Exploring Deep-Sea Microbial Processes and Element Cycling

A recent study published in Limnology and Oceanography Methods addresses the challenge of investigating how hydrostatic pressure influences microbial enzymatic activity, an important factor in understanding biogeochemical cycling in deep-ocean environments.¹ Researchers developed a stainless-steel pressure cell capable of withstanding pressures up to 110 MPa, equivalent to conditions found at depths of approximately 11,000 meters, while allowing real-time spectral absorption and fluorescence measurements through sapphire windows.¹ The system also includes temperature control from 5–50 °C and can be integrated with standard laboratory spectrofluorophotometers, making it practical for routine experimental use.¹ To validate the device, the team performed fluorometric enzyme assays and analyzed chitinase activity from both pressure-loving (piezophilic) and pressure-sensitive marine bacteria at 10°C.¹ Results showed clear, reproducible differences in enzyme responses to pressure, demonstrating the system’s usefulness for studying how hydrostatic pressure regulates deep-sea microbial processes and element cycling.

Spectroscopy sat down with Urban Wünsch and Maria Papadimitraki, the lead authors of this study, to discuss how their system’s ability to withstand pressure allows it to be used as a tool in investigating deep-sea microbial processes and element cycling.

What motivated the development of this high-pressure stainless-steel cell, and what gaps in deep-sea microbial research were you aiming to address?

Urban Wünsch: The conditions in the deepest parts of our ocean are immensely challenging for microbial life. A central part of that challenge is hydrostatic pressure experienced by cells at great depths. Are enzymes of deep-sea microorganisms generally adapted to pressure or sensitive to it? To answer these questions, many conventional enzyme activity assays already use ultraviolet (UV) spectroscopy. We wanted an analysis platform that could detect responses to pressure in real time at high temporal and spectral resolution and with high throughput.

Can you describe the key design features of the pressure cell and how they enable real-time spectroscopic measurements under extreme conditions?

Urban Wünsch: The system was designed to fit into the sample compartment of common fluorometer types that are used by oceanographers around the world. Since both the fluorometer and pressure cell are portable, the system can be used in the laboratory or on board research vessels. The cell is temperature-controlled, can simulate pressures down to 10km water depth, and only requires approximately 30 mL sample. With three sapphire windows, absorbance and fluorescence properties can be measured simultaneously, allowing the software to record changes in optical properties in real time.

What were the main technical challenges in building a system capable of withstanding pressures up to 110 MPa while maintaining accurate fluorescence and absorption readings?

Urban Wünsch: We designed the system during the Covid-19 pandemic and needed to coordinate between the two universities and the cell manufacturer, literally without room for error. The cell itself needed to be relatively big to withstand the pressure, while fitting in the sample compartment of the fluorometer. The final design shown on the article, fits the fluorometer compartment, with just a few millimeters to spare.

How does integrating this system with standard spectrofluorophotometers improve accessibility or usability for other laboratories?

Urban Wünsch and Maria Papadimitraki: Interoperability was a key design requirement from the start. If the system worked well, we wanted to have a chance to send it to other laboratories or on oceanographic expeditions. Instead of relying on highly specialized or custom-built platforms, the system is designed as an adaptable framework that can be directly coupled to commercially available benchtop spectrofluorophotometers. To match standard spectroscopic formats, we adjusted the cuvette volume to 2.6 mL and set the optical path length to 10 mm. This means that researchers do not require dedicated instrumentation or complex software to perform high pressure experiments and instead they can follow standardized data analysis routines with open research software.

Your study highlights differences in chitinase activity between piezophilic and piezosensitive bacteria. What do these differences reveal about microbial adaptation to deep-sea environments?

Maria Papadimitraki: Our findings reveal a clear functional difference in chitinase activity between piezosensitive and piezophilic bacteria, which reflects the level of microbial adaptation to deep-sea conditions. We observed that chitinase activity in piezosensitive strains drops significantly under high hydrostatic pressure and shows reduced activity after depressurization, suggesting these organisms are not equipped to function efficiently at depth. In contrast, piezophilic bacteria maintained high enzymatic activity under elevated pressure, with rates comparable to—or even exceeding—those at atmospheric conditions, an indication of inherent adaptation to pressure, likely through structural stability of proteins and possibly the use of pressure-protective compounds, such as piezolytes. More broadly, our results suggest that deep-sea microbes are optimized for extreme conditions, ensuring efficient degradation of organic matter even in environments with very limited carbon input, such as the deep-sea.

How might this system advance our understanding of biogeochemical cycling—particularly carbon and nitrogen cycling—in deep ocean ecosystems?

Maria Papadimitraki: The key advance of this high-pressure cuvette system is its ability to measure microbial activity in real time under in situ pressure conditions, rather than relying on traditional bottle incubations where samples are depressurized before analysis. Our results show that enzymatic rates can differ substantially between high-pressure conditions and after depressurization, meaning that conventional approaches may significantly over- or underestimate microbial hydrolytic activity in the deep ocean. By overcoming this methodological bias, the system allows for a more accurate assessment of how efficiently microbes degrade organic matter. The system can be utilized to further investigate the functionality and adaptation strategies of the microorganisms and enzymes involved in major nitrogen cycling pathways, such as nitrification and denitrification/anammox processes, across the water column under in situ temperature and pressure. We believe that this system can provide a more realistic picture of deep-sea biogeochemical cycling, showing that microbial processes are strongly pressure and temperature dependent and must be studied under in situ conditions.

Beyond chitinase assays, what other types of enzymes or microbial processes do you envision studying with this pressure-controlled spectroscopic setup?

Maria Papadimitraki: Beyond chitinase assays, this high-pressure cuvette system opens up a wide range of possibilities for studying key enzymatic and microbial processes that control deep-sea biogeochemistry such as the activity of various peptide- and glucose-hydrolyzing and phosphate-acquiring enzymes. For example, using substrates linked with fluorophores such as 4-methylumbelliferyl (MUF) and 7-methoxycoumarin-4-acetic acid (MCA), we can target enzymes like β-glucosidase, phosphatase, chitinase or leucine aminopeptidase. In our study, we focused on the deep-sea model microorganism, Photobacterium profundum and piezosensitive Vibrio anguillarum but the system is also well suited for testing a broader range of microbial strains or intact microbial communities to investigate pressure effects on microbial growth. It can further be utilized for isolated enzyme assays, mineral dissolution at depth, investigating how sinking particulate organic carbon is remineralized or how enzymes respond to varying substrate concentrations.

What trends are you currently seeing in how spectroscopy is being applied in oceanography?

Urban Wünsch: Spectroscopy has been integral to oceanographic research for decades at this point. For example, we have used it to characterize dissolved organic matter or quantify phytoplankton through chlorophyll fluorescence. For dissolved organic matter in particular, a lot of data covering spectral absorbance and fluorescence exists. One challenge is to make all this data openly available so new analysis tools can be trained on existing data. Besides that, we are currently seeing a whole range of different initiatives for in situ observation technologies where spectroscopy is a key ingredient. Sensors and calibration routines are getting better and our ability to detect small-scale changes in the water column will soon follow.

The study was supported by Danish National Research Foundation through the Danish Center for Hadal Research HADAL, led by Professor Ronnie N. Glud (grant DNRF145).²

References

1. Papadimitraki, M.; Wunsch, U.; Balmonte, J. P.; et al. An Ultraviolet–Visible Spectroscopy System for Studying Real-Time Pressure Effects on Enzyme Activity. Limnol. Oceanogr.: Methods 2026, e70034. DOI: 10.1002/lom3.70034
2. University of Southern Denmark, HADAL: Danish Center for Hadal Research. SDU.dk. Available at: https://www.sdu.dk/en/forskning/hadal (accessed 2026-04-28).