March 2007, Vol. 19, No.3

Cracking the Microbial ‘Black Box’ of Activated Sludge

A new research development could give water quality experts a leg up in re-engineering existing plants and designing more efficient and reliable ones in the future for removal of nutrients, particularly phosphorus.        

In the first ever metagenomic study of an activated sludge wastewater treatment process, researchers from the U.S. Department of Energy’s Joint Genome Institute (JGI), University of Wisconsin–Madison (UWM), and University of Queensland, Australia (Brisbane), were able to obtain a nearly complete DNA blueprint of the bacterial species Accumulibacter phosphatis, a key player in phosphorus removal. This step opens the door to better understanding the mechanisms involved in that removal, potentially leading to an early warning system of treatment plant failure, said Philip Hugenholtz, head of JGI’s Microbial Ecology Program and co-author of a paper on the study published in the Sept. 24 online edition of Nature Biotechnology.

Enhanced biological phosphorus removal (EBPR) is widely used at wastewater treatment plants and is one of the best studied microbially mediated biotechnology processes on the planet, according to the paper. Yet the process is not well understood at the metabolic level, and consequently, many questions surround the mechanisms that make it work.

“Engineers and microbiologists have been trying for 35 years to grow this microbe, with no success,” said Katherine McMahon, an assistant professor of civil and environmental engineering at UWM and one of the paper’s co-authors. “Remarkably, through metagenomic techniques, we were able to isolate and acquire the genome sequence of A. phosphatis without a pure culture of the organism, which, like most microbes, eludes laboratory culture.”

McMahon and her colleagues expect that the genome sequence will enable biologists and engineers to understand why and how these organisms accumulate phosphorus, allowing for major advances in optimizing and controlling the EBPR wastewater treatment process. “In particular, it makes possible further research into why some wastewater treatment plants occasionally fail,” she added.

Scope of the Issue
When EBPR works, it performs beautifully, but when it fails, excess nutrients can get washed out, potentially leading to serious pollution of lakes, rivers, and estuaries, McMahon noted. With more than 117 million m3 (31 billion gal) of wastewater being treated daily in the United States alone, any improvement in existing methods will offer treatment plants relief in meeting the increasingly stringent discharge limits for phosphorus that are expected in the future.

Microorganisms are well-equipped to do the job, but activated sludge has long been a black box mystery. EBPR systems are prone to unpredictable failures due to the loss or reduced activity of the microbial populations responsible for phosphorus removal, according to the paper. This incomplete understanding of the microbial ecology found in sludge has forced design engineers to rely on highly empirical observations.

Sequencing Step Forward
To shed some light on the challenge, the researchers compared sludge samples from wastewater plants in Madison, Wis., and Brisbane, Australia, that they maintained in laboratory-scale bioreactors to control and monitor the status of the sludge microbial communities.

Applying a metagenomic strategy directly to the sludge samples, they were able to use high throughput sequencing to get an overview of the entire microbial population contained therein and not just the sequence data for particular bacteria, as is done with genomics. In this way, “you also get DNA from organisms that aren’t doing phosphorus removal, but the key is that if you sequence enough, then ultimately you can assemble and stitch together the genome sequence of each organism in the community, rather than going organism by organism,” McMahon explained.

Subsequent analysis of the sequence data showed that despite significant differences in operating conditions, including different volatile fatty acid feeds, sludge volume, and sludge residence time, Accumulibacter species dominated both sludges, comprising roughly 80% of the biomass in the U.S. sludge and 60% in the Australian sludge, respectively, according to the paper.

Some of the initial findings reported in this study on the organism’s capabilities call into question a few previous empirical observations of how the EBPR system works, said David Jenkins, professor emeritus of civil and environmental engineering at the University of California–Berkeley. Other findings support some of these observations.

For example, in order to accumulate massive amounts of phosphorus, the organism has to shuttle the nutrient across its cell walls. It does this using transporters, which are a type of enzyme, Jenkins explained. As it turns out, the findings show that the organism has two transporters: a high-affinity one and a low-affinity one. The latter “works when it’s pretty easy to transport it — that is, when there’s a lot of phosphorus around the outside of the cell,” Jenkins noted. “When the phosphorus starts to run out, you get down to very low concentrations outside the cell,” and the high-affinity transporter, which requires a lot more energy to function, kicks in to grab the phosphorus and transport it across. “Effectively, you can get down to very low concentrations using a combination of these two transporters,” Jenkins said.

Comparatively, empirical observations have shown that plant designs using a series of compartments in the aerobic treatment zone do better than simply using one big tank of the same volume. This now makes sense, Jenkins pointed out, “because if you have a completely mixed tank, the phosphorus concentrations are the same all over.” If, on the other hand, “you have a system with a series of compartments, then at each succeeding compartment towards the effluent end, the phosphorus concentration gets lower and lower, forcing this organism to turn on its high-affinity transporter,” he said. In this way, “you go down further than you’d be able to with the low-energy one.”

What It All Means
Until now, phosphorus removal research has been limited by the lack of having a pure culture of A. phosphatis, Jenkins said. This sequence data “provides the first real way we’ve had to see what this organism can actually do, because if you know its sequence and genetic material, you can tell what enzymes it can produce and, therefore, what reactions it can catalyze,” he noted.
Of course, the genetic blueprint is just the first step toward further fundamental investigations of these bugs, but an important one.

“Now that we know all of the genes this organism has, we can use that information to design new experiments to look for the expression of the genes that we think are involved in phosphorus removal and pick apart the biochemical mechanisms that are responsible for each step in the process,” McMahon noted. “When you do that, you can begin on a much more fundamental level to understand why the bacteria store phosphorus, under what conditions they’ll stop storing phosphorus, and maybe ways to make them store more.”

Additionally, the researchers said they are hopeful the findings will lead to the development of an early warning system that could alert treatment plant operators of an impending crash or failure. “If you can get information a couple of days ahead of a crash, where you’re seeing changes in the microbial population or the organism starting to express particular shock proteins, you can maybe set about taking some actions to head it off,” Hugenholtz pointed out, allowing for a more preemptive response, rather than a reactive one.

Overall, “the findings and tools described in this landmark paper will allow the resolution of many of the questions that have arisen concerning the mechanism by which the enhanced removal of phosphate from wastewater occurs,” Jenkins said. “Understanding these mechanisms will undoubtedly lead to more efficient operation of the process and to the development of more robust designs.”

Kris Christen, WE&T