To Your Environmental Health

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BY DEBORAH KOMLOS

Environmental science and biotechnology are converging to pursue an important goal: to develop high-tech tools to address key issues such as land contamination and drinking water.

Canada’s contribution to the environmental biotechnology field includes research and development being conducted by the National Research Council of Canada’s Biotechnology Research Institute (BRI) in Montreal, Que. Founded in 1987, the BRI is the nation’s largest centre dedicated to R&D in biotechnology and comprises three focal areas: the Health Sector, the Bioprocess Platform and the Environment Sector.

Adrien Pilon, director of the Environment Sector, says a major issue driving environmental efforts has been climate change, and at a broader level, sustainability. For the past 15 years, the issue of sustainable development has been on the agenda for the OECD (Organisation for Economic Co-operation and Development) and for many developing countries, he says. With this in mind, the BRI has since participated with the OECD and several industrialized countries to prepare and compare reports on industrial biotechnology for sustainability.

“It’s very timely because now we’re moving into the Kyoto agenda and the Kyoto agenda will call for several developments and technological developments where biotechnology can play a significant role,” Pilon says. “First of all, for changing the sources of energy, reducing the energy (usage). So biotechnology can be used in industrial processes with lower energy needs and lower costs and less environmental impact and less greenhouse gas emission.”

Established in 1993, the BRI’s Environment Sector has 70 full-time employees, plus visiting scientists and guest workers, and seven research groups, Pilon says, with research activities directed toward pollution control and prevention, biomonitoring technologies and bioconversion processes for long-term sustainable development.

Pilon says an important strength of the sector’s research is that, aside from work with Environment Canada years ago that tested the fate of some modified organisms in a controlled field release, its researchers have not seen a need for introduction of engineered micro-organisms into the environment.

“We don’t look at it only from the potential of enzymes or organisms to do the job or function,” he says. “We try to understand the natural environment in which organisms evolve.”

Probing Microarrays

The sector’s Environmental Microbiology Group is divided into two basic components, says group leader Charles Greer, PhD. The first is based on fundamental research, studying bacteria capable of biodegrading environmental pollutants. The second focus is developing a toolbox of genetic techniques for use in the field.

“If we have a gene probe from a gene that’s involved in the degradation of some specific compound, then what we would do is look for the presence of this gene in the natural population in a contaminated soil, for example,” Greer says. “The presence of that gene is the first indicator that at least some of the right kinds of micro-organisms are there.

“What we’re trying to do is to develop tools and techniques that enable us to look at the microbial ecology of the entire system, how the micro-organisms are functioning in that system as a unit and trying to find out what sorts of things influence that, how the population composition and structure may change as a response to certain types of stresses,” Greer explains.

To this end, the team has developed a prototype microarray that has about 100 gene probes from various biodegradation pathways for organic pollutants. “The purpose of that work,” Greer says, “is to try to create some kind of profile of the community structure with regard to function and then try to quantitate how this changes over time due to pollution or due to actual cleanup of pollution.”

As a result, he says, predicting the loss of certain detectable functions due to polluting an environment could be possible, a capability that could then be used to predict the impact of pollution on a particular environment.

“Once the environment is polluted, perhaps there are certain functions you can’t detect anymore,” Greer says. “But if you’re actually cleaning up that environment, then you would monitor the population and see if those functions did get restored.”

Screening for function at the population level is important, Greer says, because there is a lot of built-in redundancy within some of the populations.

“Now if you have 10,000 different organisms in a gram of soil, there might be a hundred different species that are capable of performing a certain function and under certain conditions, you might wipe out 80 of them. So the question is, well, what happens with the other 20,” he says.

Among functional pathways for which the team is probing are those involved in the biodegradation of aromatic and aliphatic hydrocarbons; chlorinated, aliphatic and aromatic compounds; several heterocyclic hydrocarbons; and also for pathways belonging to the geochemical cycle for each of nitrogen, sulfur and carbon.

“So what the tool enables us to do is actually look at not only the capability for pollutant degradation in a particular system, but also to look at whether or not some of these other essential pathways and cycles are operating at present. That gives us a snapshot of the health of the ecosystem, if you want to put it that way,” Greer says. “Although we have a tremendous number of contaminated sites to deal with, ultimately what we want to do is to see this kind of approach incorporated into industry and manufacturing so that they’re not producing the (polluting) materials to begin with.”

Classifying the Culprits

Working in strong collaboration with Greer’s team is that of Roland Brousseau, PhD, who heads the sector’s Environmental Genetics Group.

The two groups have pooled resources to develop some of the genetic tools, such as the microarrays. But Brousseau’s team is focused on the taxonomy side, to detect and identify pathogens from environmental and wastewater sites.

Brousseau greatly praises the DNA microarray and related techniques that permit researchers to look at complex, poorly known communities. This is particularly important, he says, as many of the microbes — such as soil microbes that may be accustomed to living on the surface of silica particles or various dirt particles — will not grow on synthetic media.

“You put them on Petri dishes and they say, ‘I’m going to die here, I cannot breed! Where’s my rock?!’ So we cannot grow them. If we cannot grow them, we cannot study them,” Brousseau says of such microbes. “It’s the birth of a very new and exciting era in microbiology where we can look at the non-culturable, and we can look not only at the things that can grow but at things that don’t grow and also at their actual representation in the soil as opposed to in our laboratory microcosm or models of the soil,” he says.

As Brousseau and his team are concentrating on the specific bacterial functionality of pathogenicity, taxonomic identification is important. For instance, while Greer’s team may identify a gene in E. coli that can degrade naphthalene, Brousseau says his team would need to know which exact type of E. coli is present — if it were strain K12, the non-pathogenic strain that is commonly in human gut microflora, or the highly virulent E. coli O157:H7 (see images, page 15). Having this identification is crucial, he says, because people who study water need to know if the water supply has significant levels of the pathogenic strain.

Projects Brousseau and his group are working on include developing a host-pathogen model of the spruce budworm using DNA microarrays. Specifically, they have developed two types of biochips to detect the Bacillus thuringiensis (Bt) toxins that are used mainly to control forest infestations of insects such as the spruce budworm.

The chips are printed on standard-sized lab microscope slides, with the detecting region — an area of less than one centimetre squared — containing gene probes. The first biochip type has 100 gene probes corresponding to known Bt insecticidal toxins.

“So if I test a sample of unknown DNA from soil or from a culture, after that I will know right away which known genes are present. And if I see some unusual combinations I will infer, Well, maybe these new genes, these are something that don’t light up on my chip,” Brousseau says. “But if the sample is known to be toxic to insects, I need to find out what the new genes are so I can represent them on my next generation of chips.”

The second biochip, containing about 500 genes that are involved in the response of the spruce budworm to Bt, assesses the host-pathogen interaction. Response of the insect to the toxins is expressed in the various tissues of the insect. The group uses express sequence tag (EST) analysis to compare the noted patterns with those of genes found to be either over- or underexpressed when the insect is exposed to Bt toxins.

“So it helps us understand how the insect is damaged or stressed by these toxins,” Brousseau says. “And knowing that, we may be able to add other components or an insecticide to bring the stress level to a point where either we have faster kill or, contrary, where we try not to kill the insect but to really decrease its feeding ability inside, without totally messing up its reproductive success.” He adds that breeding is still desired, but while the insects are still small and eat less foliage, and without selecting for resistance in the next generation.

The Molecules of Things

The Analytical Chemistry Group at the BRI, headed by Jalal Hawari, PhD, is also working on detection methodology, but targeting the chemical side of the pollution story.

The team has developed characterization tools for on-site locations such as military sites, gas stations, refineries and commercial sites that are contaminated with hydrocarbons, and specifically, to discern whether or not these pollutants originate from petroleum products.

The methodology, initially developed in the mid-1990s but still being optimized, has provided a much-improved technique over the procedures that were in place, Hawari says, which relied on infrared radiation that could detect anything with a carbon-hydrogen bond.

“Any organic chemical on it has a C-H bond, so the results were biased. So sometimes they are about 10,000 times higher than what is coming from petroleum products,” Hawari says. Examples he gives of possible non-petroleum sources of soil organics include the residue remaining after a barbecue, or years’ worth of organic material accumulation from leaves falling and decomposing in the soil.

“So we had to develop a method specifically to tell us that this is hydrocarbon from petroleum, and these methods are now sound and used by the petroleum industry, by the pulp and paper industry,” he says.

Hawari’s group developed a separation technique that takes an extract from soil or bacteria (i.e., the biomass), and performs fractionation, chromatography cleanup and then compound separation into different families of aliphatic hydrocarbons, aromatic hydrocarbons and polar chemicals. Controls are used, Hawari explains, to make comparisons with the extract profiles from uncontaminated soil. These procedures involve GS-MS (gas chromatography-mass spectrometry) and GS-FID (gas chromatography-flame-ionization detection) protocols that the team has developed.

The fraction of organics originating from the contaminated soil is referred to as TPH (total petroleum hydrocarbon), Hawari says, and includes PAHs (polycyclic aromatic hydrocarbons), which have an aromatic ring such as benzene, xylene or toluene. BTEX is another detectable component, which represents four specific aromatics (benzene, toluene, ethylbenzene, and xylene) that can volatilize, and the gas phase can be collected and analysed by the devised methodology. Other analytical techniques the group has worked on include LC-MS (liquid chromatography-mass spectrometry) to analyse explosives, heavy metals and other pollutants.

One of the group’s current projects is a $1.7-million collaboration with the United States Air Force and Canada’s Department of National Defence to examine the degradation and environmental fate of two common explosives, RDX and HMX.

“We study how these chemicals migrate from first the soil where the training is happening, through sub-surface soil to reach groundwater, or if the plant absorbs these chemicals and what do they do with them,” Hawari says.

He likens understanding the environmental fate of chemicals to the importance of knowing the pathway that a drug follows through a human body and what the drug does to a disease, both of which influence the drug’s effectiveness.

“If a chemical engineer was to develop a process, he needs these parameters — how A goes to B, what is in the middle,” Hawari says. “Process optimization requires understanding all the reaction steps involved in the degradation process.”

Sustaining Development

Under the direction of Peter Lau, PhD, the Bioconversion/Sustainable Development Group is addressing various topics including biodegradation and working with naturally occurring microbial enzymes to create value-added products. Examples of the team’s work on biodegradation are shown on the bottom of page 16 and above.

“Sustainable development is a field that is threatening to have a small group addressing it,” Lau says. “But nonetheless, I think we have an angle through biotechnology . . . The goal is really to have a sustainable environment which includes a bioeconomy, through research that aims at the development of a toolkit of biocatalysts and, in the longer term, to have integrated bioprocesses that have attributes of ecoefficiency.” The group also hopes to deal with the energy sector and the chemical industry, to reduce both costs and greenhouse gas emissions, and have accessibility to new products, Lau adds.

A specific current project involves a group of enzymes referred to as Baeyer-Villiger monooxygenases (BVMOs), which occupy the second step in the biotransformation of ketones to lactones. While BVMOs can be used in industry to produce biopolymers, Lau says his group is not aiming to produce those materials, but instead is using the technique of directed evolution to improve the performance of naturally occurring enzymes for industrial application.

“I prefer to call it an enzyme system because with each one of them, if you go through the characterization of it, meaning that if you determine the substrate specificity of these enzymes, you will find that they are not all the same and that they will react as complementary reagents,” Lau says. “So this has the opportunity to give you a spectrum of diverse products.”

For instance, Lau envisions that this enzyme group could be used to produce stereo-specific chiral products that a chemist could then use as building blocks for further synthesis of a desired compound.

Lau says another application of the microbial toolkit is to provide what he refers to as “controlled” biodegradation, which uses bioconversion to produce usable products from the various steps in a degradation pathway. This has an advantage of creating “value-added products,” he says, because material that may otherwise be discarded as a byproduct or waste material of a particular pathway step is converted into something usable.

Echoing Pilon’s comments, Lau says his team is looking to the natural environment as a bioresource for enzymes. In fact, most of the microbes the team is using come from culture collections and many have not been worked with since their isolation.

“So for example, a couple of strains that we are studying actually have been in the ATCC collection or they have been isolated for some 30 years,” Lau says. “And, the more we look at it, the more fascinating these guys are. So these are like the oldies but the goodies. The bottom line is we simply do not know enough of the microbes around us, of their capabilities. You can isolate new organisms, but the old organisms in the culture are there screaming for characterization. It really depends what you are after.”