microbial ecology of expanding oxygen minimum...

Over geological time the ocean has evolved from being an anaerobic incubator of early cellular existence into a solar-powered emitter of molecular oxygen (O2), a transformation that has been punctuated by catastrophic extinctions followed by the iterative re-emergence of bio-logical diversity1,2. Today, the ocean is being transformed in response to human activities. Indeed, the fourth assess-ment report of the Intergovernmental Panel on Climate Change observed that the ocean is becoming substan-tially warmer and more acidic3. As these changes inten-sify, marine ecosystems will experience disturbances in the structure and dynamics of food webs, with resulting feedback on the climate system4. Oxygen-starved regions of the ocean, known as oxygen minimum zones (OMZs), are important bellwethers for these changes5.

OMZs are an intrinsic feature of water columns that arise when the respiratory O2 demand during the degradation of organic matter exceeds O2 availability in poorly ventilated regions of the ocean6–9. Increases in ocean temperature drive decreases in O2 solubility and reduced ventilation owing to thermal stratification of the water column8,10, resulting in OMZ expansion. Consistent with this, between 1956 and 2006 the O2 con-centrations in the OMZ of the northeast subarctic Pacific (NESAP) declined by 22%, and the hypoxic boundary layer (defined as ~60 μmol O2 per kg water) expanded upwards from a depth of 400 m to 300 m (REF. 11). Similar declines have been observed in the eastern tropical Atlantic12, the equatorial12 and northeast13,14 Pacific, and in the Southern Ocean8 during the past 50 years.

As O2 concentrations decline, the amount of habitat available to aerobically respiring organisms in benthic

ecosystems and pelagic ecosystems reduces, changing the species composition and food web structure in these regions15. Organisms that are unable to escape O2-deficient conditions may experience direct mortal-ity (that is, the fish in these regions die) or decreased fit-ness16,17. Even organisms that can escape to more highly oxygenated refuges are susceptible to increased preda-tion and density-dependent reductions in population size18. OMZ expansion also causes changes in the cycling of trace gases such as methane (CH4), nitrous oxide (N2O) and carbon dioxide (CO2), which are important for metabolism and can have an effect on climate. CH4 and N2O are powerful greenhouse gases with radiative forcing effects that are approximately 25 and 300 times the effect of CO2, respectively. Although oceanic CH4 emissions are minor (

Thermal stratificationA temperature-layering effect that occurs in water owing to differences in water density: warm water is less dense than cool water and therefore tends to float on top of the cooler, heavier water.

Benthic ecosystemsEcosystems residing at the lowest level of a body of water such as an ocean or a lake, including the sediment surface and subsurface layers.

Pelagic ecosystemsEcosystems residing in the region of a body of water that is neither close to the bottom nor near the shore.

Radiative forcing effectsThe change in net irradiance between different layers of the atmosphere.

Coastal upwellingThe upwards movement of deep, nutrient-rich water along a coast, caused by wind-driven currents.

OxyclineA sharp gradient in oxygen concentration that is associated with a redoxcline (a shift in electron donor and acceptor usage).

EutrophicPertaining to a body of water: rich in mineral and organic nutrients.

EutrophicationExcessive nutrient input to a lake or other body of water (frequently owing to run-off from the land), resulting in explosive plant growth and animal mortality owing to oxygen starvation.

Here, we review recent observations that have emerged from the intersection of taxonomic and functional gene surveys, gene expression studies and measurements of process rates to better formulate hypotheses regarding the metabolic interactions that drive OMZ ecology and biogeochemistry on a global scale. We focus on bacte-rial and archaeal contributions to these networks, with the understanding that microbial eukaryotes and viruses have biologically essential but as-yet physiologically uncharacterized roles in modulating matter and energy transformations in OMZs.

OMZ formation and expansionOMZs are typically found on the western boundaries of continental margins, where wind-driven circulation patterns push nutrient-rich waters upwards to the sur-face in a process known as coastal upwelling. This process effectively fertilizes surface waters and results in high levels of photosynthetic primary production. During photosynthesis, phytoplankton fix CO2. Much of the inorganic carbon that is fixed through photosynthesis is respired in surface and intermediate layers of the water column through microbial degradation processes. A fraction of the product of primary production sinks as dead organisms and particles that are exported to depth. Throughout the ocean this process, called the biological carbon pump, has a large influence on the biogeochemi-cal carbon cycle because carbon is sequestered in the interior of the ocean for long periods of time, during which it cannot influence the climate24. Estimates of carbon rain rates to carbon sediments in the northeast Pacific suggest that the presence of an OMZ greatly increases the amount of carbon exported to the deep ocean25.

Persistent O2 deficiency occurs when the amount of dissolved O2 in the water column is consumed faster than it is resupplied through air–sea exchange, photosyn-thetic O2 production and ventilation

15. Global circula-tion patterns transport younger, more oxygenated waters throughout the deep ocean, resulting in deep oxycline formation. Thus, in profile, OMZs resemble a band of O2-deficient water inserted between two O2-containing water masses (FIG. 1). The upper O2 thresholds chosen to define OMZs have been manifold, ranging from

Nature Reviews | Microbiology

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EcotypeA group of organisms within a species that are selectively adapted to a particular set of environmental conditions and therefore exhibit behavioural, structural or physiological differences from other members of the species.

NH4+ (DNRA), a process that takes place under suboxic

or anoxic conditions, has the potential to moderate the loss in fixed nitrogen and to regenerate redox couples (in the form of NO2

− and NH4+) for anammox47–49. Taken

together, these microbial nitrogen transformations constitute a distributed metabolic network linking the metabolic potentials of different taxonomic groups to higher-order biogeochemical cycling of nitrogen in the environment.

Recent studies also posit an essential role for sul-phur cycling in OMZs, coupling the production and consumption of reduced sulphur compounds to dis-similatory NO3

− reduction and the fixation of inor-ganic carbon50,51. The integration of carbon, nitrogen and sulphur cycles represents a recurring theme in the O2-deficient water column, where electron donors and acceptors are actively recycled between lower and higher oxidation states (BOX 1).

OMZ microbiotaTaxonomic survey data based on small-subunit ribo-somal RNA (SSU rRNA) gene sequences indicates that there are conserved patterns in microbial com-munity composition between open-ocean and coastal OMZs and enclosed or semi-enclosed basins experi-encing water column O2 deficiency. The most abun-dant phyla in the OMZs are (in order of abundance) Proteobacteria, Bacteroidetes, marine group A (a candi date phylum), Actinobacteria and Planctomycetes (FIG. 3). The phyla Firmicutes, Verrucomicrobia, Gemmatimonadetes, Lentisphaerae and Chloroflexi, as

well as the candidate divisions TM6, WS3, ZB2, ZB3, GN0, OP11 and OD1, are also present in OMZs (FIG. 3) (note that the taxonomy used in this Review is taken from the Greengenes database52). The distribution of these taxa varies throughout the water column, with different subdivisions partitioning along the oxycline. These patterns reflect unique and overlapping interac-tions among individual microorganisms, and among populations and communities of microorganisms.

In the oxic surface waters overlying OMZs, sequences affiliated with the SAR11 cluster and the order Rhodobacterales (class Alphaproteobacteria), with the order Methylophilales (class Betaproteobacteria), and with the SAR86 cluster and the clone Arctic96B-1 (class Gammaproteobacteria) are prevalent, as are sequences affiliated with the phylum Cyanobacteria, with the marine OM1 clade (phylum Actinobacteria), and with the clone Arctic97A-17 and the genus Polaribacter (phylum Bacteroidetes). In dysoxic and suboxic waters, prevalent sequences include those that are affiliated with the SAR11 cluster (class Alphaproteobacteria), with the agg47 cluster (also known as ESP OMZ sequence accumulation cluster II (EOSA-II)53) and the clones Arctic96B-1, ZD0417 and ZA3412c (class Gammaproteobacteria), and with the SAR324 cluster and the genus Nitrospina (class Deltaproteobacteria). Sequences affiliated with Microthrixineae (class Actinobacteria), anammox bacteria (genus ‘Candidatus Scalindua’, phylum Planctomycetes), the phylum Chloroflexi and various Verrucomicrobia are also pre-sent in dysoxic and suboxic regions. In suboxic and anoxic waters (that is, OMZs), the dominant sequences are affiliated with the SUP05 cluster (Suiyo Seamount hydrothermal plume group 5 (REF. 54); also known as EOSA-I53; class Gammaproteobacteria). In addition to these SUP05 sequences, prevalent sequences in anoxic or sulphidic waters include those affiliated with the sulphate-reducing family Desulphobacteraceae (class Deltaproteobacteria), the sulphur-oxidizing Arcobacteraceae (class Epsilonproteobacteria), the clone VC21_Bac22 (phylum Bacteroidetes), the phylum Gemmatimonadetes and the phylum Lentisphaerae. The presence of candidate divisions increases with decreas-ing O2 concentrations, with most sequences affiliated with OP11 and OD1 identified in anoxic waters. In addition to bacterial SSU rRNA genes, sequences affili-ated with NH3-oxidizing marine group I (MGI) archaea in the phylum Thaumarchaeota have been identified in several locations, where they are most prevalent in the oxycline55–59.

Taxa of emerging interest in OMZs. Although many of the taxa identified in O2-deficient waters are ubiquitous throughout the ocean, patterns of endemism emerge at the level of operational taxonomic units (OTUs) obtained by clustering SSU rRNA gene sequences together at specific identity thresholds. These OTU distribution patterns reinforce a model of ecological type (ecotype) selection in which genetically cohesive populations manifest distinct ecological or biogeochemical roles60. These distinct roles in turn form the basis of distributed

Figure 2 | O2 concentration affects ecosystem energy flow. Alternative states of the sea water (oxic, dysoxic, suboxic and anoxic) and corresponding molecular oxygen (O

2) concentrations are defined. The red–orange area indicates the range of energy

transferred from pelagic nutrients to higher-level predators under oxic conditions. With declining O

2, higher-level predation is suspended and the proportion of energy

transferred to microorganisms rapidly increases (the yellow–green–blue area). This energy is generated via microbial respiration using a defined order of terminal electron acceptors (TEAs), with O

2 as the preferred TEA, followed by nitrate (NO

3−),

manganese iv (Mn iv), iron iii (Fe iii), sulphate (SO4

2−) and, finally, carbon dioxide (CO2).

Figure is modified,with permission, from REF. 35 © (2008) American Association for the Advancement of Science.

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384 | JUNE 2012 | VOLUME 10 www.nature.com/reviews/micro

© 2012 Macmillan Publishers Limited. All rights reserved

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interaction networks that integrate the phenotypes of different taxonomic groups. Here, we focus on the four most abundant bacterial taxonomic groups identified in surveys of OMZs (the SUP05–Arctic96BD-19 group, the SAR11 and SAR324 clusters, and the candidate phylum marine group A) for OTU distribution analysis (FIG. 4).

SSU rRNA gene sequences affiliated with chemoauto-trophic, sulphur-oxidizing gill symbionts of deep-sea clams and mussels were first identified in open-ocean OMZs in the Arabian Sea, the ETSP and the Namibian upwelling53,61,62. Phylogenetic analysis indicates that these symbionts are part of a larger group of free-living symbionts (also referred to as the gammaproteobacterial sulphur-oxidizing cluster (or GSO)) consisting of two closely related, co-occurring and currently uncultivated lineages, SUP05 (REF. 54) (encompassing the clam and mussel symbionts) and Arctic96BD-19 (REF. 63) (FIG. 4).

SUP05 and Arctic96BD-19 exhibit overlapping but not identical distribution patterns, consistent with redox-driven niche partitioning. SUP05 is most abundant in the slightly to moderately sulphidic waters at the base of the sulphide–NO3

− transition zone, and the organisms in this clade derive energy from the oxidation of reduced sulphur compounds using NO3

− as a terminal electron acceptor50,62. Arctic96BD-19 is most abundant in dys-oxic and suboxic waters, as its members derive energy from reduced sulphur compounds using O2 as a terminal electron acceptor64,65. Both SUP05 and Arctic96BD-19 members have the potential to use the energy gained from the oxidation of reduced sulphur compounds to fix inorganic carbon via Rubisco50,65. Under more sulphidic water column conditions, SUP05 members are replaced by Epsilonproteobacteria that exhibit similar meta-bolic capabilities66–68. The presence of SUP05 species in

Box 1 | Redox-driven niche partitioning

Reduction–oxidation (redox)-driven niche partitioning in the molecular oxygen (O2)-deficient water column selects for

shared metabolic capabilities across different ecological scales. Consistent with this observation, the chemical gradients found in marine oxygen minimum zones (see the figure, label 1) also exist in interior oceanic waters in the form of sinking organic particles or ‘marine snow’ (REF. 105) (see the figure, label 2). Particle association provides a nucleation point for otherwise suboxic or anoxic processes in oxygenated waters owing to the formation of microscale oxyclines83,106,107 (see the figure, label 3). The sulphate-reducing potential of such particles has been demonstrated82, and a similar relationship has been identified for methane production and transport in the North Pacific Ocean108. The interplay between particle-associated and free-living bacteria creates distributed networks of metabolite exchange between community members with alternative or competing nutritional or energetic needs109 (see the figure, label 4). The recent identification of the SAR324 cluster as particle-associated bacteria with genomic potential for inorganic carbon assimilation, sulphur oxidation and methane oxidation reinforces the ecological and biogeochemcial importance of microzone formation throughout the water column65. CH

4, methane; CO

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HeterotrophicDependent on obtaining carbon for growth and energy from complex organic compounds.

Entner–Doudoroff pathwayAn alternative series of reactions for the catabolism of glucose to pyruvate, using a different set of enzymes from those used in either glycolysis or the pentose phosphate pathway.

Dark oceanThe depths of the ocean beyond which less than 1% of sunlight penetrates; also known as the aphotic zone.

non-sulphidic OMZs serves as a biomarker for chang-ing ecosystem dynamics, indicating an increased poten-tial for toxic sulphur blooms and periodic or persistent anoxia57,62,64.

SAR11 is the most abundant and ubiquitous clade of Alphaproteobacteria in the ocean, often constituting 30% of surface bacterioplankton communities69. The dominant SAR11 OTUs observed in OMZs (FIG. 4) are closely related to Pelagibacter ubique, a cultivated mem-ber of SAR11 in subgroup Ia, and there is also a minority representation for subgroups Ib and II. SSU rRNA gene surveys support the existence of several SAR11 ecotypes that exhibit geographical and depth-specific water col-umn distributions70. Ecotype selection is particularly apparent in the NESAP OMZ, where more than 30 dif-ferent SAR11 OTUs have been identified (FIG. 4). Current studies of cultivated SAR11 strains and free-living popu-lations indicate a genomic repertoire that is streamlined for rapid heterotrophic growth71. Comparative genomics analyses suggest that there are differences in the glyco-lytic potential of coastal and open-ocean SAR11 popu-lations. Consistent with this observation, carbon use and gene expression assays have measured preferential glucose use by coastal isolates that are associated with a gene cluster encoding a variant form of the Entner–Doudoroff pathway72. However, the specific metabolic capabilities that enable SAR11 members to thrive in O2-deficient waters remain unknown. The role of surface water SAR11 populations in mediating demethylation of dimethysulphoniopropionate (DMSP) to methylmer-captopropionate (MMPA) may indicate a role for this group in OMZ sulphur cycling. Although this phenotype is shared between a number of different pelagic bacte-ria73,74, SAR11 members are unable to perform dissimi-latory sulphate reduction, making them dependent on exogenous sources of reduced sulphur for growth and further reinforcing a model of distributed metabolite exchange75.

The candidate phylum marine group A was first iden-tified almost 20 years ago in northeast Pacific Ocean waters from 100 m and 500 m depth intervals76,77. Since that time, SSU rRNA gene surveys have identified marine group A as being ubiquitous in the dark ocean. The domi-nant marine group A OTUs observed in OMZs (FIG. 4) are closely related to subgroups SAR406, Arctic95A-2, Arctic96B-7 and ZA3648c, with an additional minority

representation for subgroup ZA3312c. Six additional sub-groups seem to be endemic to the NESAP, Saanich Inlet and the ETSP (information not shown). These results are consistent with previous observations indicating that there is strong habitat selection for different marine group A subgroups78. Despite these organisms being widespread in the ocean, their metabolic capabilities remain unknown.

Similarly to marine group A, the SAR324 clade (also known as marine group B) of the class Deltaproteobacteria is also prevalent in the dark ocean79–81. The most com-mon SAR324 OTUs observed in OMZs (FIG. 4) are closely related to marine group B–SAR324 clade II, and there is also a minority representation for marine group B–SAR324 clade I; two additional clades are endemic to Saanich Inlet and the ETSP (information not shown). SAR324 members have the potential to oxidize one-carbon (C1) compounds and reduced sulphur compounds, using the resulting energy to fix inorganic carbon via Rubisco65. Consistent with a functional role for Rubisco, microautoradiography linked with catalysed reporter deposition fluorescence in situ hybridization (MAR–CARD–FISH) demonstrated that SAR324 members fix inorganic carbon and undergo particle association in oxygenated waters of the North Atlantic65.

The symbiotic oceanRecent advances in microbial ecology that combine cul-tivation-independent molecular methods with process rate measurements are beginning to reveal previously unknown metabolic interactions in the O2-deficient water column. Parallel advances in exploring the dark ocean have identified similar metabolic interactions at different ecological scales. In the following case study we touch on these observations, with particular emphasis on the integration of carbon, nitrogen and sulphur cycles.

Unravelling a cryptic sulphur cycle. The identification of the SUP05–Arctic96BD-19 clade of Gammaproteo-bacteria suggested an important role for sulphur cycling in the ecology and biogeochemistry of OMZs53,57,62. Metabolic reconstruction of the SUP05 metagenome identified numerous genes encoding components of the sulphide oxidation and nitrate reduction pathways. Principal components of both pathways were found to be clustered in a 52 kb ‘metabolic island’ that also contains a gene encoding the large subunit of form II Rubisco, consistent with coordinated regulation of car-bon and energy metabolism50. More recently, single-cell techniques were used to assemble genomic scaffolds for Arctic96BD-19 from North Atlantic and Pacific waters, uncovering sulphur oxidation and CO2 fixation genes

65. The discovery of potential sulphur oxidizers in non- sulphidic waters is enigmatic, bringing into question the source of the reducing equivalents that are needed to fix inorganic carbon.

Sinking particles have been proposed to be sources of reduced compounds such as sulphide in suboxic or anoxic waters82,83. However, SO4

2− reduction in the water column is difficult to measure because sulphide rapidly

Figure 3 | Bacterial diversity in the ocean. Dot plot of the diversity of bacterial taxa at various sample points and depths in Saanich Inlet (SI), the northeastern subarctic Pacific (NESAP; labelled P4, P12 and P26), the Hawaii Ocean Time-series (HOT), the eastern tropical South Pacific (ETSP) and the Namibian upwelling (NAM; also known as the Benguela upwelling), based on small-subunit ribosomal RNA (SSU rRNA) gene sequence profiles. ‘*’ indicates a sample taken from P4 1,000 m in June 2008; all other NESAP samples were taken in 2009. Samples are organized according to the similarity of their community composition, as revealed by hierarchical clustering of the distribution of taxonomic groups across environmental samples. The molecular oxygen (O

2)

concentration is shown for each oceanic sample, and the classification of the environment as oxic, dysoxic, suboxic or anoxic is also indicated in the colour bar. Names for identifying bacterial groups were selected according to the taxonomic level at which the most relevant information was available. Data used to generate the dot plot were derived from sequences deposited in Genbank.

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A_01

MG

A_02

MG

A_03

MG

A_04

MG

A_05

MG

A_06

MG

A_07

MG

A_08

MG

A_09

MG

A_10

MG

A_11

MG

A_12

MG

A_13

MG

A_14

MG

A_15

MG

A_16

MG

A_17

MG

A_18

MG

A_19

MG

A_20

MG

A_21

MG

A_22

MG

A_23

MG

A_24

MG

A_25

MG

A_26

MG

A_27

MG

A_28

MG

A_29

MG

A_30

MG

A_31

HOT 4,000 mP26 10 mNAM 119 mNAM 90 mP12 10 mETSP 450 mSI J06 10 mSI A07 10 mHOT 130 mHOT 200 mHOT 500 mHOT 770 mP4 1,000 mETSP 60 mETSP 200 mP12 500 mSI J06 120 mSI N06 10 mSI A07 100 mSI F06 125 mSI A08 120 mSI F06 100 mSI J06 100 mSI A08 100 mSI A07 120 mP4 1,000 m*P12 2,000 mP12 1,000 mP26 500 mP4 500 mP26 2,000 mP26 1,000 mP4 1,300 mSI J06 200 mSI F06 215 mSI A08 200 mSI A07 200 mSI N06 120 mSI N06 100 mSI N06 200 m

SAR324 Marine group A

25 50 75 >100101

0

200

400

600

800

1,000

0

50

100

150

200

250

0

20

40

60

80

100

0

200

100

400

300

SUP05–Arctic96BD-19 SAR11


R E V I E W S



SyntrophyMetabolite exchange that occurs between two or more groups of organisms and is necessary for cell growth or energy production.

ChemolithoautotrophicCapable of obtaining energy from the oxidation of inorganic compounds and carbon from the fixation of carbon dioxide.

auto-oxidizes in the presence of even trace amounts of O2. Typically, when sulphide is detected in OMZs, it originates in rare pockets of NO3

−- and NO2−-depleted

water84 or is released by diffusive flux from sediments30,85. Thus, the in situ component of the sulphur cycle in non-sulphidic waters has been described as cryptic because it lacks obvious chemical expression in the water column. To resolve this enigma, researchers conducted process rate measurements of SO4

2− reduction in the ETSP OMZ using radiolabelled SO4

2− (35SO42−) after a pulse of unla-

belled sulphide to capture the formation of radiolabelled sulphide51. High rates of SO4

2− reduction were detected (between 0.28 and 1.0 nmol per m2 per day) coupled to the production of NO2

−, N2O and N2. Consistent with process rate measurements, metagenome sequenc-ing recovered a limited number of genes originating from canonical sulphate-reducing bacteria, including Desulphatibacillum, Desulphobacterium, Desulphococcus, Syntrophobacter, and Desulphovibrio spp.

Sulphur oxidation coupled to NO3− reduction in the

ETSP OMZ was supported by metatranscriptomic anal-yses, which revealed that transcripts for dissimilatory sulphite reductase (dsr) genes, sulphur oxidation (sox) genes (encoding proteins that mediate thiosulphate oxi-dation), adenosine-5ʹ-phosphosulphate (APS) reductase (apr) genes (encoding proteins that mediate the con-version of sulphur to SO4

2−) and the gene encoding the catalytic subunit of respiratory NO3

− reductase (narG) were highly expressed86. Although most of these tran-scripts originated from sulphur-oxidizing organisms, including members of the SUP05 clade and close rela-tives, a minority of aprA and dsrB transcripts affiliated with canonical SO4

2−-reducing bacteria were detected, consistent with there being an active sulphur cycle in this environment. Interestingly, in ETSP metagenomes, 32% of the top hits to aprA were affiliated with SAR11, and cognate aprA transcripts were highly expressed throughout the oxycline. Although the precise role of SAR11 in sulphur cycling in OMZs remains unknown, the expression of Apr could indicate a link between DMSP demethylation and the production of reducing equivalents for sulphur oxidation in the surrounding water column.

In addition to providing reducing equivalents for NO3

− reduction, sulphur metabolism may contribute to NH4

+ production through the process of DNRA (also termed NO3

− or NO2− ammonification). Both

SO42−-reducing and sulphur-oxidizing bacteria have

been shown to carry out DNRA, in some cases using the conversion of NO3

− or NO2− to NH4

+ as an electron sink for substrate-level phosphorylation48, and in oth-ers coupling the conversion process to generate a proton motive force49. Although most of what we know about DNRA comes from sediment incubation or laboratory experiments with pure cultures49,87, several studies using nitrogen-15 incubations have measured potential DNRA rates in the water columns of the Baltic Sea88, the ETSP22 and the Namibian upwelling89. In the ETSP OMZ, DNRA is estimated to provide a substantial portion of the NH4

+ that is required for anammox22, with up to 22% derived from SO4

2− reduction51. The potential contribu-tions of sulphur-oxidizing bacteria, including members of the SUP05–Arctic96BD-19 and SAR324 clades, to DNRA in the O2-deficient water column remain to be determined.

The existence of a cryptic sulphur cycle coupling the metabolic activities of SUP05–Arctic96BD-19 bacte-ria and sulphate-reducing bacteria in the O2-deficient water column or in association with sinking particles is reminiscent of the symbiotic associations found at oxic–anoxic interfaces90. Indeed, chemoautotrophic symbioses are a common innovation at hydrothermal vent and cold seep habitats, where eukaryotic hosts provide optimal access to the redox couples that are needed to fix inor-ganic carbon on or near the sea floor91. Similarly, symbi-otic sulphur-oxidizing and SO4

2−-reducing bacteria have been described in association with the shallow-water sand-dwelling mouthless worm Olavius algarvensis; for these bacteria, metabolite exchange between different taxonomic groups balances out the fitness costs asso-ciated with resource competition in the host milieu92. Other forms of syntrophy, including direct electron trans-fer, have been described between bacterial and archaeal cells such as acetogens and methanogens or SO4

2− reduc-ers and methane oxidizers, resulting in the production of reduced compounds that fuel subsurface metabolism and deep-sea chemolithoautotrophic communities93,94. Thus, the ecology and biogeochemistry of OMZs repre-sents one manifestation of the greater symbiotic ocean and its impact on the world around us. This impact is rooted in the collective metabolic capabilities of micro-bial cells that drive matter and energy transformations throughout the depth continuum.

Co-occurrence networksJust as cellular complexity arises through networks of genes, proteins and metabolites interacting across multi-ple hierarchical levels95–97, so ecological and biogeochemi-cal phenotypes arise from complex interactions between microbial community members. As Chisholm and Cary state, “No single organism contains all the genes neces-sary to perform the diverse biogeochemical reactions that make up ecological community function. Yet, distributed among the community, are all the functions necessary to

◀ Figure 4 | Diversity in the four most abundant bacterial groups identified in OMZs. The four most abundant groups that were identified in small-subunit ribosomal RNA (SSU rRNA) gene surveys of oxygen minimum zones (OMZs) are the SUP05–Arctic96BD-19 group, the SAR11 and SAR324 clusters, and marine group A (MGA). Each histogram bar represents a cluster of SSU rRNA gene sequences, or an operational taxonomic unit (OTU), generated at a 97% identity cutoff (clustered using the furthest-neighbour algorithm). The height of the bar is equivalent to the sum of all sequences belonging to a specific OTU across all environments surveyed: the eastern tropical South Pacific (ETSP), the Hawaii Ocean Time-series (HOT), the northeastern subarctic Pacific (NESAP; labelled as P4, P12 and P26), Saanich Inlet (SI) and the Namibian upwelling (NAM; also known as the Benguela upwelling). ‘*’ indicates a sample taken from P4 1,000 m in June 2008; all other NESAP samples were taken in 2009. Heat maps below the histograms represent the distribution of sequences in each OTU across all environments surveyed. Heat maps were clustered by row using Euclidean distance and the furthest-neighbour algorithm to highlight patterns of diversity among samples. Inset colour scales depict the colour code for the number of SSU rRNA gene sequences in heat maps. Data were derived from sequences deposited in Genbank.

R E V I E W S




Degree

Betweenness

a

b

A

B

kA = 12

kB = 8

≥0.05


Arctic96B-7

SAR324

SAR11

Microthrixineae

S23_91

Nitrospina

Cytophaga

Rhodobacter

ZA3420c

Nitrospina

SAR324

Cytophaga

SAR11

2501005 25 50


a

b

2,40010 100 500


1,200 ≥0.05

Betweenness centrality

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progressive changes in microbial co-occurrence patterns over time and uncovering potential symbiotic associa-tions102,103. On a global scale, co-occurrence analysis of 298,591 publicly available SSU rRNA gene sequences was used to define nonrandom and recurring co-occurrence networks, consistent with habitat preference between specific taxonomic groups104.

Although networks based on phylogenetic infor-mation alone cannot explain underlying mechanisms of metabolite exchange, they can help define putative metabolic interactions and enable more direct hypoth-esis testing when combined with data about envi-ronmental parameters, process rates and functional genes. We applied co-occurrence network analysis to publicly available SSU rRNA gene sequences from the Hawaii Ocean Time-series, the NESAP, Saanich Inlet and the ETSP, and identified nonrandom patterns of co-occurrence between microbial taxa associated with oxic, dysoxic, suboxic or anoxic water column condi-tions (FIG. 5). In this analysis, dysoxic, suboxic and anoxic subnetworks are dominated by OTUs representing the SUP05–Arctic96BD-19, SAR11, marine group A and SAR324 clades, consistent with members of these clades having overlapping habitat preferences and the potential for metabolite exchange. Moreover, OTUs representing these taxa were identified as hubs in the larger net-work on the basis of the number of connections run-ning through them, consistent with previous reports of ‘keystone’ connectivity in marine ecosystems102,103. Interestingly, the most abundant bacterial OTUs in the network are not typically the most connected, and dif-ferent OTUs for several taxonomic groups participated in multiple subnetworks, suggesting that the overall net-work consists of many functionally redundant modules with the potential to change over time.

Where do we go from here?We live on an ocean-dominated planet, and the collec-tive metabolic expression of cellular life in the ocean has a profound influence on the evolution of the bio-sphere. Cellular life in the ocean is in turn dominated by microbial communities that form interaction networks

which are both resilient and responsive to environmental perturbation. Over geological timescales, recurring changes in the oxygenation status of the ocean have resulted in multiple biotic crises with concomitant changes in marine ecosystems and climate balance. Available monitoring data suggest that OMZ expansion in the modern ocean is consistent with a renewed period of change. When viewed from an Earth systems perspec-tive, these observations take on immediate significance as we consider the potential impacts of OMZ expansion on marine resources and global warming trends. These impacts include reduced biodiversity and food secur-ity and increased production and transport of radia-tively active trace gases owing to changes in microbial interaction networks.

Determining how these interaction networks form, function and change over time reveals otherwise hid-den links between microbial community structure and higher-order ecological and biogeochemical processes. Indeed, over the past few years, plurality sequencing combined with process rate analyses and targeted gene surveys in coastal and open-ocean OMZs has identi-fied conserved patterns of microbial community struc-ture and function, and has uncovered novel modes of metabolic integration that couple carbon, nitrogen and sulphur cycles. These findings have important implica-tions for our understanding of the nutrient and energy flow patterns in expanding marine OMZs. Looking forwards, comparative studies are needed to define the shared or specialized metabolic subsystems that mediate microbial community responses to changing levels of O2 deficiency in the water column in different oceanic prov-inces. Additional time series monitoring studies combin-ing gene expression and process rate measurements are also needed to validate pathway predictions and pro-vide parameters for regulatory and network dynamics for more effective ecosystem modelling. An effective human adaptation and response to OMZ expansion, ranging from our environmental management to our policy towards Earth systems engineering, may depend on our collective capacity to understand and mimic the problem-solving power of the symbiotic ocean.

R E V I E W S



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AcknowledgementsThis work was carried out under the auspices of the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Canada Foundation for Innovation (CFI) and the Canadian Institute for Advanced Research (CIFAR). J.J.W. was supported by the NSERC, and K.M.K. was supported by the Tula Foundation-funded Centre for Microbial Diversity and Evolution (CMDE) at the University of British Columbia, Vancouver, Canada. We thank N. Korniyuk, P. Macoun, V. Tunnicliffe, F. Whitney, M. Robert, A. Hawley and N. Hanson for insightful commentary; D. Walsh for assistance with data visualization; P. Tortell for help with figure 1; and the anonymous reviewers for insightful commentary and guidance.

Competing interests statementThe authors declare no competing financial interests.

FURTHER INFORMATIONSteven J. Hallam’s homepage: http://www.cmde.science.ubc.ca/hallam/Center for Coastal Margin Observation and Prediction: http://www.stccmop.org/Genbank: http://www.ncbi.nlm.nih.gov/genbank/Greengenes taxonomy database: http://greengenes.lbl.govInstitute of Ocean Sciences: http://www.pac.dfo-mpo.gc.ca/sci/sci/facilities/ios_e.htmLeibniz Institute for Baltic Sea Research: http://www.io-warnemuende.de/en_index.htmlMonterey Bay Aquarium Research Institute: http://www.mbari.org/default.htmThe Eastern South Pacific Oxygen Minimum Zone: http://omz.udec.cl/The Fourth Paradigm: Data-Intensive Scientific Discovery: http://research.microsoft.com/en-us/collaboration/fourthparadigm/Victoria Experimental Network Under the Sea (VENUS): http://www.venus.uvic.ca/

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Abstract | Dissolved oxygen concentration is a crucial organizing principle in marine ecosystems. As oxygen levels decline, energy is increasingly diverted away from higher trophic levels into microbial metabolism, leading to loss of fixed nitrogen and toOMZ formation and expansionMicrobial energetics in OMZsFigure 1 | O2 concentrations in the ocean. a | Minimum molecular oxygen (O2) concentrations for different regions of the ocean. Locations highlighted in this Review are indicated and comprise the Hawaii Ocean Time-series (HOT), the northeast subarctic PacFigure 2 | O2 concentration affects ecosystem energy flow. Alternative states of the sea water (oxic, dysoxic, suboxic and anoxic) and corresponding molecular oxygen (O2) concentrations are defined. The red–orange area indicates the range of energy transOMZ microbiotaBox 1 | Redox-driven niche partitioningFigure 3 | Bacterial diversity in the ocean. Dot plot of the diversity of bacterial taxa at various sample points and depths in Saanich Inlet (SI), the northeastern subarctic Pacific (NESAP; labelled P4, P12 and P26), the Hawaii Ocean Time-series (HOT), tThe symbiotic oceanFigure 4 | Diversity in the four most abundant bacterial groups identified in OMZs. The four most abundant groups that were identified in small-subunit ribosomal RNA (SSU rRNA) gene surveys of oxygen minimum zones (OMZs) are the SUP05–Arctic96BD‑19 grouCo‑occurrence networksBox 2 | Network analysisFigure 5 | Co‑occurrence networks: correlations among bacterial OTUs in different OMZs. a | The network of interactions between the operational taxonomic units (OTUs) identified in FIG. 4 and found in various oceanic oxygen minimum zones (OMZs) described Where do we go from here?

microbial ecology of expanding oxygen minimum...

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