Mangrove Microbial Biogeochemistry

Twin Cays

belize_tcsign.jpgThis project was active from 2000-2005; additional funding is pending so that our research can resume in the near future.

Twin Cays, Belize, is an oceanic mangrove ecosystem that lies 12 miles offshore of mainland Belize. Twin Cays is comprised of two islands that are separated by a shallow channel. The Twin Cays ecosystem is intertidal and supports a diverse variety of ecological habitats. On Twin Cays, we examined the microbially-mediated transformations of important bioelements, primarily: carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur, in sediments. Our work focused on understanding elemental transformations in microbial mat communities. Microbial mats are abundant on Twin Cays, as well as other mangrove islands. Microbial mat communities are also found in intertidal mudflats and marshes in temperate regions across the globe. We conducted similar research in Belize and in coastal Georgia and South Carolina, making it possible to compare nutrient and elemental cycling in temporal and tropical microbial mat ecosystems.Study SitesThe Smithsonian Institution maintains a field station on Carrie Bow Cay, Belize (left). Twin Cays lies approximately 2 km to the west of Carrie Bow; it is a nature preserve that is protected from fishing and development by the Belizian government. Twin Cays is an oligotrophic oceanic mangrove ecosystem, which means that concentrations of major (for example, nitrogen and phosphorus) and trace (for example, iron) nutrient elements are very low and primary production is frequently nutrient-limited. The fringe trees, which lie adjacent to tidal creeks, are taller and grow faster than the dwarf trees in the interior (Feller et al. 1999, Ecology 80:2193). Previous research at Twin Cays has shown that fringe mangrove trees are nitrogen limited while interior, dwarf mangrove trees are phosphorus limited (Feller et al. 1999). Though the tall "fringe" trees and the short "dwarf" trees are the same species they exhibit different growth rates, probably as the result of nutrient limitation.

belize_dwarf.jpgThe Study Site

The biocomplexity program has three primary study areas on Twin Cays: Boa Flats, The Lair, and The Dock. At each site, other Biocomplexity PIs are conducting well-replicated nutrient enrichment experiments to examine the impact of nitrogen, phosphorus, or nitrogen + phosphorus addition on mangrove growth. Our work in this biocomplexity project strives to document and unravel the microbially-mediated processes that drive nitrogen and phosphorus cycling in mangrove environments. Oceanic mangrove islands lie in oligotrophic offshore waters where nutrients are in scarce supply. Therefore, understanding internal nutrient cycling within different mangrove sub-habitats is crucial for understanding system scale patterns of nutrient limitation.

Nitrogen and phosphorus cycling are tightly coupled to other elemental cycles, such as those of oxygen, hydrogen, and/or sulfur. We are working at the experimental sites and at two additional sites on the island, called the North West Dock and the Clear Cut site (on the South end of the island). At present, most of our work is focused on documenting patterns of carbon, nitrogen, and oxygen cycling in surface microbial communities, known more commonly as microbial mats. Our studies will document spatial and temporal patterns of oxygenic photosynthesis, carbon fixation, and oxidation, and on two processes in the nitrogen cycle: nitrogen fixation and denitrification. Examining interaction and feedback between all of these processes is a key aspect of our study.

Research questions addressed include:

  • How common are microbial mats in oceanic mangrove environments? How much variability is there in the dominant microbes (e.g., heterocystous belize_flux.jpgversus filamentous cyanobacteria versus purple sulfur bacteria) that are present in these mats?
  • What are the diel patterns and rates of nitrogen fixation and denitrification in mangrove sub-habitats?
  • Are rates of N-input and N-loss comparable? Are rates seasonably variable?
  • What controls rates of nitrogen fixation and denitrification?
  • How do rates of nitrogen fixation and denitrification relate to rates of benthic primary production (~ oxygenic photosynthesis)?
  • What are the environmental controls on nitrogen fixation and denitrification?
  • Can spatial and temporal patterns of nitrogen cycling help explain the observed patterns of N vs. P limitation in mangrove trees?

This work constituted the PhD dissertation of Rosalynn Lee (2006) and Bill Porubsky (PhD 2008) who also conducted work in Belize.

The Processes We Are Studying

Nitrogen Fixation is a microbially-mediated process that has a high energy demand. Certain bacteria that possess the enzyme nitrogenase are able to biochemically convert dinitrogen to ammonium, which is subsequently incorporated into cellular nitrogen pools (amino acids, proteins, etc.).

N2 + 8H+ + 16 ATP --> 2 NH4+ + 16 ADP + 16 Pi

belize_rose.jpgA variety of Eubacteria, including photosynthetic and heterotrophic bacteria, and Cyanobacteria are capable of fixing atmospheric nitrogen. In general, higher nitrogen fixation rates are present in photosynthetic communities, where light energy can be used to fuel the energy demanding process. Photosynthetic nitrogen fixation is commonly carried out by Cyanobacteria or by photosynthetic bacteria (e.g. purple sulfur bacteria). Rates of heterotrophic nitrogen fixation tend to be lower, often because of reductant (energy) limitation. However, heterotrophic nitrogen fixation is important in some habitats; for example, sulfate-reducing bacteria may be important nitrogen fixers in sediments.

Since nitrogen is a limiting nutrient in mangrove environments, nitrogen fixation may be an important N input term to these ecosystems. In fact, previous studies have documented significant rates of nitrogen fixation in coral reefs and carbonate sediments. However, while nitrogen limitation may favor the presence of microorganisms capable of fixing dinitrogen, energy or limitation by some other nutrient or environmental condition may limit their activity in nature. For example, nitrogenase is inactivated by exposure to oxygen so nitrogen-fixing microorganisms must have a strategy to protect their nitrogen fixing enzymes from oxygen exposure. This is a particularly tricky problem for microorganisms that are oxygenic phototrophs (like Cyanobacteria). 

Various strategies exist for supporting both oxygenic photosynthesis (or oxygen exposure) and nitrogen fixation, including specialized N2-fixing cells (a heterocyst), temporally separating photosynthesis (occurs during the day) and nitrogen fixation (occurs at night), spatially segregating photosynthesis (outer portion of tufts or clumps of filaments) and nitrogen fixation (inner part of tufts or filaments), or by secreting mucous to stimulate bacterial respiration around the cell. Another factor that can limit nitrogen fixation is the availability of iron, a critical trace metal that makes up the active complex of the nitrogenase enzyme. Iron could exert a major control on nitrogen fixation in the tropics as carbonate sediments are typically iron poor. 

Our work documents the rates of nitrogen fixation as well as the environmental and physiological controls on the process; the questions our work addresses have not previously been examined in mangrove habitats.

Denitrification is the process by which microorganisms convert nitrate to dinitrogen gas. In terms of the global nitrogen cycle, denitrification serves to balance nitrogen fixation by removing fixed nitrogen (rather than supplying it) to the biosphere. Most denitrifying bacteria are heterotrophic, utilizing organic carbon, hydrogen, or hydrogen sulfide as electron donor and nitrate as electron acceptor. The electron donor is oxidized (to CO2, water, or sulfate) and nitrate is contemporaneously reduced to dinitrogen gas (N2): 

2NO3 + electron donor (e.g., organic carbon, hydrogen)
---> N2 + oxidized product (e.g., CO2, H2O...)

Denitrifying bacteria require a source of reductant (energy) and a source of oxidant (nitrate). Denitrification also requires the absence of oxygen because most denitrifying bacteria are facultative anaerobes, meaning they will respire oxygen instead of nitrate if given a choice between the two. Having a no-to-low oxygen requirement but a need for nitrate (which is often derived from nitrification, the O2-catalyzed oxidation of ammonium to nitrate) often puts denitrifiers in a redox dilemma: they often have to survive using nitrate obtained from sources external to their immediate habitat. Tropical carbonate environments are low in organic carbon, and bioavailable nitrogen, particularly nitrate, is present at low to undetectable amounts; thus, denitrification may be either oxidant (nitrate) or reductant (organic carbon) limited. Previous studies in mangrove environments suggest that denitrification rates are low and frequently nitrate limited. Such studies have implied that denitrification is not a significant term in the system N budget. However, since data in mangrove habitats are limited, the general importance of denitrification in these ecosystems cannot be concluded at this time.

We determine rates of nitrogen fixation using the acetylene block technique. Using this method, acetylene is added as a competitive inhibitor of N2-fixation and the production of ethylene serves as a proxy for the fixation of N2. Acetylene also blocks the final step of denitrification (the reduction of N2O to N2), so monitoring the accumulation of N2O in the presence of acetylene is used to estimate denitrification rates. To examine substrate limitation effects, rates are determined in the presence of substrate amendments (organic carbon, nitrate, other nutrients) as well as under natural (i.e. control) conditions.

The impact of in situ oxygen production on rates of nitrogen fixation and denitrification are evaluated by incubating light vs. dark (thus, no photosynthesis) treatments and by including light incubations that have been amended with DCMU, an inhibitor of PSII electron transport. While DCMU blocks PSII electron transport, it does not interfere with PSI (cyclic) electron transport. So a comparison of processes in DCMU amended treatments with those in controls lacking DCMU allows us to determine whether a process is dependent on PSI or PSII derived energy.

In bioassay experiments, we evaluate the response of microalgal and photosynthetic bacterial biomass (as chlorophyll) to various nutrient additions (nitrogen, phosphorus, iron, etc.). We also monitor how short-term (days) nutrient addition impacts rates of denitrification and nitrogen fixation.

Oxygenic Photosynthesis is mediated by a variety of algae and cyanobacteria in surface biofilms (on roots or stems of plants), on the sediment surface, or in microbial mats. Typically, there are a diverse array of primary producers in microbial mats, including diatoms, green algae, and a variety of cyanobacteria (filamentous and coccoid forms). These organisms make use of the "Z-scheme" of photosynthesis, and the process culminates in the production of molecular oxygen. Photosynthesis can be limited by energy (light availability) or by nutrients, if a particular microorganism has a particular unmet nutrient or trace element requirement.

We determine rates of oxygenic photosynthesis by measuring oxygen production using sensitive oxygen microsensors and a picoammeter, which together allow us to quantify small changes in oxygen concentration. Using the "light-dark shift" technique, we determine rates of oxygenic photosynthesis in "real time.” We use a computer-controlled picoammeter to control the movement of small microelectrodes and by doing so can quantify microscale (µm) variations in oxygen production. By monitoring oxygen production over the course of a daily light cycle, we can calculate integrated rates of benthic microalgal production in different mangrove sub-habitats.

Primary production, or the fixation of inorganic carbon (HCO3-) into organic biomass, is mediated by photoautotrophic and chemoautotrophic microorganisms in microbial mats. Photoautotrophic microorganisms include microalgae (diatoms), cyanobacteria, and photosynthetic bacteria. Chemoautotrophic microorganisms include those that couple the oxidation of inorganic compounds (hydrogen sulfide, ammonium, methane) with the reduction of HCO3- into organic matter (CH2O). A diversity of photosynthetic and chemoautotrophic microorganisms thrive in Twin Cays microbial mats.

We determine rates of carbon fixation using a stable isotopic tracer, H13CO3-. Samples are incubated in the presence of H13CO3-, and incorporation of inorganic 13C into biomass is quantified on a mass spectrometer.

We also document naturally occurring patterns of 13C and 15N distribution. Examining the natural abundance isotope ratios of C and N in microbial mats can shed light on C fixation pathways, the importance of N fixation, and N recycling efficiency and retention.

See The Microbial Mats of Twin Cays

Researchers:

Rosalynn Lee

Bill Porubsky

Papers:

Joye, S. B. and R. Y. Lee, 2004. Benthic microbial mats: important sources of fixed nitrogen and carbon to the Twin Cays, Belize ecosystem.  Atoll Research Bulletin, 528: 1-24.

Lee, R. Y., and S. B. Joye, 2006. Patterns and controls on nitrogen fixation and denitrification in intertidal sediments of a tropical oceanic mangrove island.  Marine Ecology Progress Series, 307: 127-141.

Lee, R. Y., W. P. Porubsky, I. C. Feller, K. L. McKee and S. B. Joye, 2008.  Porewater biogeochemistry and soil metabolism in dwarf red mangrove habitats (Twin Cays, Belize).  Biogeochemistry, 87: 181-198, doi: 10.1007/s10533-008-9176-9.

Feller, I.C., C. Lovelock, K. McKee, U. Berger, S.B. Joye, and M. Ball, 2010. Biocomplexity in Mangrove Ecosystems. Annual Review of Marine Science, 2: 395-417.

Funders:

National Science Foundation, Biocomplexity in the Environment