SBI Research Feature: Developing a Biosensor to Increase Wastewater Treatment Efficiency

Faculty members from Botany and Plant Pathology, Biological and Ecological Engineering and Environmental Engineering have combined efforts to develop a biosensor that could increase the efficiency of wastewater treatment plants. The five-year project is funded by the National Science Foundation’s Biocomplexity in the Environment program and makes use of new genomic approaches to study bacteria that remove nitrogen from wastewater. In this short Web interview, professor Dan Arp and post-doctoral researcher Barbara Gvakharia (Botany and Plant Pathology) describe the project.

What is the overall goal of the project?

Dan: The problem that we approached has to do with wastewater treatment where one of the goals is the removal of nitrogen. One of the many steps in the treatment process is the conversion of nitrogen in the form of ammonia into nitrate. This process is carried out by ammonia oxidizing bacteria. The challenge to the wastewater treatment engineer is that while these bacteria are a critical component of the treatment process, they are also fickle. They are easily damaged by a number of things that can come along in the waste stream, such as chlorinated aliphatic compounds, metals and other contaminants. 

So, for this project, we wanted to see if we could use genomic approaches to identify “sentinel genes” in the ammonia oxidizing bacteria. These are genes that are the first ones to change their expression levels when the bacteria are exposed to a contaminant.Then, if we could do that, maybe we could create a “reporter strain” - a bacterium that would fluoresce or “light up” and serve as an early warning system when something was affecting the efficiency of the ammonia oxidizing bacteria. 

Ultimately, the goal is to be able to run wastewater treatment plants at full capacity. There are tens to hundreds of compounds that can inhibit the performance of ammonia oxidizers so, right now, treatment plants have to run at a low capacity so that they can ensure that all nitrogen is removed. But if they had a warning system to identify problems, maybe they could run the plants at a higher capacity.

The first step though, is proof of concept – can we identify the “sentinel genes” that respond to presence of the pollutants.

What experiments have you carried out so far? 

Barbara: We started out looking at the global transcription response of one ammonia oxidizing bacteria, Nitrosomonas europaea, to  two contaminants – chloroform and chloromethane.  Both of these compounds inhibit the activity of Nitrosomonas but in different ways – the bacteria can recover from exposure to chloromethane but not to chloroform, it causes irreversible damage.

We ran a series of experiments where we exposed samples of the bacteria to the contaminants and then looked at gene response using microarray analysis.  This is now a pretty common technique that allows you to look at the changes in transcription of certain genes.  It uses a silicon chip that is about the size of a matchbox and has pieces of DNA attached that include the sequences for all of the 2460 genes of Nitrosomonas europaea.

How does microarray analysis work?

Dan: It’s pretty complicated, but essentially what we have on this little chip are all these different genes represented with their DNA sequence and when we put in the messenger RNA from the sample bacteria, they go and find their complementary sequences, and stick to their unique identifier. Each of those messenger RNA sequences has been labeled with a fluorescent tag so now you put it under a camera and you ask which genes on the chip are lighting up and how much. The higher the intensity of the fluorescence, the more copies of messenger RNA that are present, and that’s an indication that a gene has been “turned on” or “upregulated.”

So what did the experiments show? Have you identified "sentinal genes"?

Barbara: We were interested in how the cell responds to the treatment and we found that it does respond in a very distinct way. As we expected, the chloroform treatment caused more damage and caused more genes to be upregulated or downregulated. The chloromethane treatment was less harmful but it also caused certain discernable changes in the overall transcription. We also found that there were 37 genes that overlapped in both treatments and were upregulated by exposure to both chloromethane and chloroform. These are the candidates to be the “sentinel genes” – they all were upregulated when the bacterium was exposed to the contaminants. We had a paper come out last spring in Applied and Environmental Microbiology that describes these experiments.

Have you taken the next step and tried to create a “reporter strain” that responds to the contaminants?

Barbara: Yes. From the list of 37 genes that were upregulated by both exposure to chloroform and chloromethane, I chose three genes to start with. Two of these code for heat-shock proteins and one for an enzyme called metallo-beta-lactamase. This last one is the gene that was most highly upregulated by both treatments so it was at the top of our list. I transformed N. europaea with plasmids (small circular DNA) carrying promoters of these genes upstream of Green Fluorescent protein (GFP). I got three transformed strains and treated them with chloroform and chloromethane in order to find out if these treatments will induce the fluorescence of GFP. Right now, I am done with the chloroform treatment and I found that one strain does respond to chloroform treatment, it increases the fluorescence six- to nine-fold. But I’ve had no luck with chloromethane – for some reason it just doesn’t respond, I don’t know why, this is biology!  I think I may need to look at some other genes which would be more specific to chloromethane. 

Dan: So the “reporter strain” Barbara created is working for chloroform and now we could look at many other chlorinated aliphatic compounds and find out what the response range is – chloroform: yes, chloromethane: no – you could continue and get a general sense of what this strain will respond to.

Who are the other people involved in the project and what are they working on?

Dan: Roger Ely, from Biological and Ecological Engineering and his graduate student, Sun Hwa Park, are looking at metals that inhibit the activity of the ammonia oxidizers. Lew Semprini and Mark Dolan from Environmental Engineering have been working with a post-doc, Tyler Radniecki, and a PhD student, Sean Sandborg, on the effect of aromatic compounds, for example Toluene, on the bacteria.  Lew Semprini also has a student, Ellen Swogger, who has been looking at what happens when these bacteria are bound to a surface. Barbara has done her work with cells that are free floating in solution, or planktonic, but in wastewater treatment plants the bacteria would either be on a surface or bound together in what’s called a flock. So the question Ellen is helping answer is – are the bacteria going to behave the same in these aggregated forms as they do in planktonic forms? And Brian Wood is helping with this part of the project since he has a lot of experience working with biofilms. In our lab, Luis Sayavedra-Soto has been working with Barbara. The project has also supported workshops on genomics and bioinformatics for high school teachers through the Science Education Partnerships Program (SEPS).

This project is in its third of five years; do you have plans for future related research projects?

Dan: We are hoping to further develop the reporter strain technology and may apply for additional grants for that effort. There are also a lot of interesting science questions that have come up – for example, Barbara’s work has brought up a lot of questions about gene function and specifically toxin-antitoxin genes. Nitrosomonas europaea has more of these genes than any other bacteria sequenced and there a lot of interesting questions about how they govern metabolism.We’re also interested in the possibilities raised by the addition of an Illumina DNA sequencing machine to OSU’s Center for Genome Research and Biocomputing. This machine can sequence an entire bacterial genome for $2000 in 2.5 days.With this capacity, we also hope to determine gene expression by counting individual messenger RNA copies rather than basing the estimate of activity on a ratio of fluorescence, as with the microchips.  Genomic tools are advancing so rapidly and they present an amazing opportunity for this type of research.