Wading through Mendota’s Mysteries

Lake Mendota is called the most studied lake in the world, but we still don’t have a clue. Katherine (Trina) McMahon and her team are exploring its microbial dark matter for answers.

It was a silly question, so Trina McMahon laughed. What’s more important: a lab coat or a Twitter handle? “Twitter handle, for sure. We don’t do anything anymore in the lab,” she says. “Probably a pair of muck boots is even more important. You’ve got to get dirty in the field and get your samples, and then maybe spend a day in the lab, but then you spend the rest of your time in front of a computer.”

Microbial ecologists like McMahon use computers as their eyes. The bacterial communities they study — microbiomes in the human gut, in a Yellowstone geyser, in Lake Mendota — are almost entirely invisible. How, then, to see? “What we’re spending so much of our time doing in microbiome research is natural history, what the plant ecologists were doing 120 years ago, running around with their field notebooks,” says the Vilas Distinguished Achievement Professor with appointments in both bacteriology and civil and environmental engineering. “Only our field notebooks are our sequencers.”

It’s the first golden age of microbiome discovery, and this generation of microbiologists has little need for a microscope. Instead they use increasingly sophisticated techniques to read the genetic code of entire ecosystems, running complex statistics on powerful computers to sketch their specimens. It’s undoubtedly a paradigm shift — in humans, for example, it’s been suggested that the human microbiome is so important to human health that it’s like discovering a new organ system.

Could the next breakthrough come from Lake Mendota?

Sam Schmitz BS’17 collects water samples near the buoy marking the Mendota Deep Hole, the deepest part of the lake (about 25 meters). (Photo by Sevie Kenyon BS’80 MS’06)

 

 

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Lake Mendota is often called the most studied lake in the world. That’s in part because Edward Birge and Chancey Juday helped launch the science of limnology at the University of Wisconsin. The Center for Limnology has been a locus of world-class ecological research for decades, developing some of the most complex ecological models in the world.

It now also happens to be the lake with the world’s most amassed microbial data thanks to 18 years of methodical sampling now overseen by McMahon’s lab. This shared focus on Lake Mendota implies a certain kinship of purpose, but it also stokes a friendly intellectual rivalry.

McMahon knows all about Lake Mendota’s fabled scholarship, but she has her critique: those models ignore microbes. The limnologists say that the microbes are always there, in pretty much the same numbers, and they always do pretty much the same thing: turn dead things back into their constituent nutrients and carbon dioxide. Why worry about them?

“I think Trina has been very bold in being willing to do the Birge and Juday thing, the pure descriptive phase of it,” says recently retired UW Center for Limnology director Stephen Carpenter. “As a basic science enterprise, I totally support it.”

At the same time, he acknowledges it wouldn’t be hard to find ecologists who would question the return on investment so far. “That kind of modeling is very important,” McMahon acknowledges in return. “But it glosses over all of the mechanism. I want to understand the mechanism.”

Just one example: over the last decade, microbial breakthroughs have rewritten our understanding of the nitrogen cycle, the natural processes that convert nitrogen in the environment into different chemical forms. “Because there may be something in the mechanism that fundamentally changes the coarser scale models in a way that you can’t predict.”

“Respect the microbes” is a motto printed on the back of one of Trina McMahon’s T-shirts. (Photo by Sharon Vanorny)

 

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Robin Rohwer winces as she opens her laptop to launch R Studio, an interface used for statistical programming. Her left middle finger is broken and bruised, the result of an epic race-day capsize in Lake Mendota. It was so windy the race was canceled, and five of the six sailboats dumped on their way back in.

A lifelong lake junkie, Rohwer knows lakes, the look and feel of them. If you told her what microbes were present, she could probably tell you the color of the water. But if you broke out mugshots of Lake Mendota’s most common bacterial species, she wouldn’t recognize a single one. For a fish biologist or a botanist, that would be unthinkable.

Rohwer uses R Studio as her X-ray spectacles. She wasn’t a programmer when she started in McMahon’s lab in 2011, but now she has a library of personal code. “I just make a loop and look at it in a ton of different ways,” she says. By season, by week, by top 10, by temperature, depth, and light intensity.

The resulting kaleidoscope of graphs are exploratory plots that guide her toward a more intuitive understanding of the data. “When I visualize them, what I see in my head is the curve over time,” she says. “Is it spiky? Is it smooth? That’s how I think. Even if you don’t see a pattern, it gives you an idea of something to start with.”

It’s a necessary perspective given the crazy diversity of microbes. Rohwer’s been trying to decode 11 years’ worth of bacterial samples collected from the deepest point in Lake Mendota between 2000 and 2011. The mission: identify everything in these 95 samples.

During this time, as many as 29 fish species were found in the lake alongside 18 species of zooplankton and 16 species of aquatic plants. For microbes, the magic number might be 17,437. That’s not 17,437 species, but 17,437 OTUs, or operational taxonomic units. “We can’t use the word species because that’s taken by the microbiologists,” she says. They have very strict definitions of a microbial species. “But we need to call it something in order to work with it.”

Microbes facilitate the cycling of almost every nutrient through the lake ecosystem, and their DNA contains signatures of these chemical reactions. Rohwer uses these signatures and other genetic fingerprints to sort the microbes into OTUs. What emerges is a rough picture of “who” is probably doing what.

While the majority of bacteria survive using fairly basic life chemistry, bacteria are so prolific and diverse that you can’t rule out the possibility of something really funky, something you couldn’t even imagine. It’s microbes, after all, that have evolved to survive temperatures above boiling and to tolerate toxic heavy metals. “Microbes are crazy diverse,” McMahon says. “We don’t know if 17,437 actually means that there are truly 17,437 different ways of making a living in the lake, or 25. That’s one of the things that we’re trying to figure out.” Those 25 OTUs are the most common threads, present most of the time, and clearly the workhorses of the lake.

Then there’s the remainder, making up the majority, called the “long tail” because that’s what their frequency of occurrence looks like when plotted on a graph. Most of these 17,437 OTUs occurred only once, on one day, but this rare biosphere makes up a huge proportion of the data set. “What is this deep diversity doing in the ecosystem?” Rohwer asks. This is a primal question driving microbial ecologists, but she just shrugs: not enough data.

Realistically, they have little idea of what even the common bacteria do. Consider AC1, by a long shot the most prosperous family of bacteria in Lake Mendota. “AC1 is just so abundant and nobody knows what it does,” Rohwer says. But here’s the kicker: Twenty-five years ago, nobody even knew that AC1 even existed.

 

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In the beginning, there was the Great Plate Count Anomaly. Early microbiologists noticed that while microbes were abundant and ubiquitous, most of them would not grow in the lab. Even today, it’s estimated that fewer than 1 percent of bacteria sampled from the environment can be cultivated using standard laboratory methods. It wasn’t until sequencing breakthroughs in the 1990s that scientists could begin to close in on these cryptic microbes.

The most amazing story of microbes hiding in plain sight is an order of tiny oceanic bacteria called SAR11. Until 1990, SAR11 was nothing more than microbial dark matter. But once its genetic signature was catalogued, SAR11 was discovered to be prolific beyond belief — numerically, its various species comprise about half of the microbes in the ocean. When they discovered a virus that infects SAR11, it took little more than a back-of-the-envelope calculation to declare it the most abundant organism on the planet. This is how a microbe shakes up the world.

Before long, the hunt was on for a freshwater equivalent to SAR11. DNA from AC1 was first recovered from an Arctic lake in 1996, and since then it has been discovered in every lake that’s been examined. Like SAR11, it is dominant, particularly in Lake Mendota, which McMahon calls an AC1 factory. “They’re so small that sometimes people probably thought they were viruses,” she says. “We knew that there was something, but we didn’t know their names or anything about them.”

When Alexandra Linz arrived in McMahon’s lab in pursuit of her Ph.D., McMahon suggested a high-risk, high-reward project: cultivating AC1. It’s never been done, and success could launch a career. Every month or so Linz collected another sample from Mendota and she’d inoculate another 96 cultures, each recipe unique. She’d return a month later, but nothing took. After a year of this — more than a thousand tries — Linz realized that the potential number of variables in play compounded to a frighteningly large number. She wanted a Ph.D. project, not a lottery ticket, and moved on to broader survey work comparing lakes in northern and southern Wisconsin.

Basic comparisons can be made by using different sets of sequencing data, such as DNA and RNA. “Looking at the genomes is like looking in someone’s toolbox,” Linz says. “You can probably tell what profession they are, a woodworker or a plumber, just by what tools they have in their toolbox. But looking at the RNA is like looking at what tools they have out on their workbench. What are they doing right now?”

Her Ph.D. work is focusing on the role of microbes in carbon cycling in lakes (an area of particular value in understanding climate change). But along the way she also got involved in looking at seasonality — how the microbial community changes from year to year. Seasonality is one of the baseline rhythms of biology, patterns that humans have probably observed since even before we became humans. Seasonal variation, particularly in lakes that ice over regularly, is a cornerstone of lake science.

Surprisingly, Linz found no seasonality in the microbes that live in a certain kind of lake in northern Wisconsin. “I’ve looked at the data every way I can think of to try and find a seasonal trend, but I haven’t been able to find one,” she says. Previous studies had found seasonality in other kinds of lakes, but they’d also noted a higher degree of variability in summer. “Maybe it’s not so surprising that we can’t predict the summer community based on the previous year,” Linz says. But still, the finding hints at a layer of difference and complexity separating microbial ecology and its coarser-scale cousins.

Is there a longer cycle or a more complex link to weather that can’t be seen because we haven’t been looking long enough? Or maybe, after another decade of research, we’ll realize that it’s just a dice roll? “We know there is an element of randomness in microbial communities,” Linz says, apparently only a little frustrated by the endless enigma. “I think it’s really fun that there is so much unknown about microbial ecology. It’s a young field, and there’s a lot we still have to discover.”

 

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Linz’s efforts to cultivate AC1 in the lab were not wasted. A few cultures produced a drastically reduced mix of microbes, including AC1, and were sequenced to figure out if cooperation was their survival secret. Then Sarahi Garcia, a visiting scientist from Uppsala University in Sweden, helped McMahon’s lab sequence a single AC1 from Lake Mendota, part of a search for the light-sensing protein rhodopsin, which had been found in other AC1 specimens. Already well understood because of its sensory role in vision, there’s also growing evidence that, in microbes, rhodopsin doesn’t just sense light but can also capture its energy.

This is not in your father’s biology textbook, and it probably wasn’t in yours, either. Microbes are infamous for all kinds of funky metabolic tricks, but this could change the way we think about lakes. “It’s a way to get energy without using chlorophyll,” McMahon says. “There could be all of this biomass and energy generation going on that we’re not accounting for in our models that assume chlorophyll is a main driver.”

The best way to prove it would be to create a pure culture of AC1 and show that it can survive on light alone. But recall the Great Plate Count Anomaly and how nobody has successfully cultured AC1. That leads you back to the genome.

AC1 has a very small genome, McMahon says: “Like crazy small, endosymbiont small.” She’s talking about bacteria that evolve in a symbiotic relationship with an organism and rely on their host for so much that they can afford to jettison many genes. “To find a genome that small in a free living organism is weird.”

So AC1 is incredibly abundant, which is to say, highly successful. It also has a very small genome, but its genes include the ability to manufacture rhodopsin. So it stands to reason that the rhodopsin is doing something. But what?

To help unravel the puzzle, McMahon approached her UW colleague Katrina Forest, a bacteriologist who studies photoreceptors — proteins that respond to light. Forest was intrigued by the science and tickled by the implication that everything we understand about the equations for carbon and energy balance in lakes, and not just Lake Mendota, may be askew. “I love it when you realize that, even in our advanced times, when you can justifiably think we’ve already solved most of the big problems, that there
is something so completely not appreciated and novel,” Forest says.

Forest’s lab has been hard at work teasing out details on a molecular level, getting closer to understanding what the rhodopsin is doing. “We still don’t have any proof that AC1 is doing primary production in the lake, but it certainly has got all of the jigsaw puzzle pieces,” she says. “This organism is encoding this phototrophy system that really is brand-new in terms of understanding how the lake ecosystem keeps itself alive.”

A similar investigation is playing out in the oceans over SAR11, which also has a rhodopsin structure. Dueling calculations disagree over whether primary production is even possible, though McMahon says that the current consensus is that SAR11 isn’t doing much of it. One theory is that the rhodopsin may help the microbe survive extreme conditions.

“Nobody has done the calculations in lakes, and I’m not even convinced that the calculations they have done in the ocean really account for everything,” McMahon says. She’s not willing to declare victory on her AC1 primary production theory, but neither will she concede.

“If they didn’t have this, then they probably wouldn’t be so ubiquitous and abundant,” she suggests. “I think it’s still open. I mean, that’s your sense of mystery, right?”

The preponderance of evidence in Lake Mendota is clear: phosphorus is the problem. Too much phosphorus leads to an overgrowth of algae, which leads to stinky, pea green lakes. Even McMahon concedes, yes, phosphorus is the driver, the catalyst, the baddest of actors.

Robin Rohwer uses a power auger on Lake Mendota to drill a hole through the ice for water sampling during the winter of 2015.
(Photo Courtesy Robin Rohwer)

 

 

And yet, she really thinks we should be paying more attention to nitrogen. Partly this is about her fascination with AC1. They clearly play a role in the nitrogen metabolism of the lake. But she’s also shown that nitrogen may well play a role in the eruption of cyanobacteria that have the ability to turn the lake from merely unpleasant to toxic.

To understand how, you need to envision summer, when the lake is stratified — warm water on top, cool down below. This happens because as water warms it expands and gets less dense. The density difference is so extreme that, once a lake stratifies, the warm and cold regions can’t be mixed until the top layer cools in the autumn. Stratification has profound effects on almost everything in the lake. The top layer keeps refreshing its oxygen by mixing with the air. Dead things drift slowly to the bottom to rot. Before long, the oxygen in the bottom layer gets used up. The microbial community switches into anaerobic decomposition.

“So down at the bottom you’ve got all these microbes cooking, breaking down the dead stuff, making those nutrients available again, but they’re trapped down there until the fall,” McMahon says. In that autumnal mix, the entire lake rapidly becomes saturated with oxygen and nutrients. Quite often there are huge, nasty cyanobacteria blooms, but these go largely unnoticed because people aren’t boating or swimming.

Then the lake, well mixed with oxygen and nutrients, freezes. There’s not much sampling under the ice, but the microbes are still active until, eventually, the spring thaw comes. Nitrogen can take many forms in the environment; in the fall, ammonia is abundant but gets gradually converted under the ice to nitrate. How early the ice forms and how long it stays influences the ratio of ammonia to nitrate at ice off.

Phytoplankton, or regular algae (not the cyanobacteria), prefer ammonia, so they’ll consume that first, then start to work on the nitrate. This ratio between ammonia and nitrate, combined with climatic conditions, seems to be a trigger for cyanobacteria bloom. Underneath the ice, the nutrients from the previous summer sit, an echo in the system. McMahon sees this nitrogen reverberate through the microbes in the lake, a rolling mix of cause and effect, and revels in the effort to untangle it all from a background of climate change, land use, and natural variability. “It’s possible there is a pattern that we haven’t seen because we haven’t been looking long enough,” she says. And then she laughs: “That’s the usual basic science cop-out.”

She dreams about finding the key to making all cyanobacteria go away, some microbial trick to starve it of phosphorus, but she knows her field is just at the very beginning of even being able to imagine such innovation. “I want to be able to do something to the system to fix it, not just study it,” she says. “But we’re pretty far from being able to do something like that.”

 

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Lake Mendota through the eyes of Trina McMahon is a bit of a paradox. The lake has its seasons, but the microbes may not. It’s got incredible diversity, yet we can’t even name what’s there. Its most common species could be doing the most uncommon things with sunlight. And it’s got a phosphorus problem — but don’t forget the nitrogen.

“It has this reputation of being the most studied lake in the world, but it’s also kind of a weird lake,” McMahon says. With high calcium and magnesium concentrations, it’s not, chemically, an average temperate lake. It also has a diverse mix of agricultural and urban influences. “Taking what we know about Lake Mendota and extrapolating it to all the lakes in the world is very difficult because it is not really a textbook lake,” she says. “But it is the one in the textbook.” And she laughs again.

Indeed, Lake Mendota is at a difficult place in its history. In the last decade, it’s been hit with a succession of shocks, including two major invasive species and increasing precipitation from climate change. Water clarity is in decline again. State and federal support for research funding and environmental regulation is in serious doubt. Carpenter — perhaps the preeminent aquatic ecologist of his generation — is stepping down.

Carpenter’s not going to give microbes or microbial ecology a free pass. Ecologists know that basically all roads lead through microbes, that they are the gatekeepers in nutrient flow through ecosystems. Yet despite an enormous amount of effort, we still don’t know how those flows are blocked or limited or enhanced by different microbial groups. “It turned out to be a lot harder than anybody knew,” he says. “If you really want to know the rate of sulfate reduction, you might just be better off to measure the rate of sulfate reduction instead of worrying about who did it.”

But he also reminds critics that breakthrough understanding was lacking in all branches of ecology for a long, long time. “If we don’t really delve into this microbial structure question, we’re never going to bridge structure and function in the microbial world,” Carpenter says.

That’s the challenge for McMahon and her colleagues. On her docket right now is a closer look at how microbes affect mercury in the lake, and she also works downstream with wastewater treatment, where microbes help remove excess phosphorus from the system. Meanwhile, rapidly advancing technology combined with the ongoing in-depth lake studies have generated a backlog of data and hypotheses to test.

“There is just so much that we don’t know,” McMahon says. “Yes, it’s awesome because it’s the most studied lake in the world, and we’re famous for that reason. But we also don’t understand it at all. It’s weird. How can that be? We should g understand everything if it’s the most studied thing, right?

 

Sidebar: Data in the Depths

Investigating Lake Mendota’s microbial mysteries requires mining the water for data, but it’s not as simple as walking to the shoreline and dunking a bucket under the waves. It’s a long, meticulous process of equipment preparation, sampling, and storage.

Students working in Trina McMahon’s lab collect samples twice a week from “ice off” (roughly late March) to lake freeze (usually December). Each boat trip takes them to the buoy marking the Mendota Deep Hole, the deepest part of the lake, where they measure water clarity, sunlight penetration, pH levels, temperature, conductivity, dissolved oxygen, and barometric pressure. Next, they collect two water samples using a 12-meter tube, which yields an integrated sample of the entire water column down to that depth. Each sample is stored in a 4-liter container for transport.

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Back in the lab, a small portion of each sample is placed in a tube with a compound that prevents organic cells from bursting when frozen. These samples are preserved so individual cells can be extracted and put through genomic analysis in the future. Larger portions of each sample undergo a filtering process.

The filtered portions are frozen and used later to determine the nutrients present in the water at the time of sampling. The filters themselves (and everything they caught) are carefully folded, placed in small tubes, and frozen for future DNA extraction and genomic analyses. The readings taken during sampling and the steps taken throughout sample processing are all painstakingly recorded in a lab notebook and also entered into a database.