Sequencing Mysterious Microbes Of San Francisco Estuary

Life in the Sacramento-San Joaquin River Delta has continued to persist amidst almost constant human interference, from intensive manipulation of the area's natural hydrology and salinity to the arrival of introduced species and high levels of agricultural runoff. But the changing climate presents a growing challenge for an already sensitive ecosystem. In 2022, higher temperatures and extended drought created the conditions for a record-breaking toxic algal bloom, which killed thousands of fish and turned the waters of the San Francisco Bay - from Emeryville to Albany - a deep reddish-brown.

That's why Lauren Lui, a research scientist with the Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab), is set on uncovering how the Delta's microscopic life impacts the ecological health of this area and the quality of California's drinking water. Using the latest sequencing technology, Lui, who conducts research in Berkeley Lab's Biosciences Area, plans to assemble a database with the complete genomes of the estuary's microorganisms. The database will help scientists develop a predictive understanding of how microbes may respond to environmental changes.

"It fascinates me that there are all these little things in the soil and the water that affect us," Lui said. "We're trying to understand what is going on out here and how everything is working together to trigger these harmful algal blooms. If we can figure out what the tipping points and levers of the system are, that can inform policy on how to manage our ecosystems."

The Micro Missing Piece of California's Central Corridor

There's a flurry of activity as the crew of the Sentinel, a research vessel run by California's Department of Water Resources (DWR) as part of their monthly environmental monitoring, prepares to cast off into the blue-green waters of the Sacramento-San Joaquin River Delta.

"The color of the water starts to tell you what's there. Microorganisms are using the sunlight to grow," said Lui, who is aboard the Sentinel to survey the microbes of the Delta. She takes in the view off the deck, where saltwater from the San Francisco Bay mixes with the Sacramento and San Joaquin rivers to create the largest estuary on the West Coast. A maze of lush islands expands out ahead, their greenery contrasting with the industrial factory stacks, storage containers, and electricity pylons lining the southern shoreline. Fishing dinghies coexist with barking sea lions, cawing gulls, and flocks of migratory birds, all momentarily gathered here over their shared taste for striped bass, catfish, steelhead, Chinook salmon, and sturgeon.

"Working on this project has really opened my eyes to how much is going on here."

- Lauren Lui

Lui not only grew up in Sacramento, but has deep familial roots in the area going back six generations. "When I take Amtrak from Berkeley to Sacramento to visit my family, I've always seen the San Pablo Bay out the window," Lui said. "But working on this project has really opened my eyes to how much is going on here."

Though it's easy to overlook from the highway, this place is the nexus of California's water infrastructure, supplying freshwater to two-thirds of the state's population and a $50 billion agricultural industry that serves the entire country. Stakeholders from the San Francisco Bay Area all the way down to San Diego depend on the careful operations of state and federal agencies in managing an intricate network of levees that propel the flow of water through the Delta and maintain the proper balance of saltwater and freshwater.

DWR, who is overseeing today's excursion, is one such state agency. And with their mandate to deliver fresh water to millions and safeguard the federally protected birds and fish of the Delta, they have a vested interest in understanding the triggers of algal blooms. Scientists generally know that blooms happen when excessive nutrients in the water cause algae to grow rapidly and then die, and that higher temperatures increase their probability. But experts still can't predict where and when the next one might occur - or how harmful it will be to people and wildlife.

Lui and Ted Flynn, a senior environmental scientist for DWR, agree that's at least in part because few, if any, surveys regularly collect data on the estuary's smallest inhabitants: microbes. Flynn leads a joint federal and state monitoring program that has conducted monthly sampling cruises along the Delta for nearly 50 years. But while the long-term water quality and biological data they've collected has helped detect large-scale shifts in the environment, the methods DWR currently uses to track zooplankton and phytoplankton provide scant information on the still-tinier organisms like bacteria, archaea, and viruses - microbes that perform the critical role of cycling nutrients like nitrogen and phosphorus through the entire ecosystem. Because levels of these key elements can regulate the formation of algal blooms, understanding which microbial communities are present and how they function could be pivotal for developing predictive models for blooms and other threats.

"There's a knowledge gap between things we can see and things we can't," said Flynn. "And we can't understand the macro without the micro."

Sequencing the Unseen

It may seem surprising that experts don't know more about these miniscule power players. But there's a reason agencies like DWR don't monitor microbes.

"One of the only ways we can study them, besides looking at them under a microscope, is by looking at their genomes," Lui said.

It's often impractical to isolate and culture individual microbes in the lab, so Lui has embraced environmental genomics, which involves collecting water and sediment samples from the environment, sequencing all the DNA in the samples, and then parsing out which species are present and what their metabolic capabilities are. But when a single drop of water may contain hundreds or thousands of microbial species, this is no cake walk.

"Think of each species and its genome as a different puzzle. Imagine taking all those puzzles and dumping them into one box. Even worse, all those puzzles then go through a blender so the pieces become just small fragments. Then try to assemble all those puzzles separately. That's basically what environmental genomics is," Lui explained.

Even with the latest long-read sequencing technology - which essentially provides bigger puzzle pieces to work with - this process takes niche expertise and specialized facilities. Fortunately, with her background in microbiology and bioinformatics combined with Berkeley Lab's support, Lui is up for the challenge.

"Being at Berkeley Lab is really ideal for this research. Not only is this helping us study and manage a resource that's in our backyard, this work literally couldn't be done without some of the Lab's computing and sequencing resources," Lui said, referring to the National Energy Research Scientific Computing Center (NERSC) and the Joint Genome Institute (JGI), both Department of Energy Office of Science user facilities at Berkeley Lab.

Digging for DNA

Lui is proud to be contributing knowledge that agencies like DWR and her other collaborator, the United States Geological Survey (USGS), can use to better manage the estuary's natural resources. Still, these partnerships are far from one-sided. She wouldn't have access to a boat, for starters, let alone one specially equipped to conduct biological monitoring. Along with the chemical reagents and neatly labeled glass beakers one would find in any wet lab, the Sentinel boasts a special intake that pumps water directly from the river into a customized laboratory where it is analyzed and collected in an on-board sink. After arriving at the day's first stop on Grizzly Bay near Suisun Slough, Lui collects her first samples from this faucet. While the water filling her plastic containers looks clear, it is full of microscopic bacteria, archaea, and viruses whose DNA will give Lui a snapshot of the microbial communities inhabiting this site.

Out on the deck, the crew deploys a crane that scoops up sediment from the river bottom and deposits it into a large filtering container. Lui and DWR's benthic specialist, Betsy Wells, peer excitedly into the wooden box now filled with precious mud.

While Wells focuses on the clams and other invertebrate animals inhabiting the sediment, Lui pursues the microscopic life hidden between particles of sand, silt, and other debris in the mud itself.

"This is awesome," said Lui as she scraped a layer of dark brown silt off the top of the mud pile before going back with another collection tube to grab material from farther down. Besides the water samples she's already collected, Lui will sequence each layer of sediment separately to zero in on the microorganisms living in different strata with distinct ecological roles.

From Mud to Models

Back at Berkeley Lab, Lui prepares the samples for long-read sequencing, the first step in assembling those genome puzzles. This approach involves repeated sequencing of as many long pieces of DNA as possible to compile complete genomes for each organism.

"Getting complete genomes rather than just fragments is crucial for understanding the full capabilities, functionality, and survival requirements for each organism," Lui said. "And the more overlapping pieces you have, the more confident you are that your genome is correct."

Once Lui knows which genes different species have in their genomes, she can hunt for clues about how those organisms may be functioning. Lui might look out for genes related to processing nitrogen, for example, because the amount of nitrogen circulating in an environment is linked to algal blooms.

Right now, Lui is focused on setting up a baseline database of the microorganisms present in the estuary. Future samples can then be matched to intact genomes in the database, significantly reducing the sequencing and computing power required to piece them together. This initial groundwork will make it more feasible for Lui to track microbial populations over time and across different locations to see how they adapt to environmental changes.

"How the microbes are responding to climate change or the salinity increasing because of drought farther up the Delta - these are all things that we can track with sequencing," said Lui.

Genomes can also provide insight as to how or why microbes are evolving.

"How species interact with each other and the environment impacts which traits are passed on, and then we can see those changes in the genome," she said. "Microbial evolution can happen on the timescale of days or weeks, especially under extreme conditions. We don't understand how what we're doing - either with climate change or what we're dumping in the water - is affecting them."

This understanding of who's there, what they're doing, and how they're responding to a changing environment will help create more detailed predictive models of estuary dynamics. Ultimately, such models could inform policy decisions that cap agricultural inputs or other sources of nutrient pollution at different times of year, or enable agencies to adjust other parameters like salinity to compensate in high-risk time periods.

"I would hope in 10 to 20 years we would be able to take a few measurements and predict where and when a harmful algal bloom will happen, where the water quality might be bad, and know what the levers are for mitigating those events," Lui said. "Doing research that's important for this area where I grew up means a lot to me. Being out on the boat, seeing the water - we take it for granted, but once the water gets bad, it could be gone. We have to take care of it."

This research was supported by Berkeley Lab's Laboratory Directed Research and Development program. Part of this work was conducted at the U.S. DOE Joint Genome Institute, a DOE Office of Science User Facility supported by the Office of Science of the U.S. Department of Energy.