Researchers at Yukon University are working on a tiny solution to a big mining problem–using local bacteria to treat nitrate-contaminated waste water.

Thanks to a $75,000 grant from the Mitacs Accelerate Program and Minto Metal Corp, along with partnerships with the National Sciences and Engineering Research Council (NSERC) and the Institut national de la recherche scientifique (INRS), Yukon University Master’s student Taylor Belansky is sampling and testing bacteria taken directly from the Minto Mine site, an underground mine which extracts copper and gold approximately 240 KM north-west of Whitehorse.

Nitrates–one nitrogen molecule bound to three oxygen molecules (NO3)–are a common compound of nitrogen, often formed naturally as part of the nitrogen cycle, the process of nitrogen re-uptake and distribution which is essential to life on earth.

Nitrates, however, have a number of industrial uses, and nitrate runoff can accumulate in water supplies and cause eutrophication, a form of nutrient pollution which throws aquatic ecosystems out of balance. This can lead to out-of-control algae blooms–often physically observable as a sheet of green sludge on the surface of the water–and oxygen depletion, rendering the water bodies uninhabitable to fish and other aquatic life. One of the most common forms of nitrate contamination in the south comes from agriculture, as nitrates are a common fertilizer.

High quantities of nitrates in drinking water can also have consequences for human health, including an increased risk of cancer, stomach issues and headaches, and are known to cause deleterious effects on babies and small children.

In the northern mining industry, the main source of nitrate runoff comes from a source a layperson might not even consider– it’s generated from blasting residue, says Guillaume Nielsen, NSERC Industrial Research Chair in Northern Mine Remediation with Yukon University. Ammonium nitrate fuel oil (ANFO) is a commonly used explosive, popular due to it’s simple production and low cost, but leaves behind nitrates as a waste product, which then need to be contained and cleaned up.

Traditionally, clean up of nitrates and other forms of nitrogen pollution has been done via “active” remediation processes, says Nielsen, a broad term which encompasses methods of purification in which continuous human oversight is required, such as the introduction of other chemicals, reverse osmosis and electrodialysis. In addition to being expensive to execute–especially in the North, where everything has to be shipped in–some of these active methods can also produce additional waste, which then itself needs to be disposed of.

By contrast, using specialized bacteria–denitrifying bacteria, which naturally uptake and break down nitrates (NO3) into nitrogen gas (NO2) as part of their respiratory process–is a kind of “passive” form of treatment, which uses “what mother nature can bring,” says Nielsen.

Passive methods of remediation are not new to the mining industry– for example, naturally-occuring bacteria are being used to clean up the Silver King site at the Keno Hill mine, where they uptake dissolved sulphates present in waste water and convert them to sulphides, which bond to toxic heavy metals, such as cadmium and zinc, making them more stable and easier to clean up.

Passive methods of nitrate removal have been previously used successfully in the South, says Nielsen, but can “be very challenging up North, because of the cold climate we have.”

“There’s a lot of potential failures, due to the freezing temperatures, which reduce microbial activities,” Nielsen says, adding that other factors, such as pressure and food availability, can impact bacterial ability to process nitrogen.

The northern biome already has bacteria in it that are used to living–even thriving–under northern conditions, and so instead of just trying to import strains that have worked in warmer climates, Nielsen and Belansky have been searching for home-grown bacteria already adapted to the harsh Yukon climate.

“Understanding how tough (these bacteria) are opens a new world of investigation,” says Nielsen. “It’s so worth it to work with these guys (the native bacteria) and understand them and make them work for us.”

It’s more complicated, however, than simply finding the “right” bacteria to do the “right” job–soil is home to a broad biome of bacteria, all working in delicate concert with each other. Not unlike your own gut biome which can host trillions of living bacterial flora composed of a multitude of species, all working and living in a complex ecosystem. So in their work, Belansky isn’t just looking to find and separate out one specific strain of denitrifying bacteria–she’s looking at entire naturally occurring biomes of bacteria, to see which one is most efficient under the conditions of the bioreactor.

In order to do this, samples are taken from the Minto Mine site and then meticulously preserved in a bacterial sample pack, a cotton pack Neilsen says looks a lot “like a tea bag,” on which “bacteria may have been growing for many years.” As soon as you remove the samples, however, the bacterial population is subject to change–some of the bacteria may be anaerobic, meaning they don’t like oxygen, or some may be temperature sensitive, or sensitive to light–which means handling them can be tricky.

“You don't want (that sample) to change–you want that sample to stay as it is exactly until you can do the DNA extraction and genetic characterization,” Nielsen says.

To keep the sampled biomes exactly as they are, samples are put into liquid nitrogen, which holds them at around -200 degrees Celsius. They’re then transported to the lab and put into another specific freezer, which holds the samples at -86 degrees Celsuis, until they’re ready to be thawed out and (hopefully) “ready to go full flow.”

“This (process) is just to tell the bacteria to stop evolving, stop changing,” Nielsen says.“They’re such tough little guys and it’s so interesting and important to work with them.”

Tough as they may be, they’re still living creatures, working within the limits of their biology. In order to utilize them to clean out nitrates, they can’t just be left in wastewater ponds, exposed to the elements. Instead, the denitrifying bacterial ecosystems are put into controlled food “bioreactors” into which waste water and food are pumped. In Yukon winters–where temperatures can easily fall to -30 or -40 degrees Celcuis, well below the freezing point of water–the reactors typically need to be maintained at between 3 and 5 degrees Celsius, says Nielsen, which is around the temperature of your refrigerator.

“Denitrifying bacteria transform nitrate–NO3–through a series of transformations into nitrogen gas. So (that’s) what we want to look for in those bacteria that we've collected from the mine,” says Belansky. “We give them the right conditions, the right kind of food and a certain known amount of nitrate, and then we sample up the bacteria, plus all that food and that nitrate over time.

“We look to see if that nitrate is going down, and (if it does) we know we've got (denitrifying) bacteria and that they're doing what we hope for them to do.”

In order to succeed in their task of breaking down nitrates into nitrogen gas, the bacteria in these bioreactors need not only the right conditions and temperature, but something to eat, along with something to cling on to to form colonies in the absence of soil, both issues for which Nielsen and Belansky are seeking unconventional and local sources.

Firstly, to feed the denitrifying bacteria in a bioreactor, you need an appropriate food source; although they need it to survive, denitrifying bacteria don’t actually “eat” nitrates–they eat carbon. Carbon from sugars is easier for bacteria to take up–it’s “way more bio-assimulatable,” says Nielsen– so in the past sugar-rich feeds like ethanol, methanol, sodium acetate and even molasses have been used.

Belanksy, however, is pioneering a different approach, and is experimenting with materials more readily available in the Yukon–even right there on the mine site–such as wood chips, waste from Yukon breweries, and even harvesting and using invasive species, such as foxtails (Hordeum jubatum) and sweetclover (Melilotus albus).

Likewise, Belansky is looking at local and ecologically friendly alternatives to the plastic beads which have been the “industry standard” as a growth medium, she says–especially given the growing concerns around microplastics and plastic pollution in the Arctic.

To be a good medium, whatever she chooses needs to have “a lot of surface area for bacteria to grow on” while allowing water to pass through, keeping the aerobic (oxygenated) system healthy. To this end, Belansky is trying out a simple, highly biodegradable and cost-efficient alternative–Arctic char (Salvelinus alpinus) vertebrae, readily available from Icy Waters, a local industrial scale fish farming operation, for whom the bones would otherwise be a waste product.

“The (vertebrae) have a similar size and similar properties (to plastic beads) that would make them suitable to host the bacteria that we want to grow,” says Belansky. “They’re a natural source in the North.”

As part of her work looking for bacterial ecosystems which might best process the offending nitrates, Belansky took 20 different samples from around the Minto Mine site, most of which were within a few kilometres of each other. Sixteen of those were sediment samples.

In her section of the lab at Yukon University, Belansky has these 16 samples set up in large glass jars, side by side, which have been fed for growth and tested for nitrate reduction capabilities. For anyone who has ever grown their own sourdough or made their own kombucha, the samples look remarkably similar to something you’d find in your own fridge–some are the colour of good tea, some are murky and thick like an old cup of coffee, some are pale and thin like brine, some have sediment and others do not–despite being taken so close to one another.

As a general rule, she says, the darker in colour these biome brews are, the more successful it was at removing nitrates, although an amazing 15 of the 16 samples achieved between 96 and 98 percent reduction of the compound.

Only one sample was unsuccessful, and in the line up it was easy to spot–clear, with a solid base of sediment.

Although she can’t be certain why that particular biome didn’t take, Belansky suspects it has to do with the conditions during sampling last year.

“It was a really high water year, with the floods and everything,” she says. “This changed the conditions and made it look like the conditions where we found (the sample) would be really good, but probably (would be different) in a normal year. That's my guess.”

Belanksy and Neilsen hope to have their first prototype denitrifying bioreactor set up and doing a test run at the Minto Mine site sometime in September. In the meantime, they’re continuing to tinker and experiment with their samples, trying to hone in on the best biomes and conditions to work with at the mine site.

Belansky says her favourite thing about working with these bacteria is just how different the samples are, even when taken from the same general area.

“I just think that what's amazing about them is the diversity just in a small area. When you do soil profiles like this… you can take one, and then like three meters that way, you get a totally different soil profile.”

Belansky has a collection of the samples behind glass on a little plaque, like a set of paint swatches, although she herself describes it as more of “a class photo.” Holding it up, she looks at the samples with obvious affection.

“I don’t think I have a favourite,” she says. “I love them all equally.”