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
Table of Contents
Acknowledgements ...................................................................................................................................... 3
Executive Summary ...................................................................................................................................... 3
Project Objectives ........................................................................................................................................ 3
Project Approach .......................................................................................................................................... 4
Project Outcomes ......................................................................................................................................... 7
Conclusions ................................................................................................................................................. 10
Acknowledgements
This project was supported by Agriculture and Food Research Initiative Competitive Grant no.
2011-38422-31204 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 (NH3) to nitrite (NO2-). 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.
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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 NO2- 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 NH3 + NH4+ shifts heavily to
NH4+. N. europaea can only oxidize NH3, thus a shift to NH4+ 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.
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Figure 2: AOB Growth Media
NOTE: 0.5 mL of 11.85 mM Phenol Red (a pH indicator) stock solution was added to Part I, to
make the final concentration of the pH indicator at 5.925 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 NO2- concentrations reached a steady-state in
the SBRs, varying slightly with each fill-and-draw event (Radniecki et al. 2011). Once this
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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 OD600, optimal density at 600
nm absorbance) and NO2- 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 NO2- 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 (OD600 ~ 0.050). The SBRs were shaken at 110 rpm at 30oC in the
dark. Once the cells reached early stationary phase (OD600 ~ 0.070), cells and growth media were
removed from the SBRs and replaced with fresh growth media as outlined below (Daily Fill-andDraw 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 AgNO3).
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 30oC.
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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 (HNO3-) over night. DI H2O was added to the dissolved cell pellet suspension to
reduce the HNO3- 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% HNO3- and
3mL DI H2O. The supernatant was filtered through a 0.2 µm syringe filter. As the cell pellets did
not dissolve in the 2% HNO3-, the concentration of HNO3- 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% HNO3- 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 0.025 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 NO2- concentrations produced vs.
time. In addition, a theoretical set of calculated data is
+
Table 1: Ag Concentrations Added
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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 NO2- production (as well as OD600), 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: NO2 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.
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Ag+ Mass (ug)
Total Ag+ Mass Accumulated in Reactor #4
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Total Ag+ in Reactor
Calculated Ag+ in Reactor
+
Figure 4: Total Ag Mass Accumulated in Reactor #4
Ag+ Mass (ug)
Total Ag+ Mass Accumulated in Reactor #5
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Figure 5: Total Ag Mass Accumulated in Reactor #5
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Ag+ Mass (ug)
Total Ag+ Mass Accumulated in Reactor #6
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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 NO2- 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
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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.
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