Tour of microbes - eating organochlorides

Bacterial remediation of contaminated sites is one of the tours available at this year’s Australian Society for Microbiology meeting.

Associate Professor Mike Manefield, in the Centre for Marine Bio-innovation and the School of Biotechnology and Biomolecular Sciences at the University of New South Wales, is tackling the legacy of unregulated industrial activity - plumes of toxic chemicals that contaminate the groundwater under our cities.

Manefield and his team have been working on the bioremediation of three main organochlorines for a number of years now, and this will be the focus of his talk at the Australian Society for Microbiology conference.

The first is perchloroethylene (PCE), which is used as a chemical solvent in the dry-cleaning industry. The lack of regulation before the 1970s meant that these chemicals were often disposed of by being tipped into gutters or onto the ground.

The second, 1,2-dichloroethane (DCA), is a chlorinated hydrocarbon primarily used to produce vinyl chloride monomer, which is the major precursor for polyvinyl chloride (PVC) production - a widely used plastic. DCA is also used as a solvent in the formation of polystyrene and latex.

And the third, chloroform, is a precursor to refrigerants and plastics as well as being used as an extractant.

All these chemicals are toxic. They kill in acute doses (chloroform also kills at low concentrations), are known carcinogens and because of their long half-life, they are recalcitrant in the environment. But Manefield is quick to point out that despite their toxicity the organochlorides have some very useful properties.

“These chemicals are soluble, volatile and stable, which are useful properties and they have utility in society,” said Manefield, “so we do not want to stop using them, but we need to handle them better and find better ways to clean them up.”

The stability of these chemicals - some have an abiotic half-life of hundreds to thousands of years - means that they can be transported and stored for extensive periods, but it also means that if they are spilled it can be disastrous.

Organochloride respiration

The Botany Industrial Park is an environment where lots of improper disposal of chemicals has occurred. A major clean-up of the groundwater at the site is underway and Manefield’s research feeds into this effort.

“The aquifer has been contaminated for a long time,” said Manefield. “Containment lines are in place and groundwater is pumped out of the aquifer and into a treatment plant to decontaminate the water. It is estimated this will take 200-300 years. It’s expensive and energy intensive, so we need to find a better way.”

Because organochlorides are denser than water, when they are spilled into the environment they percolate into the soil, into the groundwater and form an organic phase below the water.

“They are good at dissolving greases and fats, but they do not mix with water,” Manefield explained. “They just sit there in an organic phase on the bottom of the aquifer slowly dissolving into the water forming a plume downgradient - they’re called DNAPLs, dense non-aqueous phase liquids.”

It is this dissolved phase of the plume that the containment system prevents from entering Botany Bay. But Manefield and his team want to create a barrier of bacteria to replace the groundwater treatment plant.

One problem with this is that the environment the organochlorines occupy is anaerobic, so rapid aerobic biodegradation processes aren’t useful. A second problem is that the undissolved organochlorines sitting on the bottom of the aquifer can be toxic to the bacteria.

But there is a small selection of anaerobic bacteria that break down organochlorines. Manefield said it took a number of years for his team to work out how to successfully grow these bacteria, but they are now making progress. Industry has funded the development phase and continues to provide funding for this work.

“The bacteria also respire the organochlorides in the gradient up the plume,” Manefield said. “The transfer of electrons to organochlorines removes the chlorine atoms, which makes these chemicals harmless. The fully dechlorinated breakdown products are abundant hydrocarbons in the environment; for example, one breakdown product is ethene, which is used to ripen fruit.”

Serendipity

 Manefield’s team are working with the three groups of bacteria, Dehalococcoides, which breaks down PCE, Dehalobacter, which breaks down DCA and Desulfitobacterium, which degrades chloroform.

“They’re known as organochlorine respiring bacteria, or ORBs,” he said.

Manefield’s team takes samples from contaminated sites back to the lab to assess the activity of the microbial communities and their ability to break down pollutants.

“It is a slow process to strip a culture down to the single organism you want to look at,” said Manefield.

And they are not easy to grow. Manefield said they started working on reductive dechlorination in 2005 and it took two or three years to optimise growth conditions for these fastidious organisms.

Once they got the growth conditions right, they started working on the enrichment process, which involved creating an environment as favourable as possible for the ORB and as unfavourable as possible for competing microbes, such as methanogens and homoacetogens.

Chloroform is a particularly problematic organochlorine because it inhibits the ORB that can degrade other organochlorines. In another study, Dr Matthew Lee in Manefield’s team was assessing this inhibitory effect in some groundwater from the Botany site when things appeared to be going wrong.

“We thought we had a leak,” said Manefield, “because the chloroform disappeared from one of our replicate cultures. But then the chloroform disappeared from the other replicates as well.”

This led to the unexpected discovery of a chloroform-degrading bacterium.

The researchers began subculturing samples from the Botany site to find out what was causing the chloroform to disappear. They identified a Dehalobacter species that degraded chloroform into dichloromethane and then to acetate, carbon dioxide and methane. It’s the first isolate that can completely dechlorinate chloroform.

“The more we enriched the cultures the faster they consumed chloroform,” said Manefield. “And we found that this bacterium was much more tolerant to high levels of chloroform - the average bacterium can withstand 10 ppm, our culture is highly active at 200 ppm.”

The team has sequenced the genome of this species and discovered that it has a larger genome than other Dehalobacter species. They also found that it had 22 reductive dehalogenase genes that code for the enzyme responsible for the breakdown of the chloroform.

“This may link to its tolerance to higher concentrations of chloroform,” Manefield suggested.

Technology transfer

Manefield started a biotechnology company last year called Micronovo Pty Ltd, which serves to transfer technology from the lab into the hands of environmental consulting companies actively remediating contaminated sites.

The company conducts molecular-based diagnostic analyses on groundwater samples, using quantitative PCR to determine the capacity a sample has to break down a pollutant. They can then give advice on what is needed if a sample does not have the right bacteria for a clean-up operation as well as supply the appropriate bacterial cultures. Through Micronovo, Dr Joanna Koenig in Manefield’s team was responsible for the first bioaugmentation with organochlorine-respiring bacteria on the Australian continent.

The company currently has contracts with environmental consultant firms Golder Associates, Parsons Brinckerhoff and Aecom for Australian operations.

“We have four or five contracts at any one time currently. It won’t make me wealthy but it’s very satisfying seeing genuine application to an environmental cause. It also provides good contacts for funding ongoing research and attracts high-quality students into the lab.”

Dr Mike Manefield is currently an ARC Future Fellow in the School of Biotechnology and Biomolecular Sciences at UNSW, having spent 2004 to 2010 as a Senior Research Associate in the UNSW Centre for Marine BioInnovation where he remains Deputy Director. Following a PhD on the ability of algal metabolites to inhibit bacterial quorum sensing, he spent four years in the UK developing and applying RNA stable isotope probing. He was employed from 2001 on a continuing contract as a postdoctoral scientist in the NERC Centre for Ecology and Hydrology in Oxford, UK. He returned to Australia in 2004 and was employed by UNSW to lead the bioremediation program for the Environmental Biotechnology Cooperative Research Centre.