Research Feature: Developing a Biosensor to Increase Wastewater Treatment Efficiency - Part II

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Related SBI research feature: Developing a Biosensor to Increase Wastewater Treatment Efficiency -- part I, December 2007.

The journal article related to this feature: Radniecki, T.S., Semprini, L., and Dolan, M.E. 2009. Expression of merA, amoA and hao in Continuously
Cultured Nitrosomonas europaea Cells Exposed to Zinc Chloride Additions
, Biotechnology and Bioengineering 102: 2, 546-553.

Posted: February 4, 2009

A paper in the February issue of Biotechnology and Bioengineering describes the latest research efforts of an interdisciplinary OSU team working to develop microbial biosensors that could one day increase the efficiency of wastewater treatment plants. The research focuses on bacteria used to remove nitrogen from wastewater and is working to develop an early warning signal that will indicate when the bacteria’s activity is interrupted by pollutants. In the Biotechnology and Bioengineering article, OSU environmental engineers Tyler Radniecki, Lewis Semprini and Mark Dolan describe their studies of the bacteria’s response to the pollutant zinc. In this short Web interview, Tyler Radniecki, a post-doctoral researcher, describes the goals of the project and the research presented in the team’s recent paper.

What was the goal of the project?

This project is part of a larger effort at OSU led by Dan Arp, Peter Bottomley and Lew Semprini and funded by the National Science Foundation to identify sentinel genes in the bacteria Nitrosomonas europaea. Sentinel genes are genes whose expression is increased, or “turned on,” in response to a stimulus, in our case, in response to contact with specific contaminants or groups of contaminants. The increased expression of the sentinel gene results in the cell producing more of a particular protein that the cell needs to respond to the contaminant. If we can recognize when those genes are “turned on,” then perhaps we could use them not only to identify when the cells are being inhibited but more importantly we can use these genes to identify which contaminant is causing the inhibition.

An earlier OSU study by Sunhwa Park and Roger Ely, in Biological and Ecological Engineering, had identified possible sentinel genes for the presence of zinc. In this paper, we were evaluating the suitability of those genes as sentinels in a longer term study that more closely mimicked conditions the bacteria would experience in a wastewater treatment plant. The study also has relevance for soil and groundwater remediation. The bacteria we worked with are ubiquitous in soils and identification of sentinel genes could lead to better detection and monitoring of heavy metals in contaminated soil and groundwater.

This study focuses on zinc – why is this metal a concern to wastewater treatment plants?

Zinc is one of many heavy metals that have been identified as a “priority pollutant” by the U.S. Environmental Protection Agency. It can come from industrial waste as well as from stormwater runoff from streets and roofs. Once in the wastewater stream, zinc can kill or severely decrease the activity of bacteria that play an important role in wastewater treatment and the global nitrogen cycleNitrosomonas europaea, the bacteria we worked with, is an ammonia oxidizing bacteria – it carries out the first step in converting ammonia to nitrogen gas. Ammonia oxidizing bacteria, such as N. europaea, are widely considered to be the most sensitive fauna in a wastewater treatment plant. If their activity is reduced because of the presence of contaminants such as zinc, that affects the efficiency of the entire nitrogen removal system.

In your experiments you used a chemostat. What is that and why did
you use it?

A chemostat is an experimental chamber where all chemical concentrations are static – i.e. you are at a constant equilibrium. You are bringing food and nutrients, in the form of growth media, into the chemostat at a constant rate and at the same rate you are pumping the media out. The design of the chemostat is such that the growth rate of the bacteria is equal to the rate of the additions and removal of the media. Hence, the bacteria are consuming the food and nutrients as quickly as they are being pumped into the reactor and the bacteria are growing as quickly as they are being pumped out of the reactor. If the growth rate of the bacteria becomes inhibited, they cannot multiply quickly enough to maintain a constant population within the reactor and their numbers will dwindle as they are removed from the chemostat. Also, the food and nutrient concentrations will begin to increase within the reactor as there are fewer bacteria present to consume it. When this happens, we say that the bacteria have been “flushed out.” Additionally, N. europaea requires large amounts of oxygen and they acidify their environment as they grow. To keep a chemical equilibrium within the chemostat, air is bubbled through the media and the pH is kept constant by the automated addition of a base – sodium carbonate. The advantage of this set up is that it allows you to do a very controlled long-term study and gain insight into inhibition and recovery mechanisms that take place over days or weeks. These are observations that are difficult to make in batch studies that are limited to a couple of hours due to oxygen and pH constraints.

Diagram of the chemostat.
This photo shows the chemostat with its components labeled.

How were the experiments set up?

We grew the bacterial culture in a chemostat and then periodically introduced pulses of zinc into the system. We monitored the response and then when the system returned to equilibrium, we introduced another spike of a higher concentration of zinc. We ran the experiment for a total of 85 days and over that time added four different pulses of zinc. Throughout the experiment we monitored three things: (1) the concentration of ammonia and nitrite, the inputs and outputs from the reaction, (2) the activity of the enzymes that oxidize ammonia to nitrite, and (3) changes in the number of transcripts of the sentinel gene merA.

What were your results?

When we looked at the concentration of ammonia, we didn’t see very much happening – it was kind of boring – until the very last spike where we added so much zinc that we killed the bacteria. But we found that if we looked at the bacteria a little closer and looked at the activity of the ammonia oxidizing enzymes we found that their metabolic activity was immediately reduced with each addition of zinc. We didn't see the effect of this on the overall concentration of ammonia and nitrite because the bacteria recovered their ability to oxidize ammonia fairly rapidly. This was unusual because zinc is known to be an irreversible inhibitor of N. europaea.  However, when we looked more closing at the gene expression of the ammonia oxidizing enzymes we saw that N. europaea overcame zinc inhibition by increasing the expression of these enzymes and producing brand new ammonia oxidizing enzymes. This had not been documented in previous short-term studies. But where it really got interesting is when we looked at the gene expression of the sentinel gene, merA. For every spike of zinc added to the chemostat, even at concentrations that did not inhibit N. europaea, there was a spike in the amount of merA transcript produced.  So even though we saw no change in actual activity of the reactor and only a slight change in ammonia oxidizing enzyme activity, there was a huge spike in the up-regulation of the merA gene. We also found that as the zinc was flushed out of the system, the merA gene response went back down to steady-state levels. 

Why are these results significant?

From a biosensor perspective, these two points are very interesting. For one, the gene response is very large – we were seeing 30-fold and 50-fold increases in the number of gene transcripts being produced – so there is the potential to detect the contaminant at low concentrations. Also, by measuring merA gene response, we were able to detect the presence of the contaminant before seeing any negative effects on the process performance – the oxidation of ammonia.  If you want to design an early warning system that says, “Hey, there is some zinc in the system do something before it is too late,” this has potential.  Another thing that is promising is that we found that as the zinc washed out of the system, the signal also decreased, so the merA sentinel gene response is mimicking the amount zinc in the system – once the pollutant is gone, the gene is down-regulated to background levels. So a biosensor based on merA expression has potential to be quantitative as well.

Were there any surprises in the study?

Our work was based on another study by Sunhwa Park and Roger Ely that identified merA as a possible sentinel gene. They found a 48-fold increase in merA and they also found an 80% inhibition of the same bacteria when they added a relatively low concentration of zinc. We started our experiments with this low concentration of zinc but we didn’t see any inhibition of the reaction overall, or at the enzyme level, with this amount zinc. It turns out, that the difference was the media which we were using to grow the bacteria. Since Sun Hwa Park was doing short-term studies, her media included only buffer and ammonia, but for my longer-term studies I had to include trace nutrients, such as magnesium and calcium, that would foster the bacteria's growth over a longer time period. So I repeated Sun Hwa’s batch experiments and monitored the effect of the trace metals. I found that the cells became less sensitive to zinc with the addition of each individual trace nutrient, especially magnesium, and when I had added all the trace nutrients I used in the chemostat media, the bacteria wasn’t inhibited by the zinc at all. So this showed that the trace nutrients are playing a big role in the protection of the cells from zinc – especially magnesium.  Additional tests showed that increases in magnesium concentrations made it more difficult for zinc to enter into the cells. A practical aspect of this study is that if a treatment plant is experiencing problems because of zinc inhibition, the addition of more magnesium could protect the ammonia oxidizing bacteria. 

For more information about Tyler's research or the larger biosensors project visit the following links: