Geomicrobiology of the Manantial del Toro cave (Dominican Republic): a microbial “living filter”?

Project Details

Key Question

Is the dark microbial biosphere at Manantial del Toro acting like a “living filter”, controlling the release of nitrate, phosphate and iron to the marine ecosystem?


The submarine Manantial del Toro cave (Dominican Republic) belongs to a karst system that connects heavily used agricultural lands to fragile marine ecosystems (coral reefs). Abundant biofilms hosting a N- and Fe-cycling “dark” microbial community may play an important role in remediating nutrient pollution from agricultural runoff.

Aims of the Project

To improve our understanding of nutrient circulation and cycling though karst environments, and of the roles of the dark microbial biosphere in these processes

Project Description

Karst regions cover ~10% of Earth’s continents, and their aquifers are a source of drinking water for a quarter of the world’s population. These highly permeable underground drainage systems, consisting in a network of interconnected cavities and fractures, allow for the rapid circulation of underground water. In coastal areas, karst systems connect continental subsurface waters to the marine environment through submarine springs. The rapid circulation of underground water to the ocean has potential negative impacts on fragile coastal ecosystems such as wetlands and coral reefs. For instance, nutrient-rich water runoff from agricultural lands circulating through karst drainage systems can cause harmful algal blooms in marine environments.

Subsurface ecosystems such as karsts host a “dark” (non-photic) microbial biosphere, that is still largely poorly known and understood. This project aims at investigating microbial nitrogen, phosphorus, and iron cycling at Manantial del Toro, a flooded cave in the shallow karst plain developed in the La Altagracia province (Dominican Republic). The submerged Manantial del Toro cave contains more than 3 kilometers of explored passages and has a strongly stratified water column, with oxygenated, nitrate-rich freshwater overlying anoxic sea water. Abundant, fast-growing, rust-coloured biofilms forming “slime curtains” attached to the walls and ceiling of the cave thrive below the halocline. Preliminary genomic analyses revealed a microbial community specialized for nitrogen cycling, including ammmonia oxidation, nitrite oxidation, and anaerobic ammonia oxidation. This intense microbial N-cycling may result in net nitrogen loss from the cave system through the production of gaseous N species (denitrification), and thus naturally remediate nitrogen nutrient pollution circulating through the karst system.


The bright red appearance of the biofilms suggests intense iron-(oxyhydr)oxide precipitation, potentially driven by microbial iron oxidation, which may be coupled to nitrate reduction. This rare and poorly understood metabolism is thought to be a potentially important microbial metabolism for the dark biosphere of early Mars. Given the high capacity for iron-(oxyhydr)oxides to sorb inorganic phosphate, we expect that these biofilms may act as an important phosphate sink in the cave, further helping the remediation of nutrient pollution in the water.


Here we propose investigate microbially driven nitrogen, phosphate, and iron cycles within the biofilms and the waters of the Manantial del Toro cave. In particular, we will determine what role the dark microbial biosphere may be playing in controlling the release of nitrate, phosphate and iron to fragile marine ecosystems at the margin of the karst plain, and thus to act as a natural, living filter.


The shallow karst system connects the Caribbean sea to a high-use land area, traditionally dominated by agriculture, and recently affected by the rapid development of tourism in the region. Global climate change and associated increasing temperatures, decreasing precipitations, and sea level rise will cause important strains on the local agricultural systems, and impact groundwater availability and quality. In parallel, the predicted increase in the frequency and intensity of storms is likely to lead to increased flooding and runoff to the ocean. Improving our understanding of nutrient circulating and cycling though karst environments is crucial to our ability to adapt to these important environmental changes, and to protect fragile costal ecosystems.


In order to evaluate the role of the Manantial del Toro microbial community to act as a natural “living filter” for nutrient pollution, we will measure the fluxes and speciation of nitrogen, phosphorus, and iron that enter and leave the cave.

The biofilms will be imaged using electron microscopy (SEM, TEM), and the associated iron-bearing mineral phases will be characterized using X-Ray Diffraction (XRD) and Fe K-edge X-ray Absorption Spectroscopy (XAS).

In order to better understand the role of the biofilm microorganisms in Fe, N and P cycling, the abundances of these elements in the biofilms will be measured, and their distribution and chemical speciation will be characterized down to the nanometre scale using Scanning Transmission X-ray Microscopy (STXM).

Culturing and sequencing approaches will be used to identify the N- and Fe-cycling bacteria responsible for Fe-(oxyhydr)oxide precipitation within the biofilm. These analyses will potentially provide new data on microbial iron oxidation coupled to nitrate reduction, a poorly documented iron biomineralization process.

This work will be performed in collaboration and under the guidance of Prof. Jenn Macalady (Penn State University), who is an experienced cave geomicrobiologist and diver. Macalady’s group will assist with field measurements and sampling, and carry out a metagenomic analysis of the biofilm to improve our understanding of the structure and function of the N-, Fe- and P-cycling microbial community at Manantial del Toro.

Methods to be used

Geochemical measurements in the cave water and the biofilms, field sampling, microscopy (SEM, TEM, STXM), mineral characterization (XRD, EXAFS), culturing and sequencing approaches

Specialised skills required

The project requires a student with strong interests in geochemistry, geomicrobiology, and mineralogy. Additional specific training will be provided, including in field and laboratory techniques and characterization methods.

Please contact Julie Cosmidis on if you are interested in this project