Sampling focus: The Suboxic Zone

Fig. 2: Strong water column stratification results in anoxic conditions with a well defined suboxic zone,

where both O2 and H2S are absent. We consider this an ideal location to sample for microbes that have not been

well characterized (if at all).

OxygenSulfide

Fig. 1: The Black Sea

Looking at Black Sea Microbial Communities to Understand Ancient Oceans

John Kirkpatrick1, Brian Oakley2, Clara Fuchsman1, Sujatha Srinivasan2, James T. Staley2, James W. Murray11School of Oceanography and 2Department of Microbiology

University of Washington, Seattle, WA April, 2005 Abstract #987

AcknowledgementsThanks to Billy Brazelton for all of his advice; and also to Mark Rider and Audrey Harris for their laboratory assistance.This work was funded by NSF Microbial Observatories 0132101 and NSF-IGERT grant DGE-9870713.

Conclusions"Life as we know it" is defined not by 3 dimensions, but by 4. If we as Astrobiologists want to

overcome the inherent scientific difficulty of being in one place at one time, one way to start is by thinking about not only other places, but other times. By considering the unusual (yet historically important) environment of the Black Sea, we can gain insight not only into Earth's past but also on the variety of forms and functions that life takes. Here we have presented the results of a first-pass assessment of the microbes living in the Black Sea. Among other observations, we can say that:• There is a very large amount of diversity in the phylum Planctomycetes; more unknown

bacteria in this location, in fact, than previously known and characterized worldwide.• The Planctomycetes of some depths (such as σ = 15.7, 15.8) appear to be dominated by

a few strains, possibly due to the favorability of the anammox reaction in certain chemoclines.

• A few strains of Planctomycetes are ubiquitous throughout the suboxic zone. This includes one type of bacteria, previously unknown, which grows in inorganic anammox enrichment media.

• There appears to be increased diversity at the bottom of the suboxic zone; we hypothesize that there might be a correlation to the viability of S-based metabolisms at those depths.

Now that we have gathered essential 16S rDNA data on various depths of the Black Sea, along with fresh samples, we can attempt to answer questions raised by our previous work. These include: What are the dominant species? We are working on Fluorescent In-Situ Hybridization

(FISH) techniques to identify and count specific strains of bacteria. How active are these bacteria? We have incubated and collected samples spiked with

14C-bicarbonate in order to measure rates of chemosynthesis. Which species or groups are primary producers? We have collected samples for a

combined 14C and FISH analysis to determine which kinds of bacteria are fixing carbon. What are the bacteria doing? We have many samples growing in selective cultures,

including those for anammox, heterotrophic denitrification, thiodentrification, and sulfite reduction, amongst others.

The R/V Endeavor. Additional samples collected 3/26/05-4/5/05.

15.9_JK460

15.9_JK312

15.9_JK420

15.9_JK445

15.9_JK461

15.9_JK417

15.9_JK500

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15.9_JK52215.9_JK512

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15.9_JK50115.9_JK454

15.9_JK44115.9_JK519

15.9_JK34315.9_JK409

15.9_JK45015.9_JK412

15.9_JK506

15.9_JK41915.9_JK442

15.9_JK451

10%

Aquifex

Pirellula staleyi

Pirellula

Planctomyces sp.

"Scalindua wagneri""Scalindua brodae"

Anammox enrichment

15.7_JK742

IsosphaeraGemmata

"Kuenenia stuttgartiensis""Brocadia anammoxidans"

"Scalindua sorokinii"

15.5_BO83615.5_BO597

15.5_BO704

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15.8_JK63615.8_JK630

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Density (depth) dependence:

σ = 15.5

σ = 15.6

σ = 15.7

σ = 15.8

σ = 15.9

σ = 16.0

(Reference)

Pirelulla-like sequences: found at all depths

Highly divergent group, typically found at the base of the suboxic zone.

Unknown group, showing some genera-level specificity to different densities

Fig. 3: Black Sea Planctomycetes Diversity. DNA Samples from various density layers were amplified with Planctomycetes-specific primers (58f and 926r), and the entire insert sequenced. This phylogenetic tree, comparing our samples versus known Planctomycetes, indicate a plethora of uncharacterized bacteria.

Fig. 4: While anammox-type sequences were detectable at all of the mid- and lower-depths, they dominated clone libraries at σθ = 15.8. This seems odd, because the anammox reaction requires NH4

+, which approaches zero at σθ = 16.0.

10%

Aquifex

"Scalindua wagneri"

Pirellula staleyi Pirellula

Planctomyces sp.

"Scalindua brodae""Scalindua sorokinii"

"Brocadia anammoxidans""Kuenenia stuttgartiensis"

Anammox enrichment

Gemmata Isosphaera

15.8_JK530

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Identified anammox and similar sequences

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H' / H'max

Dens

ity, σ

Fig. 5: Varying levels of diversity. This figure shows a diversity index (Shannon-Weaver, normalized for all depths by dividing by H’max); a higher number indicates increased diversity. σ = 15.8, shown above, is dominated by the anammox phylotype and has low diversity. At deeper density interfaces, approaching the bottom of the suboxic zone and the onset of sulfide, Planctomycete diversity increases dramatically. Further investigation will help us determine what sort of N and / or S metabolisms are related to these various groups of bacteria. (Note that these results are PCR based, and so may reflect the biases of that technique. Samples have all been screened for chimeras using Bellerophon [see Huber et al., 2004] and also RDP’s Chimera Check.)

Project Outline As we are striving to better understand the different forms life has taken on Earth, we are particularly interested in metabolic diversity. Since N- and S-based chemistry have a wide variety of life-supporting reactions, we focused on a part of the Black Sea where there is a large potential for “atypical” biogeochemistry. This is the suboxic zone (cf. figure 2), a redoxcline spanning 10s of meters which is deficient in both oxygen and sulfide. Amongst others, anaerobic ammonium oxidizing (anammox) bacteria are known to exist here; these chemoautotrophs which can release significant amounts of N2 gas (cf. figure 3). Samples collected in 2003 on the R/V Knorr were analyzed to yield depth-specific information on the chemistry and microbiology of the water column. In order to characterize the microbial community, and relate it to the chemistry of the system, we have utilized both culture-independent and culture-based techniques. DNA extraction, cloning, and sequencing has given us the basic distributions and diversity of numerous bacteria. Here we have used specific primers (58f and 926r) to focus on the Planctomycetes, an unusual bacterial phylum characterized by intracellular membranes, a complete lack of peptidoglycan, and a diverse distribution. The results of these molecular studies are summarized in figures 4-6. Enrichment cultures, designed to select for different metabolisms based on the media composition, have also yielded some successes. Among these is anammox enrichment medium (sterile seawater with NH4

+ and NO2-) which has produced an unknown strain of

Planctomycetes (cf. figures 4,5).

Fig. 3: Chemical gradients and the suboxic zone

Anammox bacteria are known to live in the suboxic zone, and survive autotrophically by producing N2 gas. We are attempting to understand their importance in the complex

interplay of various N species (and their isotopes). The relevant reaction is:Anammox Reaction: NH4

+ + NO2- N2(g) + H2O

This graph shows the various chemical distributions around the suboxic zone. Ammonium is produced at depth and is consumed (along with nitrate) in the suboxic zone; both reach negligible values around σθ = 16.0. This depth corresponds to an N2 gas maximum, relative to saturation. Nitrite shows a more complex profile, with maxima at σθ = 15.0 and 15.9. The suboxic zone is shaded (cf. figure 2). Note the relative scale bars.

0 10 20 30 40

NH4+

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(dep

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roxy

)

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1 1.005 1.01 1.015 1.02N2/Ar Sample/Saturation Ratio

NH4+

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N2/Ar Sample/Saturation Ratio

Black Sea Central Gyre

Suboxic zone

Abstract Earth's oceans would have appeared foreign to the modern observer for much of our planet's history, as evidence shows that the oceans had oxic surface and anoxic deep layers from approximately 2 to 0.5 billion years ago (cf. Canfield, 1998; Anbar and Knoll, 2002). This is much like the current Black Sea, the world's largest anoxic basin. We are investigating the anaerobic bacterial communities of the Black Sea in order to better understand the ancient oceans. We are particularly interested in novel metabolisms, such as anaerobic ammonium oxidizing (anammox) bacteria, chemoautotrophs which can produce significant amounts of N2 gas. Using samples collected from various depth horizons focused on the suboxic zone, we have constructed and sequenced 16S rDNA clone libraries using Planctomycetes-specific primers. This has allowed us to look at diversity within the Planctomycetes group, with several interesting results.

•There are multiple unknown groups, many of which are highly divergent from known Planctomycetes.•One group of 16S rDNA environmental sequences (detected only with Planctomycetes primers) branches separately from known groups and is also found growing in a selective culture for anammox bacteria. •In addition, there are several other environmental sequences similar to known anammox bacteria. •The upper depths of the suboxic zone have a relatively low diversity, with the “basement” of the zone hosting a complex array of different groups. This coincides with the potential for various S-based metabolisms.

Altogether, this raises interesting questions about the genetic diversity and metabolism of the Planctomycetes in general and anammox bacteria in particular. It also leads us to believe that there is much yet unknown about the composition of early marine microbial communities, as well as their interactions with and contributions to the planetary environment.

ReferencesAnbar, A. D. , and A. H. Knoll. Proterozoic Ocean Chemistry and Evolution: A Bioinorganic Bridge? Science 297, 1137-1142 (2002).Canfield, D.E. A new model for Proterozoic ocean chemistry. Nature 396, 450-453 (1998).Huber, T. , G. Faulkner and P. Hugenholtz. Bellerophon; a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20 2317-2319 (2004).