Was the structure of benzene really illuminated by a chemist’s dream of a snake devouring its own tail? Historians of science are skeptical of this persistent chemistry legend. But there is new interest in devouring when it comes to benzene, a naturally occurring toxin often associated with petroleum production, storage and transport. To eliminate this toxin from contaminated environments, a research team led by Elizabeth Edwards in the Department of Chemical Engineering & Applied Chemistry at the University of Toronto is testing benzene-devouring microbes that thrive in anoxic, or oxygen-poor, contaminated groundwater. Because polluted environments tend to quickly become oxygen-starved, a microbial clean-up crew capable of doing its job in anoxia is a promising development.

Elizabeth Edwards of the University of Toronto.

Elizabeth Edwards of the University of Toronto. Photo credit: Sean Caffrey

Benzene is part of a family of compounds known by the acronym BTEX, which refers to benzene, toluene, ethylbenzene and xylene. BTEX compounds comprise about one to five percent of crude oil and gasoline. But whereas most components of gasoline and crude oil are not appreciably water soluble, the BTEX compounds are. 

The molecular arrangement of benzene in a symmetrical ring structure makes it “a little bit more polar than your typical hydrocarbon,” says Edwards. This polarity makes it more soluble in water, which means two important things. First, this toxic molecule is transported easily within living organisms. Second, whereas in a gasoline or crude oil spill the heavy, tarry components stick to the soil and stay put, benzene, toluene and xylene do not. These compounds evaporate into the air and can dissolve into groundwater, moving wherever it flows, spreading and creating a plume of contaminated water.

Benzene, with its symmetrical ring structure, is a “beautiful molecule,” says toxicologist Peleah Black Moher, an environmental exposures lead with CAREX Canada, a multi-institutional carcinogen surveillance research project. But, referring to its links to a host of adverse human health impacts, benzene is also “a beast,” Black Moher says. “It’s able to form covalent bonds in a variety of macro-molecules in the body — like big proteins and DNA.”
The precise mechanisms of its biological damage are still being investigated but “because it’s binding with proteins, it’s messing a wide variety of things up within the body,”

Black Moher says. Chronic environmental exposure over a period of years can be detrimental because when attached to DNA, benzene can cause cancers like leukemia. CAREX defines benzene as “a ‘non-threshold toxicant,’ where adverse health effects may occur at any exposure level.” Says Black Moher, “For cancer, there is no ‘safe level’ or ‘threshold’ because every exposure has an associated risk.”

Health Canada, on the other hand, has established maximum allowable exposure levels. 

Non-cancerous effects of benzene exposure include bone marrow degradation, decreased red and white blood cell production, anemia, bleeding disorders and a hampered immune system. Short-term acute exposure to benzene, inhaled at levels of between 50-150 parts per million over a period of several hours, can cause neurological symptoms like dizziness, headaches, drowsiness and tremors. And while the most common exposure to benzene for Canadians is from breathing it in, especially from air that is polluted by exhaust fumes and cigarette smoke, benzene also shows up at low but measurable amounts in food and drinking water. According to Health Canada, in surface waters benzene is not generally a concern because it evaporates into the atmosphere. Its levels in water generally fall below their established maximum allowable concentration of 0.005 milligrams per litre. 

U of T graduate student Johnny Xiao with post-doctoral fellow Fei Luo check a bioreactor containing microbes that break down benzene anaerobically.

U of T graduate student Johnny Xiao with post-doctoral fellow Fei Luo check a bioreactor containing microbes that break down benzene anaerobically. Photo credit: Sean Caffrey

When it comes to water, a bigger challenge is when benzene gets trapped in groundwater. Safely removing benzene from groundwater environments is the scientific challenge Edwards is tackling. It was during graduate school in a large bioremediation laboratory at Stanford University in the late 1980s and 1990s that that she fell in love with the science behind how living organisms could clean up contaminated groundwater. “Our groundwater is so precious and so difficult to clean,” says Edwards. Unlike a river or a lake that flushes out, allowing it to recover after contamination, groundwater is extremely difficult to remediate because of its long residence time. “Things can spend hundreds or thousands of years underground moving very slowly,” she says. That means that in groundwater, contamination can take decades or longer to clean up.

Edwards has found a helping hand in nature. Crude oil and natural gas are natural compounds and lots of microbial communities can degrade hydrocarbons under a diversity of conditions, she explains. By taking naturally occurring microbes from field to lab, Edwards is working to enrich those microbes. Once identified, she can rear them in larger quantities, selecting those that do their benzene-busting job best. 

The most common use of bioremediation today, explains Edwards, involves actively turning soil to oxygenate it, stimulating aerobic degradation of contaminants by microbes. In wastewater treatment too, oxygen is actively bubbled into the water. “But when contamination gets deeper, it’s really difficult to get enough oxygen in,” she says. Once oxygen is depleted, it’s hard to re-oxygenate groundwater because oxygen has to diffuse downwards from the surface, through soil, into the water. That’s a slow process. And once groundwater is anoxic, it puts aerobic benzene-eating bacteria out of business.

Edwards describes benzene’s molecular configuration as “like a frying pan with no handle.” That structure makes it difficult, at a chemical level, for enzymes to get hold of it and react with it, in the absence of a powerful oxidant like oxygen. But during her PhD work, Edwards turned up evidence that microbes could degrade benzene, very slowly, in the absence of oxygen. “Originally it was discounted as an error of the laboratory,” she says. But follow-up work confirmed it was no error — anaerobic microbes capable of breaking down benzene did exist. Her curiosity was sparked. For 25 years, Edwards has been focused on identifying and understanding these munching microscopic lords of the ring.  
Turns out this benzene breakdown machinery is mediated by not just one but a whole suite of microbes. In nature, these microbes occur at low abundance and work very slowly. So by growing cultures of anaerobic microbes in laboratory flasks, feeding them pure benzene and selecting the microbes that break it down best, she is using evolution by artificial selection in an effort to develop cultures that break benzene down more speedily. 

Edwards has discovered via genomics that a few key members, particularly from the class known as the Deltaproteobacteria, dominate this microbial community. It’s not the first time that Edwards has harnessed tiny clean-up crews in the aid of neutralizing the impact of pernicious pollutants. In the past, she successfully identified and commercialized a microbial culture named KB-1, marketed in collaboration with Ontario’s environmental remediation laboratory SiREM, for cleaning up contamination by chlorinated solvents such as those used in dry cleaning. 

Edwards is not the only researcher on a mission to find biological solutions for benzene clean up. Carsten Vogt, an expert in anaerobic bacterial degradation of groundwater pollutants at Germany’s Helmholtz Centre for Environmental Research in Leipzig, says that of all the hydrocarbons, benzene is “more or less the most stable compound, which is why it’s very complicated to degrade. From a chemical point of view,” Vogt says, “it’s a very difficult reaction to activate in the absence of oxygen.” 

As yet, the degradation pathway for microbial breakdown of benzene is not completely understood. In Vogt’s work using stable isotopes of carbon to follow the progress of benzene breakdown, he has identified gram-negative bacteria similar to those found by Edwards as key players in a “consortium” of microbes. These microbes are difficult to work with, Vogt explains, and in anaerobic conditions they are doing very complicated reactions. “That’s why I’m fascinated.”

In her lab in Toronto, working with samples from multiple hydrocarbon-contaminated sites, Edwards has been identifying, enriching, culturing and selecting the microbes that break down benzene anaerobically, converting it to methane and carbon dioxide. In partnership with SiREM and with funding from Genome Canada, Mitacs and the Ontario Ministry of Research and Innovation, team members have increased growth rates and abundance to the point where they are scaling up from one-litre growth flasks to 100 litre reactors.

By culturing these do-gooder microbes, Edwards’s hope is that they can eventually be used to speed up decontamination of polluted soils and groundwater via bioaugmentation. Just like a microbial culture added to starter mixtures to make yogurt or beer, in bioaugmentation, contaminated soil or water is seeded with the microbes best suited to eat up toxins. Once inoculated, she says, these specialized microbes will grow where benzene is plentiful and die off once their benzene food is consumed, leaving the groundwater cleaner.  

Sandra Dworatzek, senior manager at SiREM, is “very excited about the promise of this research and development initiative. The development of this culture is a perfect fit for SiREM as we continue to increase the types of cultures available for bioaugmentation and bioremediation of recalcitrant chemical contaminants,” Dworatzek says. 
Like the speed at which anaerobic microbes consume benzene, scaling up to full commercialization may take some time. But Edwards is determined to move forward by throwing her hat — along with her chemistry — into the rings.