Probing nitrogen metabolism in the redox gradient of the Black Sea

The Black Sea, earth's largest anoxic water body, serves as a model system for studies of marine chemistry and biology. The article by Lam et al. (1) in this issue of PNAS describes a new approach to assessing the activity of Black Sea nitrifying bacteria and archaea that are globally important in nitrogen metabolism.

The Black Sea was formed after the retreat of the glaciers in the most recent warming period of the Pleistocene, ≈10,000 years ago. As water levels rose, water flowed from the Mediterranean Sea through the Sea of Marmara into the isthmus between what is now termed the Eastern and Western worlds, thereby forming the Bosporus Strait. The seawater flowed into a large freshwater lake that received runoff from the Danube and other rivers of Eastern Europe and Asia. The more dense seawater from the Bosporus sank to the lowest depths of this prehistoric lake, resulting in the formation of a salinity gradient and density stratification.

The aerobic, fresh surface waters of the Black Sea sustain a fishery that extends to a depth of ≈85 m. Beneath that depth, oxygen is depleted to a concentration of <5 μM in a redox zone that is referred to as the suboxic zone (2). The suboxic zone, which is ≈30 m in depth, harbors important nitrogen cycling metabolisms including nitrification, denitrification, and anammox (anaerobic ammonia oxidation).

Below the suboxic zone lies the sulfidic zone, dominated by sulfate reduction, which extends to the profundal depth of 2,200 m. Methanotrophic bacteria and archaea responsible for the anaerobic oxidation of methane are sulfate-reducers that reside in the sulfidic zone (3) and completely use the methane released in bubbles from gas seeps from the Black Sea's sediments (4).

Why Is the Black Sea Important?

There are two principal reasons why the Black Sea is uniquely important to science. First, it is an ideal place in which to study redox processes. Redox processes occur in sediments all over the world's oceans, but they are confined to very narrow sedimentary bands of millimeter or centimeter intervals that cannot be readily separated from one another. In contrast, the biogeochemical processes of the redox gradient in the Black Sea are spread over meters so samples can be readily collected for each redox process of interest. Furthermore, the broad redox gradient of the Black Sea is highly stable (5).

The second major reason for the study of the Black Sea's redox zone relates to astrobiology, the study of life in the universe. The Black Sea is analogous to the oceans of earth's Proterozoic period (6). During this period, which occurred from ≈2.3 to 1.0 giga-annum B.P., cyanobacterial oxygen was produced but was insufficient to saturate the world's oceans. The result was aerobic surface waters overlying anoxic waters beneath, thereby mimicking the Black Sea. The study of the Black Sea will also provide important clues to better understanding the evolution of life and metabolism on other planetary bodies such as Jupiter's moon Europa, which may have anoxic oceans.

Redox Metabolisms of the Black Sea

The redox reactions in nitrogen cycling are particularly important and interesting in the Black Sea. The chemistry of the Black Sea provides some clues about the microbial processes that are likely occurring (Fig. 1). Until now, many of the organisms responsible for the redox processes in the Black Sea were inferred from their distributions and that of functional genes that are uniquely responsible for specific enzymatic reactions. An exception is the anammox (anaerobic ammonia oxidation) reaction originally identified in wastewater treatment (7). In this process, ammonia is oxidized as an energy source, and its oxidation is coupled with nitrite reduction. The result is the stoichiometric formation of dinitrogen, N₂, with one atom derived from each of the nitrogenous substrates, ammonia and nitrite. Importantly, like denitrification, this process results in a loss of fixed nitrogen from the communities in which it lives.

Fig. 1.

Nitrate, nitrite, and ammonia concentrations in the suboxic zone of the Black Sea during April 2003. Note that the expression of the crenarchaeal ammonia (CA) oxidizing gene is greatest at the nitrate peak above the suboxic zone. In contrast, the Gammaproteobacteria ammonia oxidizing bacterial (γAOB) genes are expressed in the suboxic zone as well as with the CA above the suboxic zone. Depth is provided in sigma theta density units (e.g., sigma theta 14.0 is equivalent to a density of 1.014 g/cm³), and the corresponding depth in meters from Lam et al. (1) is also listed for the suboxic zone boundaries. Clara Fuchsman provided the figure.

Although the anammox bacteria cannot be cultivated in pure culture, they can be highly enriched under continuous culture conditions, sufficiently so that a genome sequence of a wastewater consortium is now available (8). The anammox bacteria are members of a large, diverse phylum of unusual budding bacteria, the Planctomycetes, which lack peptidoglycan and the usual bacterial cell division protein, FtsZ (9). Just recently, the anammox bacteria were found to carry out nitrate ammonification that produces ammonia from nitrate reduction (10).

Evidence for anammox activity in the Black Sea was provided by Kuypers et al. (11). They identified and localized the responsible bacteria, a Scalindua species, by 16S rRNA gene sequencing, analyses for their unique ladderane lipids, and fluorescence in situ hybridization (FISH) to the zone where the process was occurring. They also showed that additions of ¹⁵N-labeled ammonia to waters from that depth resulted in the formation of ²⁹N₂ as predicted by the anammox reaction.

Evidence for denitrification has also been reported in the Black Sea (12). This corroborates evidence for denitrifying bacteria based on identification of nirS and nirK genes that exhibit a surprisingly low diversity, suggesting that relatively few species reside in the suboxic zone (13).

The loss of fixed nitrogen through denitrification and anammox is a globally important process in sediments and in oceanic oxygen minimum zones (14, 15). The importance and distribution of anammox relative to that of dissimilatory denitrification in nitrogen loss is currently under debate (16, 17).

Some of the metabolisms in the Black Sea's suboxic zones depend on oxygen, although oxygen is found in very low concentration (2). Recent evidence from ammonia monooxygenase gene (amoA) sequences supports the occurrence of ammonia-oxidizing archaea in the Black Sea's suboxic zone where oxygen is available for this process (18, 19). These archaea are thought to be capable of oxidizing ammonia to nitrite, the substrate for anammox (20).

Dyanamics and Stratification of Nitrification in the Black Sea

Lam et al. (1) provide intriguing new information on the dynamics of the redox metabolisms in the Black Sea. For the first time, evidence is given for the expression of some of the key enzymes that are responsible for carrying out nitrification in the redox gradient of the Black Sea. In particular, data are provided that archaeal, or more specifically, crenarchaeal, ammonia oxidation is occurring based on the expression of the ammonia monooxygenase gene subunit A (amoA). The maximum expression of this gene, as determined by quantitative RT-PCR of messenger RNA (mRNA), was found at the nitrate maximum, which occurs in the nitrification zone (Fig. 1). This location is at the bottom of the oxic zone, just above the suboxic zone of the Black Sea. Because mRNA has a very short half-life in cells, its presence in a sample is indicative of active metabolism.

Interestingly, Lam et al. (1) also report evidence for nitrification by Gammaproteobacteria. This group of ammonia-oxidizing bacteria (γAOB) was found at two locations. Some of the AOB were located with the crenarchaeal nitrifiers in the nitrification zone. However, they also report, based on amoA expression, that the Gammaproteobacteria AOB are also active in the suboxic zone (Fig. 1). Because this zone has low concentrations of oxygen, it is inferred that these bacteria are microaerophilic nitrifiers. Evidence that this process occurs in this zone came from use of ¹⁵NH4+ incubations in which ¹⁵NO2− and ³⁰N₂ were produced as predicted by the sequential nitrification and anammox processes, respectively. The finding of γAOB in the suboxic zone contrasts with two other recent reports of crenarchaeal amoA sequences in the suboxic zone (18, 19). However, it should be noted that these latter studies relied solely on amoA sequences and not activity measurements.

Lam et al. (1) also quantified the contribution of the respective nitrification processes based on the levels of expression. Almost 75% of the nitrite produced in the upper oxic zone was attributable to the crenarchaeal nitrifiers. Based on this, they report that approximately half of the nitrite required for the anammox is derived from the crenarchaea and half from the bacteria, with the bacteria more likely directly associated with the anammox organisms.

The fact that there is sufficient nitrite produced for the anammox reaction by nitrifying bacteria and archaea raises the question of whether denitrifiers play a significant role at all. Therefore, the relative role of these two processes in the Black Sea needs to be more fully resolved.

The work of Lam et al. (1) is groundbreaking in that it illustrates that the activity of organisms can be demonstrated through in situ analyses of transcription in the redox zone of the Black Sea. Their work provides further evidence that scientists can successfully use molecular procedures to help untangle processes and the roles played by the individual organisms that are responsible for them.