fill cuvette with DI and select "Measure Blank". f. Measure the samples that are in 50 ml falcon tubes #1-6. ICP-MS Samples Inductively coupled plasma mass spectrometry (ICP-MS) is a type of mass spectrometry that is capable of detecting metals and several non-metals at concentrations as low as 1 part per trillion (ppt). It does this by ionizing the sample with plasma, and then using a mass spec to separate and quantify these ions. In the case of this project, ICP-MS was used to quantify the Ag + present absorbed to the cells and the Ag + still found in the supernatant. The six 30 ml falcon tubes from the above fill-and-draw procedure were centrifuged at 9000 rpm for 30 minutes. The cell pellet and 2 ml of the supernatant were collected in 1.5 ml tubes and frozen until ICP-MS analysis could be conducted. To prepare the samples for the ICP-MS analysis, the cell pellets were dissolved in concentrated nitric acid (HNO 3 - ) over night. DI H 2 O was added to the dissolved cell pellet suspension to reduce the HNO 3 - concentration to 2%. The cell pellet suspension was filtered through a 0.2 µm syringe filter the next day. The supernatant was soaked overnight in a mixture of 2% HNO 3 - and 3mL DI H 2 O. The supernatant was filtered through a 0.2 µm syringe filter. As the cell pellets did not dissolve in the 2% HNO 3 -, the concentration of HNO 3 - was increased to 64% and placed on the shaker to mix overnight. After the cell pellet suspension dissolved, the solutions were then diluted to the required 2% HNO 3 - concentration, and filtered. Project Outcomes Disclaimer: This project is still in progress, therefore the data and charts provided here are not final. SBRs 1-3 were set up as controls, with no Ag + present. The starting concentration of Ag + added to SBRs 4-6 was ppm, on day 22 of the experiment, when the reactor hit steady state (Figure 3). The concentrations of Ag + that were added to SBRs 4-6 are detailed in the Table 1 on the right along with what day they were first added to the SBRs. The same concentration is added continuously in the days that follow. Figure 3 shows NO 2 - concentrations produced vs. time. In addition, a theoretical set of calculated data is Table 1: Ag + Concentrations Added El Haddad Page 7

8 shown to represent what would be expected to occur if the cells were completely inhibited. However, while the expected result was that N. europaea would start dying off at the moment the Ag + was added, until they eventually die out completely, the actual results of the experiment were that N. europaea showed a slight decrease in NO 2 - production (as well as OD 600 ), even at Ag + concentrations as high as 1.3 ppm. Previous results have shown that as little as 0.2 ppm Ag + can completely inhibit N. europaea cells in 3-hour batch toxicity tests. It is unknown why the cells show such high tolerance to Ag + in the SBRs, but possible factors include the presence of trace metals and the slower growth rates of cells in the SBRs compared to the simplified test media and exponentially growing cells used in previous acute batch assays. Figure 3: NO 2 - vs. Time As the experimental results did not match the theoretical results, further analysis is necessary to figure out why. Figures 4-6 represent data acquired through the ICP-MS. The working hypothesis expected higher Ag + concentrations in the cell pellets than the supernatant. Additionally, the Ag + concentration in the cell pellet was hypothesized to increase over time as more Ag + was added to the SBRs. El Haddad Page 8

9 Ag+ Mass (ug) Ag+ Mass (ug) Total Ag+ Mass Accumulated in Reactor # Time (Days) Ag+ (Cells) in Reactor Total Ag+ in Reactor Ag+ (Supernatant) in Reactor Calculated Ag+ in Reactor Figure 4: Total Ag + Mass Accumulated in Reactor #4 Total Ag+ Mass Accumulated in Reactor # Time (Days) Ag+ (Cells) in Reactor Total Ag+ in Reactor Ag+ (Supernatant) in Reactor Calculated Ag+ in Reactor Figure 5: Total Ag + Mass Accumulated in Reactor #5 El Haddad Page 9

10 Ag+ Mass (ug) Total Ag+ Mass Accumulated in Reactor # Time (Days) Ag+ (Cells) in Reactor Total Ag+ in Reactor Ag+ (Supernatant) in Reactor Calculated Ag+ in Reactor Figure 6: Total Ag + Mass Accumulated in Reactor #6 As expected, the mass of Ag + found associated with the N. europaea cells increased throughout the experiment as the concentration of Ag + added to the SBR media increased (Figure 4-6). However, as indicated by the slight decrease in NO 2 - production (Figure 3), this amount of adsorbed Ag + was not enough to severely inhibit N. europaea. Surprisingly, the concentration of Ag + in the SBR supernatant (i.e. the Ag + not associated with the cell mass) did not increase with increasing Ag + dosing. This led to a poor Ag + mass balance as indicated by the gap between the Total Ag + and the Calculated Ag +. Further modeling of the SBR system using Visual MINTEQ is currently being conducted to try and determine what Ag-species were found during our experiments and if they may have precipitated out of solution thus leading to a poor mass balance and a less than expected toxicity to N. europaea. Conclusion As noted throughout the experiment, there is still a lot of data analysis currently being conducted. Furthermore, there is also more research to be done to support the results of this experiment. The experiment will be re-done in Spring 2013 to validate the ability to reproduce the data. Further tweaks to the experiment will be introduced, such as adding Ag-NPs and other macromolecules to determine their influence on N europaea activity. The overall project was an important learning experience for me. Perhaps one of the most important aspects that I got out of the internship is that it taught me responsibility. There is a lot of responsibility and delicacy involved in performing experiments; I have to be careful not to break any glassware and be accurate when adding chemicals or measuring samples. Time management is another important factor. I have to manage my time wisely while performing El Haddad Page 10

11 experiments and measuring samples, making weekly presentations for our weekly lab meetings, and keeping on top of my project by keeping my data up to date. I have acquired a lot of critical thinking and problem solving skills through setting up experiments and optimizing them by a method of trial and error, as well as by analyzing data and making necessary adjustments to my work. Finally, my communication skills were improved. I had to interact with other members in the laboratory, ask for advice and help, or give advice and help. I also had to present my data either by oral presentations or written reports. All of these skills are necessary for my future career as an engineer, I will be required to manage my time wisely, be responsible, interact with colleagues, and perform my project tasks with accuracy and attention to detail. El Haddad Page 11