Analyzing Biofilm in the Inner Harbor

There's a plethora of life in Baltimore City, but what about what's living beneath the water's surface in the Inner Harbor? With the help of a microscope, let's explore the National Aquarium's process for analyzing biofilm.

  • Conservation

From local shops and restaurants to the National Aquarium, there's so much to discover in Baltimore's Inner Harbor. But what about what lives beyond the attractions, beneath the water's surface? In preparation for the installation of the National Aquarium Harbor Wetland presented by CFG Bank scheduled for later this year, the Aquarium actually began collecting data on our campus's biodiversity over ten years ago. In addition to documenting observations of terrestrial animals, we've focused on gathering information on what lives in the Inner Harbor. By collecting this baseline information, we can follow how the environment changes over time and better understand how to tailor our Harbor Wetland in the future.

Cataloging biodiversity in and around the Inner Harbor can be quite a feat! Schools of fish or encrusted mussels may be easier to see and count, but their numbers don't give us the whole picture. Biofilms are another key part of the ecosystem that can be much harder to see with the naked eye. They are dynamic communities comprised of macro and micro fauna, algae, fungi and bacteria. Examining biofilm species' abundance and diversity with specialized equipment lets us account for even the smallest aquatic creatures.

Making Biofilm Count

With the help of a microscope, seemingly nondescript biofilms come to life. Cataloging many of these biofilm-forming organisms is difficult due to their small size. However, the process has been made possible with the help of two research partnerships, the Institute of Marine and Environmental Technology (IMET) and the Baltimore Underground Science Space (BUGSS).

"Biofilms are the cornerstone of food webs, essential for strengthening a resilient and balanced ecosystem," said Director of Field Conservation Charmaine Dahlenburg. "Diverse biofilms foster healthy ecosystems."

By analyzing biofilms, we can figure out what organisms make up the communities, from the slimy algae and bacterial mats to the larger, sessile (immobile) animals like mussels, anemones and barnacles that settle more easily on the film. High biodiversity at the microscopic scale is important for the ecosystem as a whole.

Taking Science On the Go

To survey biofilms, we must first collect samples from the harbor. With our partners at IMET, we use settling discs to provide a surface where the bacteria, algae and sessile organisms can grow. Five acrylic settling discs are deployed in the harbor from April through December. During that time, they are pulled from the water once a month and put under a microscope, where we observe biofilm communities and capture video to identify and count species.

Before the COVID-19 pandemic, we collected this footage in an IMET lab, but during the pandemic, it was better to work outdoors along the pier. Initially, we would set up a foldable table outside the Aquarium, complete with a microscope and other tools for data collection. While it wasn't the fanciest set-up, it allowed us to continue our work and piqued the interest of those who walked by the Aquarium.

This public interest helped start a partnership with IMET's artist-in-residence, Stephen Bradley, who created the BioBuggy. This portable yellow and blue lab makes working outside even easier and lets us share samples with those who walk by. With the BioBuggy, we can easily collect samples on the harbor's edge and project them on a TV screen so that passersby can see them, too!

"There is always a wow factor when people see the BioBuggy," Charmaine said. "The 'muck' becomes charismatic under the microscope, and we see critters, like barnacles, working hard to filter water."

Testing Biofilm DNA

Once biofilm samples are collected, we examine their DNA makeup with the help of a team of volunteer scientists at BUGSS specializing in harbor biodiversity. With their impressive laboratory, the BUGSS team helps us determine what is living in the harbor through a process known as DNA barcoding.

The process starts by scraping a sample from the acrylic settling disc and grinding it up. As cell walls break down (a process known as lysis), DNA is released. The scientists filter and isolate the DNA from the cellular debris and put it through a polymerase chain reaction (PCR) to create copies of various DNA fragments. In PCR, the DNA goes through heating and cooling cycles during which a PCR "primer" attaches to certain DNA sequences while a DNA-replicating enzyme makes copies of these primed DNA sequences. As a result, the reaction creates millions of DNA copies from all the organisms from the acrylic disc's biofilm sample.

Two Sets of Hands Scrape Brown Muck Off of a Settling Disc to Collect Biofilm Samples from the Inner Harbor

The next step reveals whether the PCR worked. If successful, the PCR should have made enough copies to be visible via gel electrophoresis. Gel electrophoresis is a technique that separates pieces of DNA based on size. DNA samples are marked with dye, placed onto a gel and exposed to an electric current. Since DNA pieces have a negative charge, they move toward the positive charge and filter through the gel. Larger segments get stuck sooner, while smaller ones travel further. If there are enough copies, bold lines will appear in the gel. If none appears, the PCR has failed, and the team tries again.

Once our teams have enough copied or amplified DNA, we send it to a commercial company for sequencing. We are sent back millions of DNA sequences that the team plugs into a database, attempting to find matches to known sequences already in the system. If we get a match, then we have identified an organism!

Setting the Stage for the Harbor Wetland

Fourteen years ago, very little was known about the species living in the harbor. By working with scientists and the local community, we went from having zero documented species to now knowing over 400 documented species around our campus. But all this information came from more than just analyzing biofilm.

We also utilize the app iNaturalist, a free platform that allows anyone to upload images of living things for scientists to identify and use in scientific research. Through the app, we have established a project boundary around our campus, so all observations taken within our campus directly contribute to our data. The combination of iNaturalist observations and biofilm data has provided great metrics for creating a baseline of biodiversity of the harbor, allowing us to properly evaluate the success of the Harbor Wetland project over time. The next time you visit the Aquarium's campus, consider using the iNaturalist app to document the local species you see hanging around outside.

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