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CITATION Joye, S.B., S. Kleindienst, J.A. Gilbert, K.M. Handley, P. Weisenhorn, W.A. Overholt, and J.E. Kostka. 2016. Responses of microbial communities to hydrocarbon exposures. Oceanography 29(3):136–149, http://dx.doi.org/10.5670/oceanog.2016.78.

DOI http://dx.doi.org/10.5670/oceanog.2016.78

COPYRIGHT This article has been published in Oceanography, Volume 29, Number 3, a quarterly journal of The Oceanography Society. Copyright 2016 by The Oceanography Society. All rights reserved.

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Responses of Microbial Communities to Hydrocarbon Exposures

By Samantha B. Joye, Sara Kleindienst, Jack A. Gilbert, Kim M. Handley, Pam Weisenhorn, Will A. Overholt, and Joel E. Kostka

136 Oceanography | Vol.29, No.3 The impact of hydrocarbon pollution on the composition, structure, and function of microbial communities is evident in the responses“ of taxa able to use hydrocarbons as sources of carbon and energy.”.

ABSTRACT. The responses of microbial communities to hydrocarbon exposures are natural gas to the deep waters (~1,500 m) complex and variable, driven to a large extent by the nature of hydrocarbon infusion, of the Gulf of Mexico (Joye, 2015). Some local environmental conditions, and factors that regulate microbial physiology seven million liters of chemical disper- (e.g., substrate and nutrient availability). Although present at low abundance in the ocean, sants, mainly Corexit 9500 and 9527A, hydrocarbon-degrading seed populations are widely distributed, and they respond were applied as a response measure at the rapidly to hydrocarbon inputs at natural and anthropogenic sources. Microbiomes sea surface and at the discharging well- from environments impacted by hydrocarbon discharge may appear similar at a head. Of the discharged oil and gas, all of higher taxonomic rank (e.g., genus level) but diverge at increasing phylogenetic the low molecular weight alkanes (meth- resolution (e.g., sub-OTU [operational taxonomic unit] levels). Such subtle changes are ane through propane) and half of the dis- detectable by computational methods such as oligotyping or by genome reconstruction charged oil were entrained in a deep- from metagenomic sequence data. The ability to reconstruct these genomes, and water plume at a depth of approximately to characterize their transcriptional activities in different environmental contexts 1,000 m (Joye, 2015). The microbial through metatranscriptomic mapping, is revolutionizing our ability to understand the response to this hydrocarbon infusion, diverse and adaptable microbial communities in marine ecosystems. Our knowledge especially at low deep-ocean tempera- of the environmental factors that regulate microbial hydrocarbon degradation and tures, was swift and remarkable (Joye the efficiency with which marine hydrocarbon-degrading microbial communities et al., 2014; Kleindienst et al., 2015a). bioremediate hydrocarbon contamination is incomplete. Moreover, detailed baseline Oil is a mixture of hydrocarbons, descriptions of naturally occurring hydrocarbon-degrading microbial communities which are organic molecules consisting and a more robust understanding of the factors that regulate their activity are needed. of carbon atoms bonded to each other and to hydrogen atoms. Some com- INTRODUCTION water hydrocarbon discharge to date. On plex hydrocarbons contain nitrogen and Oil is introduced into the marine envi- April 20, 2010, operators lost well con- sulfur residues (Seidel et al., 2016), as well ronment through natural seepage and trol on the DWH mobile offshore drilling as metalloids; oxygen is introduced into as a result of human activities, includ- unit. A subsequent gas-fueled explosion hydrocarbons during biodegradation ing pipeline and tanker leaks and spills, resulted in the sinking of the platform and weathering (Aeppli et al., 2012). The for example, the Exxon Valdez oil spill in two days later. Upon sinking, the riser major hydrocarbon classes include sat- 1989, and in some cases large acciden- pipe separated from the drilling platform, urates (e.g., linear, branched, and cyclic tal ocean discharges, for example, the generating an uncontrolled oil well blow- alkanes), aromatics (where single and Ixtoc blowout in the southern Gulf of out at the seafloor. double bonds exist and help to stabilize Mexico in 1979 and the BP/Deepwater The DWH well blowout discharged the compound), resins, and asphaltenes. Horizon (DWH) discharge in 2010, approximately five million barrels of oil A number of aromatic hydrocarbons which ranks as the largest marine open and at least 250,000 metric tonnes of are toxic, making it pragmatic to

Oceanography | September 2016 137 understand the potential of microbial impacts (Figure 1). Microbial hydro- anaerobic pathways are more novel and populations to moderate the impacts of carbon degradation occurs under oxic, complex (Widdel et al., 2010). hydrocarbon pollution. Identifying the microaerophilic, and anoxic conditions The impact of hydrocarbon pollution microorganisms responsible for oil bio- (Head et al., 2003). Complete hydro- on the composition, structure, and func- degradation and understanding the fac- carbon oxidation is achieved though tion of microbial communities is evident tors that regulate bioremediation in the the collective action of associated, inter- in the responses of taxa able to use hydro- marine environment is critical. dependent microorganisms. Though the carbons as sources of carbon and energy. The ability to degrade hydrocarbons metabolic pathways of hydrocarbon oxi- Many of these organisms exist as part of is widespread among the bacteria, meth- dation are similar at the genus level, pri- the “rare biosphere,” a “seed bank” of taxa anogenic archaea, and fungi (Leahy and mary pathways are linked to more taxo- (Gibbons et al., 2013) that are ecologically Colwell, 1990; Head et al., 2003, 2006). nomically diverse secondary pathways noncompetitive, except when exposed to These microorganisms degrade oil (Heider and Rabus, 2008). Aerobic bio- hydrocarbons (Kleindienst et al., 2015a). and gas, either partially or completely, degradation has received more atten- Spatiotemporal investigations of micro- and reduce negative environmental tion than anaerobic biodegradation, but bial community responses to oil pollu- tion revealed the influence that blooms of conditionally rare, opportunistic taxa Biological network of oil, dispersed oil, and dispersant degradaon have on community structure and func- tion (Lu et al., 2012; Mason et al., 2012; Fe Kleindienst et al., 2015a). Subsequent Nutrients N P studies explored how community changes altered the broader ecological proper- ties of polluted environments; for exam-

Biologically ple, metagenomic analysis of oil-polluted Bio- dispersed sediments showed that their microbial oil Chemical surfactant dispersants communities had an elevated potential for anaerobic ammonium oxidation, or anammox (Scott et al., 2014). Large-scale hydrocarbon inputs stim- ulate oxygen consumption as a conse- quence of accelerated aerobic microbial activity. When oxygen is depleted, anaer- Chemically obic hydrocarbon metabolism is coupled dispersed oil to sulfate and nitrate reduction, which fundamentally shifts the nitrogen, sulfur, and carbon cycles, and promotes further changes in microbial structure and com- Metabolic Metabolic position as a function of breakdown prod- products products ucts and cross-feeding (Kleindienst et al., 2015b). After hydrocarbon exposure, the Grazers Viruses community may return to its original eco- logical functional state or be altered, with certain taxa increasing in abundance fol- lowing hydrocarbon bioremediation and persisting on a time scale of years post-​

FIGURE 1. Biological network of oil, dispersed oil, and dispersant degradation. disturbance (Kleindienst et al., 2015a). Hydrocarbon-oxidizing microbes with the capability to produce biosurfactants to In this article, we describe the path- facilitate oil degradation are shown in blue. It remains a question as to whether ways of hydrocarbon degradation in the the activity of these microorganisms is stimulated or inhibited by chemical disper- sants. Different types of hydrocarbon degraders, shown in red, have the ability to environment, the methods used to quan- degrade chemically dispersed oil as well as dispersants (e.g., Colwellia sp. RC25). tify hydrocarbon degradation rates and Secondary metabolite consumers of compounds produced during oil biodegrada- the microorganisms that mediate these tion, for which dispersant impacts are largely unknown, are shown in gray. Parts of this network (nutrient availability, viruses, and grazers) likely influence all the above reactions, and how microbial populations types of microorganisms. Illustration based on Head et al. (2006) respond to hydrocarbon inputs.

138 Oceanography | Vol.29, No.3 PATHWAYS OF HYDROCARBON Aerobic Degradation groups of metalloenzymes, particulate DEGRADATION The aerobic degradation of alkanes, par- methane monooxygenase (pMMO) and Petroleum is a complex mixture of ticularly n-alkanes, is well documented soluble methane monooxygenases, and ~15,000+ compounds formed from (Wang and Shao, 2013; Figure 2a). their homologs (e.g., propane mono- thermogenic alteration of organic mat- Alkanes are distributed ubiquitously, and oxygenase and butane monooxygenase). ter deposited in sediments tens of mil- a number of mechanisms activate them Particulate methane monooxygenases lions of years ago (Marshall and Rodgers, by breaking strong C-H bonds, an ener- use a di-copper active site and can oxi-

2004). Heterotrophic microorgan- getically demanding process. The initial dize up to C5 n-alkanes. Soluble methane isms (microbes that obtain their meta- step in aerobic alkane degradation trans- monooxygenases lie within the large bac- bolic energy and cellular carbon from forms the terminal carbon into a pri- terial multicomponent monooxygenase organic carbon compounds) use a pleth- mary alcohol, which is subsequently oxi- family (BMM), which have a non-heme ora of metabolic pathways for consum- dized to the corresponding aldehyde via di-iron active site, and can oxidize up to ing hydrocarbons in the largely nutrient- an alcohol dehydrogenase, followed by C8 alkanes, including branched alkanes, limited marine environment. The major- oxidation to a fatty acid by an aldehyde cycloalkanes, and even small aromatics. ity of research to date has emphasized the dehydrogenase. Fatty acids are then pro- Mid-length alkane (C5–C16) hydroxy- pathways for degradation and/or trans- cessed via beta-oxidation or converted to lases fall into two main classes, membrane formation of aliphatic and aromatic phospholipids and incorporated into the associated non-heme di-iron mono- hydrocarbons, since these dominate cellular membrane. Most alkane hydrox- oxygenases (AlkB) that share no homol- crude oils and are gas chromatography ylases are metalloenzymes that incorpo- ogy to bacterial multicomponent mono- (GC) amenable and thus easier to study rate metal species into the active site to oxygenases, and heme-based cytochrome (Fuchs et al., 2011; Abbasian et al., 2015; activate oxygen and attack the C-H bond. P450 (CYP153) enzymes. Both classes

Ladino-Orjuela et al., 2016). Short chain alkanes (C1–C4, gaseous are highly diverse, often found together n-alkanes) are oxidized by two known in hydrocarbon-degrading bacteria, and

Key Pathways of Hydrocarbon Degradation a R R Alcohol R Aldehyde R Metalloenzymes dehydrogenase dehydrogenase

Formation of Alcohol to aldehyde Oxidation to fatty acid primary alcohol

b

Mono- or Di-oxygenases Dioxygenases Various

Peripheral pathways Ring cleavage Rearrangements to convert to intermediate TCA intermediates

FIGURE 2. Generalized aerobic alkane and aromatic hydrocarbon degradation pathways. (a) Degradation of alkanes from primary alcohol formation on a terminal or subterminal C via metalloenzymes (e.g., pMMO, AlkB) followed by conversion of the alcohol to an aldehyde via alcohol dehydrogenase, and finally oxidation of the aldehyde to a fatty acid via aldehyde dehydrogenase. (b) Degradation of a sample aromatic hydrocarbon, toluene, to a cen- tral intermediate (e.g., 3-methyl-catechol) followed by ring cleavage and final rearrangement to TCA cycle intermediates. The first two steps in aromatic hydrocarbon degradation are performed by mono- or di-oxygenases.

Oceanography | September 2016 139 have extensive overlapping substrate benzylsuccinate synthase involved in of benzoyl-CoA, the aromatic ring is ranges. These two enzyme classes per- toluene degradation (see below). After susceptible to reduction reactions out- form terminal alkane oxidation, result- fumarate addition, Coenzyme A (CoA) lined in Harwood et al. (1998). The first ing in a primary alcohol. The majority of is added via a CoA transferase, followed step is catalyzed by a class I benzoyl- AlkB alkane hydroxylases preferentially by carbon skeletal rearrangements via CoA reductase (BcrABCD), which act upon C10 to C18 alkanes, although the a mutase, followed by decarboxylation, requires 2 ATP. An ATP-independent best-studied AlkB from Pseudomonas analogous to carbon rearrangements mechanism that employs a non-​​ putida GPo1 preferentially uses C5 to C13 mediated by methylmalonyl-CoA mutase homologous class II benzoyl-CoA reduc- n-alkanes (van Beilen and Funhoff, 2007; (Wilkes et al., 2002). tase (BamBCDEFGHI) is likely driven by Koch et al., 2009). Many model alkane-​ A diverse set of peripheral pathways electron bifurcation (Fuchs et al., 2011). degrading microorganisms contain mul- transform aromatic compounds into one Little is known about the meta- tiple AlkB homologs (van Beilen and of a few key central intermediates (Fuchs bolic pathways involved in asphaltene Funhoff, 2007). Genes encoding AlkB are et al., 2011; Ladino-Orjuela et al., 2016; and resin degradation (Lavania et al., found ubiquitously in the ocean. Figure 2b). Under aerobic conditions, 2012). These very high molecular weight, Heme-containing cytochrome P450 these are typically monooxygenases or heteroatom-containing polar struc- enzymes are found in all domains of dioxygenases that hydroxylate the aro- tures are resistant to biodegradation life. Bacterial alkane hydroxylase cyto- matic compound to produce catechol, and accumulate when crude oil is bio- chrome P450s are soluble and primar- primarily protocatechuate, gentistate, degraded (Head et al., 2006). A few ily act upon n-alkanes between C6 and or homogentistate (Fuchs et al., 2011). microorganisms, including Garciaella C15 (van Beilen and Funhoff, 2007). The aromatic ring component of these petrolearia TERIG02 (bacterium) and Long-chain alkane (C17+) oxidation intermediates is cleaved by two oxygen- Neosartorya fischeri (fungus), degrade enzymes are not well characterized and dependent strategies. Dioxygenases cleave asphaltenes in heavy crude oils. only a few pathways are known. The best hydroxyl-substituted aromatic rings; the G. petrolearia preferentially degraded described are two enzymes that share β-ketoadipate pathway is a well-known asphalt under anaerobic conditions, pro- no apparent homology and use a flavin example (Ornston and Stanier, 1966). ducing CO2, H2, as well as organic acids, cofactor. AlmA, a flavin-binding​ mono- Alternately, the hydroxylated aromatic smaller aromatics, and n-alkanes (Lavania oxygenase, is thought to act upon C20 ring is further substituted with CoA fol- et al., 2012). The underlying mechanism to >C32+ n-alkanes and was first iden- lowed by ring cleavage using expoxidases and genetic pathways involved are not tified in Acinetobacter sp. DMS17874. belonging to the bacterial multicompo- yet known. The fungus N. fischeri also Homologues have been identified in nent monooxygenase family, including grows on asphaltenes as a sole carbon other oil-​degrading bacteria (Throne- benzoate and phenylacetate epoxidation source (Hernández-López et al., 2016), Holst et al., 2007; Wang and Shao, 2013). (Fuchs et al., 2011). The resulting com- possibly using cytochrome P450 mono- LadA is a member of the SsuD bacte- pounds are often incorporated into cen- oxygenases to process asphaltenes. Other rial luciferase subfamily that oxidizes C15 tral metabolism as acetyl-CoA, succinyl- fungal isolates degrade high molecular to C36 alkanes; this gene has been doc- CoA, and pyruvate, and fed into weight polycyclic aromatic hydrocarbons umented in a thermophilic Geobacillus the TCA cycle. (PAHs) using cytochrome P450s (Syed genus (Feng et al., 2007). Anaerobic aromatic hydrocarbon deg- et al., 2011). Interestingly, the resistance radation pathways are diverse and repre- of asphaltenes and resins to degradation Anaerobic Degradation sent different mechanisms that generate may be due to their low solubility in sea- Under anaerobic conditions, there are a few key central intermediates, of which water rather than to their high molecular two mechanisms for alkane activation benzoyl-CoA is the most well known weight and chemical complexity (Marin- involving fumarate addition to a sub- (Harwood et al., 1998; Foght, 2008; Fuchs Spiotta et al., 2014). terminal or terminal carbon (in a case of et al., 2011). For example, toluene deg- propane activation) to produce a substi- radation is initiated by fumarate addi- QUANTIFYING BIODEGRADATION tuted succinate compound. Enzymes in tion through benzylsuccinate synthase RATES AND MICROBIAL this pathway are known as alkylsuccinate (BSS) via a glycyl radical, homologous POPULATIONS synthases (ASS) or 1-methylalkyl suc- to anaerobic alkane degradation through Quantifying microbial oil degradation cinate synthases (MAS), and they most fumarate addition, as mentioned above. rates in environmental samples is compli- likely function through the generation Unsubstituted aromatics may be methyl- cated due to the composition range and of a glycyl radical (Widdel and Rabus, ated, directly carboxylated, or hydroxyl- differential volatility of the hydrocarbon 2001). These enzymes share homol- ated before conversion to benzoyl-CoA pool. Direct and indirect approaches are ogy and a similar mechanism to the (Foght, 2008). Following the generation used to estimate hydrocarbon oxidation

140 Oceanography | Vol.29, No.3 rates. Direct rate measurements involve particular focus on the bioremediation enrichment by organisms consuming the tracking 14C, 13C, or 3H labeled sub- of oil spills in the environment, exists. degradation products of primary hydro- strates into oxidized products (14C or A number of studies examined how carbon degraders (i.e., cross-feeding). 13 3 C-CO2, or H-H2O; Richnow et al., crude-oil-associated bacteria metabo- However, DNA-SIP has the advantage of 1998). Indirect rate assessments involve lize fractions of complex hydrocarbon overcoming the uncertainty associated the use of proxies—such as cell counts, mixtures to optimize refining processes. with interpreting the putative function of

CO2 production rates, the rate of oxidant Microbial processes such as biodesulfur- environmental genes resembling known consumption (e.g., oxygen, nitrate, sul- ization, biodemulsification, biodenitro- hydrocarbon degradation genes and the fate), the rate of oil depletion (approx- genation can enhance oil recovery, con- substrate promiscuity of many enzymes imately, the concentration change over trol souring, and enhance remediation. involved in hydrocarbon degradation time), or bacterial production rates fol- Methods used to identify these organ- (e.g., van Beilen et al., 1994). A suite of lowing exposure to a specific hydrocar- isms range from culture-dependent -omics approaches demonstrated that bon (e.g., hexadecane or naphthalene) approaches used to grow and isolate hydrocarbon-infusion-induced enrich- or to bulk crude oil (Kleindienst et al., particular organisms to metagenomic- ment of expressed genes associated with 2015c)—to estimate hydrocarbon oxi- derived assembly of genomes of organ- aliphatic hydrocarbon degradation, and dation rates. Proxy metrics are not spe- isms associated with these processes from plume-derived representatives of abun- cific and, as such, these data should be complex microbial populations. dant Oceanospirillales and Colwellia bac- interpreted with caution. In particular, Culture-dependent studies provide teria, had the genetic capacity to degrade using changes in cell counts or bacterial access to a viable organism for which these hydrocarbons during the DWH production over time can be mislead- the genome can be characterized, and incident (Mason et al., 2012, 2014). ing because bottle effects and availabil- then the specific functional potential is Amplicon sequencing offers only a ity of other carbon substrates, for exam- validated based on functional tests, for snapshot of the taxonomic and phyloge- ple, chemical dispersants, could alter example, enzymatic activity and transfor- netic breadth of microbial community these parameters in the absence of ele- mation of specific compounds. However, structure. Generating a detailed assess- vated hydrocarbon degradation rates our ignorance of the conditions neces- ment of the functional potential of key (Kleindienst et al., 2015b,c). sary for successful cultivation of many organisms requires characterization of Direct measurement of the turn- organisms, coupled to a lack of under- the metagenome, the sum of genomic over of specific hydrocarbon substrates standing of how ecological factors such information for all organisms within an using radiolabeled tracers provides a as competitive exclusion and niche dif- ecosystem. Normally, metagenomic anal- robust means of documenting the pat- ferentiation influence growth in vitro, yses are restricted to virus or microbial terns of hydrocarbon degradation and mean cultivation-dependent techniques genomes, owing to their small genome elucidating the environmental factors likely underestimate the range of micro- sizes (Gilbert and Dupont, 2011; Knight that drive these patterns (Kleindienst organisms that can directly and indirectly et al., 2012). Validation of the functional et al., 2015b,c; Sibert et al., 2016). access hydrocarbon mixtures for energy role of these microorganisms, espe- During the DWH response, dissolved and biomass production. cially with relevance to specific func- gas (e.g., methane, ethane, and propane) Application of amplicon sequencing tional genomic potentials, requires that oxidation rates were directly measured approaches is now routine, while single- multiple -omics technologies be applied using stable and radio-labeled isotopic cell genomic, metagenomic, and tran- to the same sample and/or that direct substrates (Valentine et al., 2010, and scriptomic (“-omics”) approaches are fast rate assays be carried out in concert with Crespo-Medina et al., 2014, respectively). becoming routine for exploring microbial -omics studies (Kleindienst et al. 2015b). However, oil degradation rates were system dynamics (Knight et al., 2012). An The application of metatranscriptom- inferred from concentration changes over alternative approach for determining the ics to communities of organisms reveals time (Hazen et al., 2010, for alkanes in biological contribution to hydrocarbon which genes are being transcribed into deep waters) or through measurements degradation is 13C DNA-based stable iso- mRNA by community members under of oxygen and bulk hydrocarbon con- tope probing (DNA-SIP). Studies during specific conditions. Metaproteomics sumption (Edwards et al., 2011, in surface the DWH response (Gutierrez et al., takes this analysis one stage further to ask waters); rates were not measured directly 2013) identified a wide range of bacte- the question as to whether the proteins using isotopic tracers, making it diffi- ria in the isotopically heavy DNA frac- predicted to be produced from genes and cult to constrain the fate of hydrocarbons tion that were potentially responsible for mRNAs by a community of cells have during the DWH incident (Joye, 2015). degrading alkane PAHs. actually undergone post-transcriptional Considerable knowledge of the micro- DNA-SIP may be susceptible to modification and appropriate folding to bial biodegradation of crude oil, with the effects of indirect heavy isotope produce a potentially active molecule; this

Oceanography | September 2016 141 technique has been used to great effect The environment locally selects the (re)assessment of bioremediation and to validate predictions of potential pro- type of microorganisms that are active, response strategies in the event of tein production by communities in com- and these microbes boost their activity/ anthropogenic hydrocarbon discharges. plex soil systems. Finally, the outcome abundance in response to hydrocarbon Natural hydrocarbon seep communities of microbial activity is captured by the inputs. Crucial factors for enriching harbor distinct bacterial and archaeal metabolome, the metabolites and signal- hydrocarbon-degrading microorganisms taxa linked to key biogeochemical func- ing molecules generated and consumed include the availability and concentra- tions, such as hydrocarbon degradation. by the community. These approaches tions of hydrocarbons and the types of Within these core groups, high diversity can be combined through computa- bioavailable hydrocarbons (e.g., short- was observed at natural seeps (Ruff et al., tional modeling techniques to predict chain and longer-chain alkanes, PAHs). 2015) and also during anthropogenic how microbial communities will change, Petroleum- or natural gas-derived oil spills (Kleindienst et al., 2015a,b), as well as the mechanisms by which they hydrocarbon mixtures contain simi- underscoring the activity of specialized influence the turnover of hydrocarbons lar constituents, although the relative subpopulations or ecotypes. Because in the environment (Gilbert and Henry, abundance of hydrocarbons, includ- the environmental parameters at natu- 2015), and they have been used to deter- ing potentially toxic BTEX (benzene, ral seeps are substantially different than mine the impact of the DWH spill on toluene, ethylbenzene, and xylenes) and those existing during an anthropogenic seafloor nitrogen cycling in the Gulf of PAH compounds, varies significantly. hydrocarbon release, the taxa and eco- Mexico (Scott et al., 2014). Furthermore, abiotic processes such as types endemic to natural seeps may not weathering, absorption, and diffusion be active during oil spills (Kleindienst MICROBIAL RESPONSE TO influence the concentrations and bio- et al., 2015b) and vice versa. HYDROCARBON INPUTS availability of hydrocarbons. To examine the ecological roles of rare Hydrocarbon Degradation in The availability of electron accep- keystone taxa that provide essential eco- Waters, Muds, and Sands tors is another factor that determines system functions requires cultivation-​ Opportunistic microorganisms with the the type of hydrocarbon-degradation independent 16S rRNA gene-based biochemical ability to aerobically or anaer- metabolism (e.g., aerobic or anaerobic approaches in combination with obically degrade hydrocarbons (Head respiration), typically favoring the most next-generation sequencing technolo- et al., 2006; Widdel et al., 2010) occur thermodynamically favorable process. gies. Typically, 16S rRNA gene sequences ubiquitously across marine ecosystems However, if electron donors (i.e., hydro- are clustered into operational taxonomic in the water column, in sediments, and carbons) are present in excess, compet- units (OTUs), based on a sequence sim- in beach sands and marsh muds (Atlas ing respiration processes may occur con- ilarity threshold (e.g., 97%). However, et al., 2015). Present at relatively low temporaneously rather than in series, rare microbial hydrocarbon degraders abundance, these key microbial players dictated by the electron acceptor energy may not be identifiable on the OTU level are members of the rare biosphere (Sogin yield. Additional important selecting and, consequently, may remain hidden et al., 2006; Kleindienst et al., 2015a) factors include the availability of nutri- in large sequencing data sets. To resolve and typically comprise <10% of micro- ents (e.g., phosphorus, nitrogen, essen- environmentally relevant differences bial communities in the Gulf and else- tial trace metals), pH, temperature, and between sequences of closely related where (Yang et al., 2014). Hydrocarbon- pressure. Biotic processes further influ- microbial taxa that respond to fluctuat- degrading seed populations can respond ence hydrocarbon-​degrading microbial ing geochemical conditions (e.g., eco- with incredible speed to massive per- responses. Hydrocarbon degraders are types), bioinformatics approaches that turbations (Kleindienst et al., 2015a) part of a biological network composed of allow sub-OTU resolution are required and even natural seepage (Ruff et al., additional microbial community mem- (Eren et al., 2013). 2015). Microbial hydrocarbon degrad- bers, viruses, and grazers (Head et al., ers fall within the Gammaproteobacteria 2006) and thus are likely affected by inter- FIGURE 3. Phylogeny of 125 hydrocarbon- (e.g., the Oceanospirillum, Colwellia, actions such as syntrophic relationships, degrading bacteria (HCD), including isolates and bacteria enriched by the Gulf of Mexico Cylcloclasticus, Pseudoalteromonas, Alkan- competition, transfer of genetic material, Deepwater Horizon oil spill (DWH) or by DNA- ivorax, Alteromonas, and Marinobacter), and predation (Figure 1). based stable isotope probing. Maximum the Betaproteobacteria (e.g., Acidovorax, The detection and identification Likelihood tree of 16S rRNA gene sequences > 1,248 bp long constructed using ClustalW Burkholderia), the Alphaproteobacteria of key microorganisms that respond alignments and 500 bootstrap replicates (e.g., Roseobacter), numerous Delta- to hydrocarbon inputs is essential for (MEGA v.6.06). Sequence GenBank accession proteobacteria, as well as Actinomycetales understanding the environmentally rel- numbers are given in parentheses. Where indi- cated in parentheses and in bold, DWH beach (e.g., Acinetobacter), Bacillus, and other evant biogeochemical processes at nat- and water isolates are represented by proxy taxa (Figure 3). ural hydrocarbon seeps and for the sequences 98.6% to 100% identical (ID).

142 Oceanography | Vol.29, No.3 Phylogenetic Tree of Dominant Hydrocarbon Degrading Microorganisms Alteromonadales/Vibrionales 90 ••••• Ethane SIP bacterium clone 4-4-07 [DWH distal plume] 92 ••••• Propane SIP bacterium clone 6-4-14 [DWH distal plume] ••••• Propane SIP bacterium clone 6-4-09 [DWH distal plume] Gaseous alkanes Colwellia rossensis strain ANT9247 61 72••••• Benzene SIP bacterium clone 10-4-01[DWH distal plume] n-alkanes ••••• Methane SIP bacterium clone 2-7-14 [DWH distal plume] 98 ••••• Propane SIP bacterium clone 6-7-06 [DWH distal plume] Aromatics 100 Colwellia psychrerythraea 34H Colwellia psychroerythrus IC064 PAHs 50 ••••• Colwellia SIP clone SWPHE03 [DWH shallow seawater] 99 ••••• Colwellia sp. RC25 [oil enrichment DWH uncontaminated deep seawater] 94 ••••• Colwellia isolate B11 [DWH proximal plume] Crude oil 100 ••••• Pseudidiomarina maritima 908087 [99.9% ID, isolate DWH oiled beach sand] 100 ••••• Altermonas SIP clone SWNAP06 [DWH shallow seawater] Aerobic ••••• Altermonas sp. TK46(2) HEXPHENAP [DWH plume/shallow seawater] 100 ••••• Vibrio hepatarius UST950701-002 [98.6% ID, isolate DWH oiled beach sand] ••••• Vibrio plantisponsor MSSRF64 [99.9% ID, isolate DWH oiled beach sand] Anaerobic ••••• Vibrio sp. NAP-4 [PAH contaminated sediment] ••••• Shewanella algae MAS2741 [99.2% ID, isolate DWH oiled beach sand] 67 ••••• Pseudoalteromonas sp. EPR 2 [99.8% ID, isolate DWH oiled beach sand] ••••• DWH HCD ••••• Pseudoalteromonas sp. TK105 [PHE,NAP] [DWH plume/shallow seawater] 69 100 97 ••••• Pseudoalteromonas isolate B17 [DWH distal plume] Marine HCD 99 92 ••••• Pseudoalteromonas isolate B15 [DWH distal plume] 77 ••••• Ethane SIP bacterium clone 4-7-56 [DWH distal plume] 70 ••••• Benzene SIP bacterium clone 10-4-04 [DWH distal plume] Terrestrial HCD Profundimonas piezophila strain YC-1 ••••• Propane SIP bacterium clone 6-7-34 [DWH distal plume] Oceanospirillale Source unknown HCD Bacterium symbiont of Osedax sp. clone Rs2 100 98 Neptunomonas naphthovorans NAG-2N-126 [creosote contaminated sediment] Reference sequences Neptunomonas japonica JAMM 1380 Neptunomonas antarctica strain S3-22 Thalassolituus marinus strain IMCC1826 74 100 Oleispira antarctica RB-8(T) [antarctic coastal seawater] Oleispira lenta strain DFH11 51 94 Oceaniserpentilla haliotis strain DSM 19503 Spongiispira norvegica strain Gp 4 7.1 99 ••••• Propane SIP bacterium clone 6-7-41 [DWH distal plume] 97 ••••• Oceanospirillales bacterium clone M580104-10 [enriched in DWH proximal plume]

••••• Ethane SIP bacterium clone 4-7-24 [DWH distal plume] s 100 ••••• Methane SIP bacterium clone 2-7-22 [DWH distal plume] ••••• Propane SIP bacterium clone 6-4-24 [DWH distal plume] ••••• Oceanospirillales bacterium clone OV01102/03-20 [enriched in DWH proximal plume] G ammaproteobacteri 81 62 ••••• Ethane SIP bacterium clone 4-4-06 [DWH distal plume] 96 ••••• Methane SIP bacterium clone 2-5-03 [DWH distal plume] 92 ••••• Methane SIP bacterium clone 2-5-49 [DWH distal plume]

Methylococcaceae bacterium SF-BR Thiotrichale 88 Bathymodiolus brooksi methanotrophic gill symbiont clone GoM Chap 16S 2.1 Methanotrophic endosymbiont of Idas sp. clone M3.33 99 Methylobacter albus BG8 100 93 Methylophaga sp. SM14 [surface seawater] Methylophaga thalassica strain YK-4015 ••••• Methane SIP bacterium clone 2-5-07 [DWH distal plume] ••••• Cycloclasticus SIP clone SWNAP12 [DWH shallow seawater] ••••• Cycloclasticus sp. strain E [GoM sediment] Cycloclasticus pugetii PS-1(T) [sediment] ••••• Cycloclasticus sp. strain G [GoM sediment] s ••••• Cycloclasticus sp. TK8 PHENAP [DWH plume/shallow seawater] 100 ••••• Cycloclasticus sp. strain W [GoM sediment] Pseudomonadales

Cycloclasticus spirillensus M4-6 [animal burrow] a 96Cycloclasticus spirillensus isolate P1 [deep-sea sediment] 100 ••••• Acinetobacter venetianus ZX-PKU-001 [99.8% ID, isolate DWH oiled beach sand] ••••• Acinetobacter sp. MSIC01 (100% ID, isolate DWH oiled beach sand] Acinetobacter sp. ADP1 ••••• Pseudomonas pachastrellae PTG4-14 (100% ID, isolate DWH oiled beach sand] Pseudomonas pseudoalcaligenes KF707 100 Pseudomonas sp. OX1 [sludge] Alteromonadales 90 Pseudomonas putida mt-2 Pseudomonas putida GPo1 ••••• Pseudomonas stutzeri GAPP4 [99.7% ID, isolate DWH oiled beach sand] 100 ••••• Pseudomonas isolate B53A [DWH proximal plume] 99 ••••• Marinobacter SIP clone DWHEX95 [DWH plume] 100 ••••• Marinobacter sp. TT1 HEX [DWH plume/shallow seawater] ••••• Marinobacter sp. TK36 HEX [DWH plume/shallow seawater] Marinobacter hydrocarbonoclasticus SP.17 [oil contaminated sediment] 99 ••••• Marinobacter hydrocarbonoclasticus MARC4F [99.4-100% ID, isolate DWH oiled beach sand] ••••• Microbulbifer maritimus RV1[98.7% ID, isolate DWH oiled beach sand] Porticoccus hydrocarbonoclasticus MCTG13d [dinoflagellate culture] 65 ••••• Halomonas shengliensis SL014B-85 [99.7% ID, isolate DWH oiled beach sand] Halomonas sp. 2MN-1 [deep-sea sediment] Oceanospirillale 100 ••••• Halomonas sp. GOS3a PHENAP [DWH plume/shallow seawater] 68 100 ••••• Halomonas sp. GOS2 PHENAP [DWH plume/shallow seawater] 70 ••••• Halomonas sp. TGOS10 HEXPHENAP [DWH plume/shallow seawater] 71 Alcanivorax borkumensis SK2(T) [seawater or sediment] 100 Alcanivorax sp. Strain NBRC 101098 [seawater] ••••• Alcanivorax SIP clone DWHEX05 [DWH plume] 99 ••••• Alcanivorax sp. TY4 HEX [DWH plume/shallow seawater] ••••• Alcanivorax sp. TY6 HEX [DWH plume/shallow seawater] ••••• Alcanivorax sp. 2A75 [99.9% ID, isolate DWH oiled beach sand] 100 Alcanivorax sp. 521-1 [deep-sea sediment] 98 Alcanivorax sp. TK23 HEX [DWH plume/shallow seawater]

•••••Alcanivorax dieselolei B-5(T) [oil contaminated surface seawater] s 75 ••••• Alcanivorax dieselolei (100% ID, isolate DWH plume/shallow seawater] Alcanivorax dieselolei NO1A [deep-sea sediment] 86 100 Alcanivorax sp. Strain 2B5 [oil contaminated mud] gammaproteobacteria HdN1 [activated sludge] Arhodomonas sp. Rozel [hypersaline sediment] Methylococcus capsulatus (Bath) 100 Polycyclovorans algicola TG408 [diatom culture] Betaproteobacteria Fontimonas thermophila strain HA-01 Hydrocarboniphaga daqingensis strain B2-9 Achromobacter xylosoxidans isolate 2MN-2 [deep-sea sediment] 70 Brachymonas petroleovorans CHX [sludge] Nitrosomonas europaea (ATCC 19178) 100 100 Dechloromonas aromatica RCB [river sediment] betaproteobacteria OcN1 [ditch sediment] 100 Thauera aromatica K172 DSM 6984 [sludge] Thauera butanivorans IAM 12574 [activated sludge] 69 Azoarcus toluclasticus MF63 ATCC 700605 [aquifer sediment] 70 Aromatoleum aromaticum EbN1 Azoarcus buckelii B5-1 [soil] 99 betaproteobacteria PbN1 58 100 betaproteobacteria HxN1 [ditch sediment] 82 Desulfoglaeba 87alkanexedens ALDC(T) [sludge oily wastewater facility] 100 Desulfoglaeba alkanexedens Lake [oilfield production water] Syntrophobacter sulfatireducens strain TB8106 Desulfacinum hydrothermale strain MT-96 Desulfatimicrobium mahresensis strain SA1 64 100 ••••• Bacillus sp. BZ85 [99.2% ID, isolate DWH oiled beach sand]

Fictibacillus barbaricus strain NIOT-Ba-23 A 94 92 Bacillus nanhaiensis strain K-W9 ctinobacteria 100 ••••• Benzene SIP bacterium clone 10-4-05 [DWH distal plume] Aquihabitans daechungensis strain CH22-21 71 Aciditerrimonas ferrireducens strain IC-180 ••••• Microbacterium schleiferi 2PR54-18 [99.9% ID, isolate DWH oiled beach sand] 100 Rhodococcus sp. DK17 [oil contaminated soil] 97 Gordonia sp. TY-5 [soil] 99 Gordonia terrae CC-NAPH129-6 [diesel contaminated soil] 99 Mycobacterium sp. 6PY1 [PAH contaminated soil] 100 Mycobacterium austroafricanum JOB5 [soil] 100100 Flavobacterium rakeshii strain FCS-5 Salibacter luridus type strain KSW-1T 100 Flavobacterium sp. W6-14 [deep-sea sediment] 100 ••••• Propane SIP bacterium clone 6-7-27 [DWH distal plume] 50 ••••• Benzene SIP bacterium clone 10-7-18 [DWH distal plume] Sneathiella glossodoripedis strain MKT133 Magnetospira thiophila strain MMS-1 ••••• Methane SIP bacterium clone 2-7-11 [DWH distal plume] 100 ••••• Propane SIP bacterium clone 6-7-17 [DWH distal plume] 64 ••••• Benzene SIP bacterium clone 10-7-06 [DWH distal plume] Alphaproteobacteria ••••• Ethane SIP bacterium clone 4-7-07 [DWH distal plume] Cohaesibacter gelatinilyticus strain CL-GR35 100 ••••• Labrenzia aggregata 2PR58-2 [99.3% ID to isolate DWH oiled beach sand] Parvibaculum lavamentivorans strain DS-1 63 Pseudaminobacter sp. W11-4 [deep-sea sediment] Methylosinus trichosporium OB3b Rhodobium orientis strain JA208 100 Rhodobium orientis JA208 Lutibacterium anuloederans LC8 [animal burrow] Hyphomonas jannaschiana isolate W6-15 [deep-sea sediment] ••••• Benzene SIP bacterium clone 10-7-22 [DWH distal plume] 98 ••••• Propane SIP bacterium clone 6-7-05 [DWH distal plume] 87 69 ••••• Benzene SIP bacterium clone 10-7-11 [DWH distal plume] ••••• Ethane SIP bacterium clone 4-7-14 [DWH distal plume] 100 Halocynthiibacter namhaensis strain RA2-3 67 Marinosulfonomonas methylotropha clone SE69 Celeribacter indicus P73(T) [deep-sea sediment] 84 ••••• Rhodobacteraceae isolate B39 [DWH proximal plume] 0.05 55 Sulfitobacter pseudonitzschiae strain H3 69 Pelagicola litoralis strain CL-ES2 82 ••••• Ethane SIP bacterium clone 4-7-02 [DWH distal plume] 73 ••••• Propane SIP bacterium clone 6-4-04 [DWH distal plume] 82 Oceanography | September 2016 143 Several tools and approaches are avail- plume oxygen depletion was due to aer- the first few months to years after oil able to detect rare taxa, including oligo- obic oxidation of short chain alkanes, from the DWH spill came ashore onto typing, which distinguishes subtle nucle- propane, and butane (Valentine et al., beaches (Hayworth et al., 2015) and wet- otide variations within 16S rRNA gene 2010). Also, metabolic genes involved in lands (Mahmoudi et al., 2013; Atlas et al., amplicon reads and clusters sequences hydrocarbon degradation were highly 2015). Whereas alkanes and low molecu- into so-called oligotypes (Eren et al., enriched in the plume (Lu et al., 2012). lar weight PAHs were largely depleted in 2013). The similarity thresholds for oli- Stable-isotope probing laboratory stud- coastal sediments, high molecular weight gotypes can be as low as 0.2%, which is ies suggested that Colwellia oxidized PAHs (e.g., chrysene) persisted and could remain for many years. Oil is degraded at much reduced rates when buried; thus, submerged oil mats, tens to hundreds of meters long and up to 20 cm thick, have been reported along the inner shelf of [O]ur ignorance of the conditions necessary the northern Gulf of Mexico (Dalyander et al., 2014), and tar balls, typically for successful cultivation of many organisms, 0.5–5 cm in diameter and containing coupled to a lack of understanding of how 5% to 10% hydrocarbons by weight con- “ ecological factors such as competitive exclusion tinue to wash up on northeastern gulf shores. Chronic exposure to oiled sedi- and niche differentiation influence growth in vitro, ments has severe adverse effects on juve- mean cultivation-dependent techniques likely nile benthic fish (Brown-Peterson et al., 2015), suggesting that buried oil poses underestimate the range of microorganisms that a long-term ecological risk to coastal can directly and indirectly access hydrocarbon Gulf of Mexico ecosystems. mixtures for energy and biomass production. . Oil contamination from the DWH spill had a profound impact on the abun- dance, structure, and metabolic poten- tial of sedimentary microbial commu- nities along beaches (Kostka et al., 2011) and marshes (Mahmoudi et al., 2013; more than an order of magnitude lower ethane, propane, and butane” (Redmond Atlas et al., 2015) of the northern Gulf than the dissimilarity threshold used by and Valentine, 2012), while single-cell Coast. A time series study conducted most OTU-clustering methods (3%). genomics revealed that Oceanospirillus at Pensacola Beach, Florida, where Such subtle ecotype variations distin- has the potential to oxidize cyclohexane total petroleum hydrocarbons reached guish key hydrocarbon degraders that (Mason et al., 2012). 11,000 mg kg–1, revealed a bloom of bac- respond to hydrocarbon inputs in the A substantial, yet unconstrained, por- teria during the first four months after environment (Kleindienst et al., 2015a) tion of DWH discharged oil reached oil came ashore, with microbial abun- and serve to reveal the taxa responding coastal ecosystems, polluting a large dance in oiled sands 10 to 10,000 times to hydrocarbon and dispersant amend- (~1,800 km) swath of shoreline from that of clean sands (Kostka et al., 2011). ments (Kleindienst et al., 2015b). East Texas to West Florida (Michel et al., Geochemical evidence confirmed the In the DWH deepwater plume, the 2013). Oil was transported high onto role microorganisms play in the degrada- infusion of oil and dispersants enriched the supratidal zone of beaches by waves tion of weathered oil (Ruddy et al., 2014), for bacteria related to Oceanospirillum, and tides associated with storms (Michel and the succession of indigenous micro- Cycloclasticus, Colwellia, Rhodobacterales, et al., 2013), and a portion of the oil bial populations paralleled the chemical Pseudoalteromonas, as well as to methylo- was deposited in the intertidal and sub- evolution of the petroleum hydrocarbons trophs (Mason et al., 2012; Reddy et al., tidal zones near the beach. Because of (Rodriguez-R et al., 2015). 2012). Preferential microbial utilization the dynamic nature of coastal sediments, The most extensive metagenomic time of short-chain and higher-weight alkanes storms often resulted in the rapid burial series describing microbial hydrocarbon was inferred from compositional changes of oil in these environments. degradation, which was collected from in the hydrocarbon complex (Valentine Total petroleum hydrocarbons, and these Pensacola Beach sands, showed a et al., 2010). Localized dissolved oxygen aliphatic and aromatic compounds were similar progression of microbial popula- anomalies indicated that up to 70% of highly weathered and depleted within tions linked to hydrocarbon degradation

144 Oceanography | Vol.29, No.3 observed in other coastal sediments. dilution, dissolution, advection by ocean temperature strongly regulates the capac- Oil deposition led to a decrease in tax- currents, particle flocculation and aggre- ity and efficiency of petroleum hydrocar- onomic diversity. The bloom was dom- gation, sedimentation, and evaporation, bon degradation in seawater (Bagi et al., inated by members of the Gamma- and along with biodegradation. Similar to 2013). However, kinetic constraints do Alphaproteobacteria, and the abundance the breakdown of terrestrially or marine- not appear to be as important as pre- of genes for hydrocarbon degradation sourced organic matter, microbial com- viously perceived. For example, Hazen pathways closely paralleled microbial munities biodegrade the majority of et al. (2010) observed half-lives of C13 to population dynamics. A clear succession petroleum hydrocarbons (oil and gas) C26 alkanes to be from one to eight days pattern was observed, with early respond- that enter the marine environment. Local at low temperatures (4°C to 6°C) in DWH ers to oil contamination (Alcanivorax) temperature, oxygen levels, and nutri- deepwater plume samples. Subsequently, likely degrading aliphatic hydrocarbons, ent availability limit the rate and extent Brakstad et al. (2015) observed half-lives being replaced after three months by of hydrocarbon degradation or weather- of one to two weeks for alkanes and two populations capable of aromatic hydro- ing (Leahy and Colwell, 1990; Head et al., to four weeks for PAHs in low tempera- carbon decomposition (Hyphomonas, 2006); these factors are determined by ture (5°C) waters. Although these data Parvibaculum, Marinobacter). After one physical processes that mix and ventilate indicate that temperature was not the year, a typical beach community had water masses within the ocean. Although overriding factor limiting degradation, in reestablished that showed little to no few data are available, pressure may also many cases, the temperature response was evidence of oil hydrocarbon degrada- impact biodegradation rates through quantified under nutrient replete condi- tion potential, but it differed significantly effects on chemical solubility and/or the tions. Therefore, synergies between tem- from the community present before the physiology of hydrocarbon-degrading perature and nutrient limitation should oil spill, indicating that beach microbial bacteria (Schedler et al., 2014). Relatively be further explored. communities respond to crude oil per- few studies have been conducted under Oil is an unusual carbon substrate for turbation according to the specialization high pressure and low temperature con- microbial growth. Not only is it largely disturbance hypothesis. ditions that mimic deepwater conditions. insoluble, it also lacks major nutri- In intertidal wetlands, fine-grained This fundamental gap in understand- ents (N, P), a stark contrast to marine- sediments accumulate under relatively ing microbial hydrocarbon degrada- derived planktonic organic matter. A quiescent tidal and current conditions, tion at pressure is remarkable, given the large pulse of oil into any ecosystem could producing heterogeneous, organic-rich, petroleum industry’s trend of increas- thus lead to nutrient limitation of micro- and anoxic conditions near the sedi- ing oil and gas production in ultradeep bial metabolism. A substantial body of ment surface. Hydrocarbons accumu- (>1,500 m) water, which presents the research shows that nutrient availability lated in marsh sediments were largely implicit risk of future deep-sea oil well determines the rate of microbial oil deg- degraded within the first few years after blowouts. Further, the impacts of chem- radation in marine systems (Leahy and oil came ashore (Mahmoudi et al., 2013; ical dispersants and their influence on Colwell, 1990). These observations serve Atlas et al., 2015). Oxygen supply dic- biodegradation has not been studied as the basis for bioremediation strategies, tated the extent of hydrocarbon degra- across the full range of oceanographic such as that employed in response to the dation, and anaerobic microbial popula- conditions. More information is available Exxon Valdez spill. However, more than tions such as sulfate-reducing members of on the environmental controls on hydro- 25 years after the Exxon Valdez disas- the Deltaproteobacteria and methanogens carbon degradation in marine water col- ter, evidence remains equivocal regard- increased in relative abundance in sed- umns than in seafloor sediments. This ing nutrient limitation of hydrocarbon iments where hydrocarbons were lack of knowledge regarding oceano- degradation in studies surrounding the degraded (Atlas et al., 2015). Oil degrada- graphic controls on oil transport and deg- DWH discharge. tion genes associated with anaerobic path- radation, especially in the deep sea, is a A study conducted using mesocosms ways increased dramatically at oiled sites, critical obstacle to effective parameteriza- containing Gulf of Mexico surface sea- and even the higher molecular weight tion of oil plume models, which is critical water found that nutrients appeared to PAHs were substantially biodegraded. to improving model prediction. limit hydrocarbon degradation and res- Information from the DWH discharge piration rates, and microbial biomass Regulation of Microbial Processes indicated that oxygen is rarely com- did not increase in response to the addi- The fate and transport of discharged pletely depleted in an oil-​contaminated tion of Macondo oil (Ortmann and Lu, oil is determined by a complex inter- water column, meaning that tempera- 2015). However, under severely nutrient- play among hydrocarbon chemistry, the ture and nutrients are likely the key lim- limited conditions near the DWH well- microbial food web, and ambient ocean- iting factors for hydrocarbon degra- head, Edwards et al. (2011) observed ographic processes, including dispersion, dation. Laboratory studies show that enhanced respiration rates and a half-life

Oceanography | September 2016 145 of 26 days for oil degradation in the sur- dispersant applied (~7 million liters) and LOOKING FORWARD – face mixed layer. Because bacterial bio- by the location of dispersant application. CONCLUDING THOUGHTS mass levels did not appear to differ in Chemical dispersants are believed to Natural oil seepage and anthropo- the surface slick relative to surrounding stimulate biodegradation by generat- genic oil discharges are commonplace waters, these authors suggested that top- ing high oil-seawater interfaces that are across the world ocean. Microbes are down processes, such as grazing or viral more readily accessible to hydrocarbon- adept and efficient at degrading hydro- lysis, prevented biomass accumulation. degrading microorganisms; further, the carbons, even under nutrient-stressed Data from ultra-high-resolution mass small droplet size is assumed to relieve conditions. Developing a deeper under- spectrometry documented that oil- nutrient or oxygen limitation of oil bio- standing of the regulation and capac- derived organic matter could serve as a degradation. However, available data ity for microbial hydrocarbon remedia- nutrient source (namely N) for oil deg- provide conflicting and contradictory tion in a range of environments and over radation in deep waters collected near results: some studies suggest disper- a reasonable suite of environmental con- an active Gulf of Mexico hydrocarbon sant stimulation of biodegradation while ditions is critical. While much has been seep (Kleindienst et al., 2015b; Seidel others conclude that dispersants either learned over the past few decades, there is et al., 2016). Finally, a metagenomic time make no difference or inhibit biodegra- still more to discover. In particular, doc- series from coastal sediments exposed to dation (Kleindienst et al., 2015a). umenting the efficiency of the microbial oil from the DWH discharge shows that The effects of dispersants on micro- hydrocarbon biofilter in the presence and the abundance of genes associated with organisms might be taxa-specific absence of chemical dispersants is a key nutrient scavenging (nitrogen fixation, (Figure 4) and dependent on disper- area of future research. iron chelation) correlates positively with sant concentrations. For instance, cer- Likewise, the DWH incident revealed the abundance of genes for hydrocarbon tain Colwellia taxa responded to disper- a previously unrecognized rare biosphere catabolism (Rodriguez-R et al., 2015). sants or oil-dispersant mixtures (Bælum that rapidly responds to hydrocarbon Together, these data indicate that the et al., 2012; Kleindienst et al., 2015b), infusion (Kleindienst et al., 2015a). The ocean environment dictates the efficiency while Marinobacter (Kleindienst et al., use of -omics techniques has revealed a and capacity of microbial communi- 2015b) and Acinetobacter (Overholt et al., great deal about the diversity and phys- ties to degrade hydrocarbons. However, 2016) were suppressed by dispersants. iology of responding microorganisms, we have yet to discern how environ- Alcanivorax borkumensis, a model obli- but we do not know how effective these mental factors interact to regulate the gate hydrocarbon-degrading bacterium, microbes are in situ. For example, some final catabolic outcome of hydrocarbon was shown to be negatively impacted key microbially mediated hydrocarbon bioremediation. Thus, despite an exten- by Corexit 9500A and all anionic dis- degradation processes appeared to be sive knowledge base on hydrocarbon persants (Bookstaver et al., 2015). limited by environmental or physiolog- degradation, a quantitative understand- Another Alcanivorax strain isolated from ical factors (e.g., methane oxidation; ing is lacking, which makes it critical to Macondo oil contaminated beach sands Crespo-Medina et al., 2014). Further, incorporate microbial biodegradation demonstrated greater oil transformation rates of complex hydrocarbon oxidation pathways and regulation(s) into numer- efficiency on dispersed oil, albeit with a were not measured using sensitive iso- ical models of oil fate and transport. slight lag in growth (Overholt et al., 2016). topic tracer assays, making it impossi- Such information is necessary to accu- It seems clear that chemical disper- ble to constrain the fate of discharged oil rately construct and constrain hydro- sants result in a wide variation of bacte- during the DWH incident (Joye, 2015). carbon fate budgets. rial responses through multiple mech- Similarly, it is unclear whether chemical Chemical dispersants emulsify oil anisms, including physically changing dispersants stimulated or had no effect and break up surface slicks, generating the oil-water interface, disruption of cell on hydrocarbon degradation rates. These dispersant-​stabilized oil micro-droplets membranes causing toxicity, increasing open questions and many others must that dissolve into surface waters, effec- entrained oil concentrations, and likely be answered before the next open-ocean tively increasing the volume of water pol- changing bacterial metabolic responses oil spill occurs so that a more effective luted with discharged oil (MacDonald influencing cell growth (Kleindienst et al., response can be employed. et al., 2015). By breaking up surface slicks, 2015b). The presence of dispersants can The DWH blowout was a large- dispersant utilization can reduce the further influence the whole food web, as scale environmental perturbation that amount of thick oil stranded along shore- indicated by reduced or blocked carbon led to rapid and remarkable micro- lines and increase the oil-seawater inter- flow to higher trophic levels (Ortmann bial community shifts, raising the ques- facial area. During the DWH oil spill, et al., 2012). Assessing dispersant impacts tion as to whether, and on what time the dispersant application was unprec- across different habitats remains a crucial scale, these communities returned to edented, both because of the amount of topic for future research. the pre-discharge baseline. Available

146 Oceanography | Vol.29, No.3 evidence suggests that while the popu- imperative to determine hydrocarbon the world ocean where oil and gas explo- lation returned to baseline at the “class” degradation rates directly using isotopic ration and drilling are ongoing. We can- level (e.g., Kleindienst et al. 2015a), subtle tracers, and full documentation of system not afford to live in an “invisible present” changes in ecotype distributions per- response requires detailed spatiotempo- (Magnuson, 1990). Ecological changes sisted, meaning there could have been ral collections. Most importantly, envi- occur slowly or sporadically and are only fundamental shifts in hydrocarbon met- ronmental baselines were sorely lacking apparent and quantifiable through con- abolic dynamics in the system. The time for the Gulf of Mexico ecosystem, partic- sistent long-term observation. In what scale of full recovery to the pre-spill base- ularly in the deepwater areas, at the time is now a classic contribution, Magnuson line remains unknown. of the DWH oil well blowout (Joye, 2015). (1990) noted that the absence of long- Lessons learned from the DWH and While large amounts of data have been term monitoring data hamstrings the other oil spills have advanced hydro- collected in the wake of the Macondo ability of the scientific community to carbon microbiology and pointed to incident, background data are lacking for assess natural environmental change, data that must be collected to prop- much of the Gulf of Mexico system, par- manage the environment in a sustain- erly describe the microbial community ticularly where ultra-deepwater drilling able fashion, and document anthro- response in terms of microbial com- is now occurring. Such data are likewise pogenic perturbations. position, activity, and efficiency. It is generally unavailable for other parts of

Methylophaga

Oceaniserpentilla

JQ627834, Colwellia sp. RC25 Response to dispersant-derived PRJNA253405, Colwellia_Type02 compounds PRJNA253405, Colwellia_Type05 PRJNA253405, Colwellia_Type01 Methylococcus AF001375, Colwellia psychrerythraea PRJNA253405, Colwellia_Type10 Response to oil-derived PRJNA253405, Colwellia_Type03 compounds Cycloclasticus

Methylococcales Amphritea

Marinobacter

Alcanivorax Pseudomonas 10%

FIGURE 4. Phylogenetic tree of Colwellia species, highlighting environmental selection of physiologically distinct ecotypes. The figure shows subpopulations that respond to oil- (blue) and dispersant-derived (red) compounds in relation to gammaproteo- bacterial taxa. Responding Colwellia subpopulations were enriched in Gulf of Mexico deepwater microcosms, amended with oil-only, dispersants-only, or oil-dispersant mixtures (Kleindienst et al., 2015b). Colwellia subpopulations, representing poten- tial ecotypes, were identified from 16S rRNA gene next-generation sequencing data using oligotyping (Eren et al., 2013). Dispersant-degrading capabilities for most marine microorganisms are largely unknown, although Colwellia sp. RC25 was shown to utilize hydrocarbons and dispersants as growth substrates. Globally relevant and widely distributed aerobic hydro- carbon degraders of the Gammaproteobacteria affiliate, for instance, withAlcanivorax , Marinobacter, and Cycloclasticus. The bar represents 10% sequence divergence.

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