The Deep-Sea Microbial Community from the Amazonian Basin Associated with Oil Degradation

13 Pages·2017·2.96 MB·English

fmicb-08-01019 June9,2017 Time:15:44 #1
ORIGINALRESEARCH
published:13June2017
doi:10.3389/fmicb.2017.01019
The Deep-Sea Microbial Community
from the Amazonian Basin
Associated with Oil Degradation
MarianaE.Campeão1†,LucianaReis1†,LucianaLeomil1,LouisideOliveira1,
KokoOtsuki1,PieroGardinali2,OliverPelz3,RogerioValle4,FabianoL.Thompson1,4*and
CristianeC.Thompson1*
1InstituteofBiology,FederalUniversityofRiodeJaneiro,RiodeJaneiro,Brazil,2DepartmentofChemistry,Florida
InternationalUniversity,Miami,FL,UnitedStates,3BPExploration&ProductionInc.,Houston,TX,UnitedStates,
4SAGE/COPPE,FederalUniversityofRiodeJaneiro,RiodeJaneiro,Brazil
One consequence of oil production is the possibility of unplanned accidental oil spills;
therefore, it is important to evaluate the potential of indigenous microorganisms (both
prokaryotesandeukaryotes)fromdifferentoceanicbasinstodegradeoil.Theaimofthis
Editedby: studywastocharacterizethemicrobialresponseduringthebiodegradationprocessof
TélesphoreSime-Ngando, Brazilian crude oil, both with and without the addition of the dispersant Corexit 9500,
CentreNationaldelaRecherche
using deep-sea water samples from the Amazon equatorial margin basins, Foz do
Scientifique(CNRS),France
Amazonas and Barreirinhas, in the dark and at low temperatures (4◦C). We collected
Reviewedby:
SwingsJean, deep-sea samples in the field (about 2570 m below the sea surface), transported
GhentUniversity,Belgium the samples back to the laboratory under controlled environmental conditions (5◦C
FlorenceSchubotz,
UniversityofBremen,Germany in the dark) and subsequently performed two laboratory biodegradation experiments
*Correspondence: thatusedmetagenomicssupportedbyclassicalmicrobiologicalmethodsandchemical
FabianoL.Thompson analysistoelucidatebothtaxonomicandfunctionalmicrobialdiversity.Wealsoanalyzed
[email protected]
several physical–chemical and biological parameters related to oil biodegradation. The
CristianeC.Thompson
[email protected] concomitantdepletionofdissolvedoxygenlevels,oildropletdensitycharacteristictooil
†Theseauthorshavecontributed biodegradation, and BTEX concentration with an increase in microbial counts revealed
equallytothiswork.
that oil can be degraded by the autochthonous deep-sea microbial communities.
Specialtysection: Indigenous bacteria (e.g., Alteromonadaceae, Colwelliaceae, and Alcanivoracaceae),
Thisarticlewassubmittedto archaea(e.g.,Halobacteriaceae,Desulfurococcaceae,andMethanobacteriaceae),and
AquaticMicrobiology,
eukaryotic microbes (e.g., Microsporidia, Ascomycota, and Basidiomycota) from the
asectionofthejournal
FrontiersinMicrobiology Amazonian margin deep-sea water were involved in biodegradation of Brazilian crude
Received:23December2016 oil within less than 48-days in both treatments, with and without dispersant, possibly
Accepted:22May2017
transformingoilintomicrobialbiomassthatmayfuelthemarinefoodweb.
Published:13June2017
Citation: Keywords:hydrocarbon,biodegradation,deep-sea,Amazon,bacteria,archaea,fungi
Campeão ME,Reis L,Leomil L,
de Oliveira L,Otsuki K,Gardinali P,
Pelz O,Valle R,Thompson FLand INTRODUCTION
Thompson CC(2017)TheDeep-Sea
MicrobialCommunityfrom
Deepwater,offshoreoilproductionmayresultindirectoilexposuretothemarineenvironment
theAmazonianBasinAssociatedwith
by unplanned (oil spills) and planned release (as dispersed oil with operational discharge of
OilDegradation.
produced water). Thus, it is important to evaluate the potential of indigenous microorganisms
Front.Microbiol.8:1019.
doi:10.3389/fmicb.2017.01019 (bothprokaryotesandeukaryotes)fromdifferentoceanicbasinstodegradeoil(Hazenetal.,2016).
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Campeãoetal. AmazonianDeep-SeaOilDegraders
Microbes from all three domains of life can exploit certain (Leahy and Colwell, 1990; Harms et al., 2011; Passarini et al.,
compounds from the ∼600 1000 tons of oil that are annually 2011).
released globally from (a) natural seeps on the seafloor, (b) Environmental approaches designed to accelerate oil
delocalized runoff from human activities, and (c) oil spills biodegradation are required to mitigate damage caused by oil
(National Research Council (US) Committee on Oil in the Sea: spills. Dispersants are important oil spill-response tools for
Inputs, Fates, and Effects, 2003). In 2011, an oil well leak in enhancing the biodegradation that are required to mitigate
the Campos Basin, 120-km off the coast of Rio de Janeiro, ecosystemimpactcausedbyoilspills.Theyspeedupthenatural
caused 3,700 barrels of oil to be released into the ocean (ANP, process of microbial oil degradation by dispersing oil into
2012).Recentstudieshavedemonstratedthecommonpresence micron-sized droplets that become buoyant and dilute enough
of oil-degrading bacteria in several different deep-sea oil basins in the water column that the natural levels of biologically
(Prince, 2005; Jurelevicius et al., 2013; Hazen et al., 2016). available nitrogen, phosphorus and oxygen are sufficient to
However, most studies have focused on assessing the bacterial start and sustain microbial growth. Dispersant application
community structure in the North Atlantic, Gulf of Mexico, reduces the surface tension of the oil and creates droplets
andMediterraneanSea.Becauseeachoceanicbasinhasitsown smaller than those formed by natural dispersions or releases
physicochemicalandbiologicalfeatures,thegeographiclocation from natural seeps, allowing for quicker biodegradation. The
maythusinterfereindifferentwayswithoilbiodegradationrates. small size of the oil droplets formed also increases the surface
Inaddition,onlyafewstudieshaveaddressedtheroleofdeep-sea area available for microbial colonization and significantly
eukaryotes(e.g.,fungi)andarchaeainbiodegradation,asbacteria enhances biodegradation; this was one of the most important
areconsideredthemainplayerinoilbiodegradationintheocean processes for the removal of the oil released from the DWH
(Hazenetal.,2016).Themajorityofstudiesaddressingtheroleof spill (Atlas and Hazen, 2011; Vilcáez et al., 2013; Meckenstock
fungi,protozoa,andarchaeahavefocusedontheterrestrialrealm et al., 2014). The United State District Court for the Eastern
(McGenityetal.,2012). District of Louisiana (2010) found that 3.19 million barrels
Despite the ability of Halobacteriaceae to degrade oil of oil were released from the Macondo well in the Gulf of
compounds, including alkanes and polycyclic aromatic Mexico in 2010 (The United States District Court for the
hydrocarbon (PAH), some archaeal groups appear to have Eastern District of Louisiana, 2015). Approximately 1.8 million
reducedgrowthanddiversityinthepresenceofoil(Rölingetal., gallons of chemical dispersants, primarily Corexit 9500, were
2004). For example, Nitrosopumilus maritimus was sensitive also injected at wellhead or applied to water surface. After a
to oil in experiments performed with seawater affected by the test period, dispersant was injected at the wellhead between
Deep Water Horizon (DWH) oil spill in the Gulf of Mexico in May 15 and July 15 in an effort to prevent the formation of
2010 (Urakawa et al., 2012). However, Redmond and Valentine large surface slicks until the well could be capped (Operational
(2012) did not find a clear effect of oil on archaea growth and Science Advisory Team, 2010). Shortly after two test periods,
diversity(RedmondandValentine,2012),suggestingthatfurther dispersant was injected continuously at the MC252 wellhead
studiesareneededtobetterunderstandtheroleofarchaeainoil after May 15th, until the well was capped 61 days later on
degradation(Al-Mailemetal.,2010;Tapilatuetal.,2010;Bonfá July 15th to retain the oil in the water column and prevent
etal.,2011;Wangetal.,2011;Erdogˇmu¸setal.,2013;Jurelevicius the formation of large surface slicks (Kujawinski et al., 2011).
etal.,2014). Recentlaboratorystudieshavedemonstratedthatoveraperiod
Fungi are known for their capacity to metabolize oil of ∼60 days, the indigenous microorganisms in the Gulf of
compounds, such as alkanes (Van Hamme et al., 2003), BTEX Mexico were able to completely degrade oil in presence
(Yadav and Reddy, 1993), and PAH (Cerniglia and Sutherland, of Corexit 9500 under controlled laboratory conditions
2001, 2006, 2010). The major phyla known to metabolize oil (Wang et al., 2016). Corexit 9500 seems to accelerate the
compoundsareMicrosporidia,Ascomycota,andBasidiomycota biodegradation process, increase the number of oil-degrading
(Harms et al., 2011). Filamentous fungi make bacterial oil microorganisms, such as Oceanospirillales and Cycloclasticus,
utilization by bacteria more efficient through the production leading to the rapid degradation of hydrocarbons (Brakstad
of hyphae that act as continuous pathways for motile bacteria, et al., 2015b). However, other recent studies indicated that
linking oil aggregates (Kohlmeier et al., 2005; Wick et al., dispersant may negatively affect oil-degrading bacteria at
2007; Banitz et al., 2011). Lignolytic and non-lignolytic fungi concentration of ∼19 µg/L of Corexit 9500 (Kleindienst
could metabolize PAH, but a few are able to grow with these et al., 2015). Thus, from previous studies it is not clear if
compoundsasthesolecarbonandenergysource(Cernigliaand Corexit9500 would and interfere in microbial community
Sutherland,2010).Theassemblageoffungiandbacteriaappearto diversity and speed up oil biodegradation in the Equatorial
facilitatePAHbiodegradation(Boonchanetal.,2000;Wangetal., margin.
2012). However, a majority of studies on fungi that metabolize The potential for biodegradation of oil by indigenous
oil compounds were conducted in soils and sediments. The microorganisms (both prokaryotes and eukaryotes) has been
major phylum known to be a metabolizer of oil compounds, evaluated in different oceanic basins but not in the Equatorial
Basidiomycota, inhabits terrestrial environments and is rare in AtlanticOcean(Hazenetal.,2016).MostoilproductioninBrazil
marine systems (Shearer et al., 2007). Little is known about (>2Millionbarrelsperday)occursindeep-wateroilfields,such
fungithatcanbiodegradeoilindeep-seawaterandthepossible as the Campus Basin (ANP, 2012). Oil companies are seeking
ecologicalrelationshipsbetweenfungiandbacterialcommunities new frontiers for oil exploration and production in Brazilian
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waters. Among these regions, the Brazilian equatorial margin, the experimental unities. Despite the numerous literature on
which spans from Rio Grande do Norte to the Amapa States, anaerobic degradation of oil (e.g., Zengler et al., 1999, pp
is currently being investigated through large environmental 266–269; Kniemeyer et al., 2007, pp 898–901; Musat et al.,
surveysthatsupportthenewoilexplorationactivities(Lourenço 2009, pp 209–219), we sought to reproduce similar oxygen
et al., 2016). Oil exploration in the Amazonian continental concentration normally observed in the ocean. The 10-L WAF
shelf began in the early 1960s, but oil production has been preparations were placed in a refrigerated chamber kept at
focused on the continent. An example is the Urucu Basin, 4◦C in the dark and gently mixed (no-vortex) during 96 h.
which is known to produce high quality oil, mainly for the After settling with no mixing for 4 h, the WAF samples were
production of diesel and naphtha in Brazil. Gas is also a taken without disturbing the oil slick on top and transferred
major product from the Amazon region (ANP, 2009). In the to 500 mL capped flasks. The CEWAF treatment was prepared
last few years, major exploration operations were initiated on in a similar fashion but the Itaipu oil was premixed with the
the Amazonian continental shelf and basins by oil companies. Corexit9500 dispersant before it was added to the saltwater
In support of environmental risk assessments (fate of the container (1 mL dispersant to 25 mL oil; DOR 1:25). The
oil) it is important to evaluate the potential of these deep- CEWAF treatment was also kept in the refrigerated chamber
sea waters to biodegrade oil. The aim of this study was at 4◦C in the dark and mixed with a magnetic stirring rod
to characterize the microbial response (growth, composition, for 18 h, followed by 4 h settling (no stirring) before samples
diversity) related to the process of oil degradation (with and weretaken.Controlsconsistedofseawaterwithouttheaddition
withoutthedispersantCorexit9500),usingdeep-seawaterfrom of (dispersed) oil. After settling, all samples (WAF, CEWAF,
the Amazon equatorial margin. We performed two 48-day and Controls) were distributed in 500 mL capped flasks and
experiments and used metagenomics to uncover the microbial incubated in the dark at 4–5◦C for a period of up to 48 days.
(taxonomic and functional) diversity. We also linked these Inaddition,1-Lsampleswerecollectedformicrobialcommunity
molecular microbiology data and information to conventional assessment.Noadditionalnutrientswereaddedtotheseawater
analysesofvariousphysical–chemicalandbiologicalparameters samples.AliquotsofWAF,CEWAF,andControlwereplatedon
relatedtooilbiodegradation. the BD DifcoTM Bushnell-Haas mineral medium supplemented
with 1% Itaipu oil. Inoculated plates were incubated at 4–5◦C
for 7 days (Supplementary Figure 5). Physical–chemical and
MATERIALS AND METHODS
biologicalanalyseswereperformedatfourtimepointsduringthe
experiment,usingsacrificialbottlesforeachanalysis.
Sampling Location
For the biodegradation experiments, seawater samples Microbial and Oil Droplets Counts
containingindigenousBrazilianoff-shoremicrobialassemblages Prokaryotic cytometry counts were performed in triplicate as
were collected under semi-sterile conditions from two described previously using the fluorescent nucleic acid stain
geographical locations (Foz do Amazonas: E 587.672 and N 4(cid:48),6-diamidino-2-phenylindole(DAPI)(Gregoraccietal.,2012).
600.161; Barreirinhas: E 216.294 and N 9.800.847) according to EukaryoticcellsandoildropletswerecountedusingaFlowcam.
methodologies described previously (Wang et al., 2016). From Samples (18 mL) were fixed with 2 mL of fixer (20% buffered
each location, two water samples were collected using a rosette formaldehyde).
as follows: (i) a deep-water sample below the chemocline, close
to the seabed, at about 2570 m below the surface and (ii) a Inorganic Nutrients, Dissolved Oxygen,
samplecollectedatabout10mbelowthesurface.Waterfromat
and Hydrocarbon Concentration (BTEX,
about10mbelowthesurfaceandadeepseawereimmediately
Alkanes, and PAH)
transferredtosterilecontainersandtransportedtothelaboratory
inthedarkat4◦C. Nitrate and phosphate were measured as described previously
(Gregoraccietal.,2012).Dissolvedoxygenwasmeasuredusing
Experimental Design an electrochemical electrode. The analysis of volatile aromatic
Two identical experiments were performed using the deep-sea hydrocarbons [Benzene, Toluene, Ethylbenzenes, and Xylene
waterfromFozdoAmazonasandBarreirinhas.Theexperiments (BTEX)], alkanes, and PAHs was conducted by liquid-liquid
wereperformedat4◦Cinthedark,lastedfor48daysandwere extraction of the water samples using methylene chloride
monitored periodically at four time points (T0 = arrival in followingbyquantitativedeterminationbygaschromatography–
the laboratory; T1 = Day 0, the beginning of the experiment; mass spectrometry (GC–MS) as described previously (Wang
T2=Day8;T3=Day24,andT4=Day48).Eachexperiment etal.,2016).
consistedofthreetreatments[(I)water-accommodatedfraction
Metagenomic Analysis
(WAF),(II)chemicallyenhancedwater-accommodatedfraction
(CEWAF), and (III) Control] prepared as described previously Chemically enhanced water-accommodated fraction, WAF, and
(Brakstad and Faksness, 2000). Briefly, the WAF treatment control samples (1-L) were filtered through a 0.2 µm Sterivex
consisted of deep-sea water supplemented with Itaipu oil (at andextractedusingtheNucleospinkitprotocol.Illuminapaired
a loading rate of 0.1 mL/L). The relatively low oil rate was end sequencing was performed for all samples. The generated
chosen to avoid total depletion of oxygen concentration in data were pre-processed with fastq-mcf and paired-ends were
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FIGURE1|Featuresofthewatermasses.CTDmeasurementsoftemperature,salinity,anddissolvedoxygenalongdepth.WatercolumnprofilesatFozdo
Amazonas(E587.672andN600.161)duringtheApril,2015collection(A)andatBarreirinhas(E216.294andN9.800.847)duringtheMay,2015collection(B).
merged with PEAR (Zhang et al., 2014). Taxonomic analysis 15.3µMinthebeginningoftheexperimentto7.9µMintheend.
was carried out using Focus (Silva et al., 2014). The data were Orthophosphatevariedfrom∼1µM(Day0)to∼0.6µM(Day
submitted to Mgrast (Meyeret al., 2008) to annotate sequences 48)andfrom3.1µM(Day8)to5µM(Day48)intheWAFand
andafunctionalanalysis(SupplementaryTables1,2). CEWAFtreatments,respectively.IntheBarreirinhasexperiment,
nitratedecreasedfrom16.7to4.2µMandfrom24.5to3.6µM
in the WAF and CEWAF treatments, respectively, toward the
RESULTS
endoftheexperiment(Figure2B).Orthophosphatehadasimilar
trend,decreasingfrom0.8to∼0.1µMandfrom1.1to0.2µMin
Environmental Physical–Chemical and
theendoftheexperiment,intheWAFandCEWAFtreatments,
Biological Analyses respectively. The hydrocarbon concentration (BTEX, alkanes,
Temperature, salinity, and oxygen profiles of both locations andPAH)droppedsignificantlyinbothtreatments,whereasthe
(Foz do Amazonas and Barreirinhas) had typical patterns with half-lifeofBTEXwas9daysforCEWAFtreatmentand18days
decreasing temperature and increasing oxygen concentration forWAFtreatment(SupplementaryTable3).
as the depth increased (Figure 1). The water samples used in
thisstudycorrespondtothewatermassnamedDeepAntarctic Prokaryotic Counts
Waters(Wust,1933)andhadrelativehomogeneitywithrespect
The abundance of prokaryotic cells in the Foz do Amazonas
to temperature, salinity, and dissolved oxygen. The microbial
experiment varied from 3.85 × 105 to 3.41 × 106 cell/mL and
community in the sea surface sample and deep-water sample
from 9.72 × 105 to 5.01 × 106 cell/mL in WAF and CEWAF,
at both locations was composed of different taxa related to oil
respectively, with higher values occurring toward the end of
degradation,includingAlteromonadaceae,Alcanivoracaceae,and
the experiment (Figure 2A). In the Barreirinhas experiment,
Colwelliaceae(SupplementaryFigure1).
prokaryoticcountsvariedfrom5.31×105to2.28×106cell/mL
Biodegradation Experiments and from 1.67 × 105 to 3.36 × 106 cell/mL in the WAF and
CEWAF treatments, respectively, with the maximum count on
Two experiments (Foz do Amazonas and Barreirinhas) were
day24.MicrobialcountswerehigherintheCEWAFthaninthe
performed to evaluate the potential of deep-sea microbes to
WAFtreatmentinbothexperiments.
degradecrudeoilduringa48-dayincubation.Thedynamicsof
physico-chemical and biological parameters were monitored by
analyzingfourtimepoints(T0=arrival;T1=Day0;T2=Day Eukaryotic Counts
8;T3=Day24,andT4=Day48). In the Foz do Amazonas experiment, the total number of
eukaryotesincreasedfrom72cell/mLand17cell/mL(Day1)to
Physico-Chemical Analysis
324cell/mL(Day48)and642cell/mL(Day24)intheWAFand
Oxygen concentration varied from ∼8 mg/l at T0 of the CEWAFtreatments,respectively(Figure2A).IntheBarreirinhas
experiment to ∼6.2 and ∼6.6 mg/L at the end of the Foz and experiments,thetotalnumberofeukaryotesdroppedfrom1754
Barreirinhasexperiments,respectively.IntheFozdoAmazonas cell/mLand310cell/mL(Day1)to369cell/mLand80cell/mL
experiment, nitrate increased until day 8 (12.5 to 22.7 µM) (Day 24), in the WAF and CEWAF, respectively. An increase
and decreased to 12.9 µM after 48 days of the WAF treatment ineukaryotesnumberwasobservedinCEWAFtreatment(1466
(Figure 2A). In the CEWAF treatment, nitrate dropped from cell/mL)inDay48.
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Campeãoetal. AmazonianDeep-SeaOilDegraders
to 0% (T4) in the WAF treatment of Foz do Amazonas
experiment. On the other hand, Pseudomonadaceae increased
from 5.03% (T1) to 20.12% (T4). Alteromonadaceae peaked at
T1(35.50%)andT3(39.08%).Otherlessabundanttaxa,suchas
Piscirickettsiaceae (2.92 to 0%), Shewanellaceae (2.12 to 0.50%),
and Joneasiaceae (1.86 to 0%) reached the lowest values at the
end of the experiment, whereas Ectothiorhodospiraceae (1.13 to
5.17%) and Vibrionaceae (2.48 to 3.72%) tended to increase.
Alteromonadaceae decreases from 80.98 to 39.3% in WAF
treatment of Barreirinhas, whereas Hyphomonadaceae (0.64 to
4.84%),Oceanospirillaceae(0.68to2.88%),andPiscirickettsiaceae
(14.23 to 22.74%) increased along time. Alcanivoracaceae and
Acetobacteraceae peaked in T3 (5.41 and 2.22%, respectively);
PseudomonadaceaepeakedatT2(5.57%)andinT4(4.87%).The
controltreatment(T0andT4)inbothexperimentshadalower
proportion of Alteromonadaceae and Alcanivoracaceae than in
WAF/CEWAFtreatments.
IntheFozdoAmazonasexperiment,CEWAFtreatmenthad
a greater proportion of Marinobacter than the WAF treatment
(p-value < 0.01). Marinobacter increased from 38.89% (T1) to
47.17%(T4)intheCEWAFtreatment;itpeakedatT3(39.08%)
intheWAFtreatment.Colwelliarangedfrom10.32to1.42%in
theCEWAFtreatmentandfrom20.40to0%atT4intheWAF
treatment. Pseudomonas increased from 4.65% (T1) to 18.24%
(T4) in WAF and from 4.26% (T1) to 7.83% (T4) in CEWAF.
AlcanivoraxwasmoreabundantintheWAFtreatmentoftheFoz
doAmazonasexperiment(p-value<0.01).
The CEWAF treatment in the Barreirinhas experiment
showed that Alcanivoracaceae (11.69 to 3.79%) and
Pseudomonadaceae (8.47 to 0.68%) decreased over time
whereasPiscirickettsiaceaeincreasedfrom0.007%(T1)to11.07%
(T4). Colwelliaceae increased at T2 (46.20%) and T4 (26.92%),
FIGURE2|Microbialgrowth,oildroplets,nutrientsandgrowthofmajor
generaatFozdoAmazonas(A)andatBarreirinhas(B).Prokaryoticcell Alteromonadaceae increased at T1 (40.96%) and T3 (27.46%),
counts(I),eukaryoticcellcounts(II),oildroppletscounts(III),nitrate RhodobacteraceaeandShewanellaceaeincreasedatT2(5.97and
concentrations(IV),orthophosphateconcentrations(V),metagenomicprofiles 2.46%, respectively), and Flavobacteriaceae, Hyphomonadaceae,
ofmajorgenerainWAFtreatment(VI),metagenomicprofilesofmajorgenera
Joneasiaceae,andOceanospirillaceaeincreasedatT3(9.93,6.85,
inCEWAFtreatment(VII).
1.42, and 8.80%, respectively). In the WAF, the most abundant
groupwasAlteromonadaceae(∼80%atT1and∼45%atT4)and
Piscirickettsiaceae(∼18%atT3and∼25%atT4).
Oil Droplets
Considering genera, in the CEWAF of Barreirinhas
The number of oil droplets dropped throughout both
experiment, Colwellia ranged from 6.52% (T1) to 46.20%
experiments, reaching lower values in the CEWAF treatments
(T2).ThalassolituuspeakedatT4(7.02%).Cycloclasticuspeaked
(Figures 2A,B). In the Barreirinhas experiment, oil droplets
at T4 (10.88%) in the CEWAF and at T3 (21.17%) in the
dropped from 91 to 28 and from 39 to 0 droplets in WAF and
WAF treatment. Alcanivorax decreased from 11.61% (T1)
CEWAFtreatments,respectively.
to 3.79% (T4) in CEWAF treatment whereas in the WAF it
peakedat day24(5.41%)and droppedto4.33%at day48. The
Taxonomic Assignment of Metagenomic
control treatment (T4) had a different microbial community
Sequences
profile in both experiments, with lower Alteromonadaceae and
Overall,metagenomicsequencesrelatedtooildegradingbacteria, Alcanivoracaceaesequenceabundance.
those are previous reported in the literature, corresponded
Archaeal Groups
to more than 60% of the total in WAF/CEWAF treatments
of both experiments (Figures 3A,B). The major groups were In the Foz do Amazonas experiment, the major groups
Alteromonadaceae (Alteromonas e Marinobacter), Colwelliaceae were Thermoproteaceae, Desulfococcus, Halobacteriaceae,
(Colwellia), Alcanivoracaceae (Alcanivorax), Piscirickettsiaceae and Methanobacteriaceae in both WAF and CEWAF
(Cycloclasticus), and Oceanospirillaceae (Thalassolituus) in both treatments (Figure 4A). The proportion of Desulfococcus
WAF and CEWAF. Alcanivoracaceae decreased from 14.51% decreased from 47.66% (T1) to 19.69% (T4) and proportion
(T1) to 8.93% (T4) and Colwelliaceae from 20.40% (T1) of Halobacteriaceae increased from 35.04% (T1) to 52.0%
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FIGURE3|Bacterialcommunitycompositionatfamilylevelforwatertreatedwithoil(WAF)andwithdispersedoil(CEWAF)atdays8,12,24,and48determinedby
metagenomicanalyses.Bacterialcommunityfordeepwatercontrolsamples(nooil)areshownatdays0and48.BacterialprofilesatFozdoAmazonas(A)andat
Barreirinhas(B).
(T4) along time in the WAF treatment of the Foz experiment. Nitrosopumilaceae. Desulforucoccaceae increased in both WAF,
Thermoproteaceae (17.29%), Nitrosopumilaceae (21.35%), from16.94%(T1)to45.28%(T4)andinCEWAF,from23.30%
Methanobacteriaceae (10.09%), and Methanosarcinaceae (T1) to 64.25% (T2) (Figure 4B). Sulfolobaceae (4.51%) and
(27.40%) peaked in T1, T2, T3, and T4, respectively, in the Nitrosopumilaceae (13.72%) appeared only at T1 in WAF.
WAF treatment. In CEWAF treatment, Thermoproteaceae Thermoproteaceae increased from 5.95% (T1) to 25.30%
(from 30.34 to 21.79%), Acidilobaceae (from 12.49 to 0%) (T3) whereas Halobacteriaceae and Methanobacteriaceae
and Methanobacteriaceae (from 8.62 to 0%) decreased, decreased from 28.91% (T1) to 0% (T4) and from 20.21%
reaching the lowest values at T4 whereas Halobacteriaceae (T1) to 0% (T3), respectively. In the CEWAF treatment,
increased from 26.76% (T1) to 60.50% (T4) over time. Nitrosopumilaceae peaked at T1 (13.72%) and T2 (18.15%),
In the control treatment, the major groups at T0 were whereasMethanobacteriaceaedecreasedalongtimefrom26.72%
Nitrosopumilaceae (35.21%), Halobacteriaceae (16.27%), and (T1) to 0% (T4). Halobacteriaceae (19.36%), Thermoproteaceae
Methanobacteriaceae (16.75%), corresponding to ∼68% of total (11.98%) and Methanocellaceae (14.55%) peaked at T3. In the
archaeal sequences, whereas the control treatment at T4 was controltreatment,Nitrosopumilaceaedroppedfrom27.93%(T0)
dominantlyMethanoregulaceae. to 4.17% (T4), Halobacteriaceae from 20.23% (T0) to 15.44%
In the Barreirinhas experiment, the major groups were (T4) whereas Methanosarcinaceae (58.22%), Desulfococcus
Desulfococcus, Thermoproteaceae, Halobacteriaceae, and (8.78%),Thermoproteaceae(12.31%)andpeakedatT4.
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FIGURE4|Archaealcommunitycompositionatfamilylevelforwatertreatedwithoil(WAF)andwithdispersedoil(CEWAF)atdays8,12,24,and48determinedby
metagenomicanalyses.Archaealcommunityfordeepwatercontrolsamples(nooil)areshownatdays0and48.ArchaealprofilesatFozdoAmazonas(A)andat
Barreirinhas(B).
Eukaryotes to9%andfrom3to16%inT4intheWAFFozandBarreirinhas
Apicomplexa, Ascomycota, Bacillariophyta, Basidiomycota, experiments,respectively).
Chlorophyta, and Microsporidia were the major groups found
(Figures5A,B).TherewasaclearincreaseinAscomycota(from Functional Identification of
22to31%intheWAFtreatmentand22to25%intheCEWAF Metagenomics Sequences
treatmentinseawaterfromFozandfrom7to34%intheWAF Thefunctionalprofileofbothtreatments,WAFandCEWAF,was
treatment and 7 to 17% in the CEWAF treatment in seawater verysimilar(SupplementaryFigures2A,B).Themajorfunctional
from Barreirinhas) and Microsporidia (from 1 to 10% in the groupswerecarbohydrates,aminoacids,proteins,cofactorsand
WAF treatment and 1 to 15% in the CEWAF treatment in enzymes, representing over 60% of the metagenomic sequences
seawater from Foz and from 0.1 to 12% in the WAF treatment with known function in both experiments. The number of
and 0.1 to 1% in the CEWAF treatment in seawater from sequences associated with membrane transport, metabolism
Barreirinhas)atT1ofboththeWAFtreatmentandtheCEWAF of aromatics, motility and chemotaxis, virulence and disease
treatmentinthetwoexperimentscomparedtotheT0.Thesame increased over the time in both experiments while the number
wasobservedforBasidiomycota(from4to13%atT4ofCEWAF of sequences associated with carbohydrates and photosynthesis
intheFozdoAmazonasexperiment)andApicomplexa(from3 metabolismdecreased(Figure6).
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Campeãoetal. AmazonianDeep-SeaOilDegraders
FIGURE5|Eukaryoticcommunitycompositionatfamilylevelforwatertreatedwithoil(WAF)andwithdispersedoil(CEWAF)atdays8,12,24,and48determined
bymetagenomicanalyses.Eukaryoticcommunityfordeepwatercontrolsamples(nooil)areshownatdays0and48.EukaryoticprofilesatFozdoAmazonas(A)
andatBarreirinhas(B).
The analysis related to genes for oil biodegradation showed water masses (Silva et al., 2005). Oil biodegradation has been
that the abundance of sequences related to BTEX degradation reported in different oceanic basins (e.g., Mediterranean Sea,
increases along time in both experiments for both treatments, Gulf of Mexico), under high oxygen concentrations and low
with more percentage of sequences in WAF treatment than in temperatures (Hazen et al., 2010; Bargiela et al., 2015; Brakstad
CEWAFandwithagreaterdifferencebetweenWAFandCEWAF et al., 2015a,b), as observed in this study, indicating that
at Barreirinhas experiment. Alkanes proportion are constant oceanic conditions of these Brazilian deep-sea waters allow
along time in both treatments at Foz and have a peak at T2 for oil degradation. The microbial communities disclosed
and at T3, respectively, in WAF and CEWAF for Barreirinhas. in the sub-surface and deep-sea water of both locations
PAHpercentageofsequencesinturnisgreaterforBarreirinhas, analyzed here included several oil-degrading taxa (Yakimov
especiallyforWAF(SupplementaryFigures3A,B,4A,B). et al., 2007), suggesting that indigenous microorganisms
have the potential for biodegradation of oil. Despite slight
differences in the taxonomic patterns between the Foz and
DISCUSSION Barreirinhas locations, major oil-biodegrading bacteria were
found in all samples (e.g., Alcanivoracaceae, Alteromonadaceae,
The physico-chemical parameters of deep-sea water from both Colwelliaceae, Pseudomonadaceae, Oceanospirillaceae, and
locations examined were similar to the Antarctic deep-sea Piscirickettisiaceae).
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studiesmayberelatedtotheindigenousmicrobialcommunities
of each location, differences in experimental set up, and the
nature and composition of the oil used. Nevertheless, a clear
positiveresponseofoilonmicrobialbiodegraderswasobserved
inallstudies.
In this study, the significant drop in oxygen concentration
(from ∼8 mg/L at T0 to ∼6.2–6.6 mg/L at T4), possibly due
to microbial respiration and degradation, with a concomitant
increase in prokaryotic and eukaryotic counts, indicate the
consumption of dispersed oil. Colwellia strains are known to
degrade BTEX and alkane compounds (Methe et al., 2005)
while Alcanivorax and Marinobacter are well-known alkane-
degraders (Gauthier et al., 1992; Yakimov et al., 1998). During
our experiments, Marinobacter abundance seemed to increase
whenColwelliadroppedandviceversa;bothgeneraareableto
degrade aliphatic hydrocarbons and could compete for similar
resources.Alkanebiodegradationrelatedsequencesproportions
along time seems to show a trend similar to Marinobacter
growth. Alkanes and BTEX are among the first oil compounds
to be degraded (Hazen et al., 2010; Brakstad et al., 2015a;
Wang et al., 2016); whereas PAH are the least easily degraded
oil compound compared to alkanes and BTEX (Mason et al.,
2014b). Cycloclasticus (Piscirickettsiaceae), an important PAH-
degrading bacteria (Dyksterhouse et al., 1995), was mainly
observed toward the end of our experiments. In turn, PAH
biodegradation related sequences proportions along time is
similar to growth pattern of Cycloclasticus. The capacity of
FIGURE6|Majordifferencesbetweensubsystemsprofilesdeterminedby microbialcommunitiestometabolizehydrocarbonsisalsolikely
metagenomicanalysesatFozdoAmazonas(A)andatBarreirinhas(B).T0 to be coupled with nitrogen availability (Mason et al., 2014b).
correspondstocontroldeepwatersamples(nooil)andT1,T2,T3,andT4
AdropinnitrateconcentrationinboththeFozandBarreirinhas
correspondstodays8,12,24,and48,respectively.
experiments suggest an intense denitrification process, possibly
mediated by Colwellia (Methe et al., 2005; Mason et al.,
2014a) and Marinobacter (Gauthier et al., 1992). In addition, a
WAF and CEWAF Treatments Induce the
major component of the dispersant Corexit is dioctyl sodium
Growth of Oil Degrading Microbes
sulfosuccinate(DOSS)(Placeetal.,2010,2014;Kujawinskietal.,
The comparison of control with WAF/CEWAF treatments 2011).DOSSisasubstratethatcouldbemetabolizedbyColwellia
demonstrated the increase of microbes implicated with oil (Kleindienstetal.,2015).
degradation throughout the 48-day experiments. For instance,
Oil-Degrading Archaea
during CEWAF treatment of Barreirinhas seawater, Colwellia
boomed at day 8 (T2) and Flavobacteriaceae at day 24 (T3), Halobacteriaceae was one of most important groups in our
whereas Alcanivoracaceae and Alteromonadaceae boomed experiment in line with previous studies (Tapilatu et al., 2010;
at T1. Some groups peaked only after 24 days, for example, Bonfá et al., 2011; Wang et al., 2011; Al-Mailem et al., 2012;
Piscirickettsiaceae/Cycloclasticus, and Oceanospirillaceae/ Erdogˇmu¸s et al., 2013; Jurelevicius et al., 2014). However, this
Thalassolituus.Duringa64-daybiodegradationexperimentwith family was not the most abundant among the most important
deep-sea water collected from the vicinity of the MC252 well groups in both experiments. Some chemolithotrophic archaea
in the Gulf of Mexico that was then amended with 10 µm oil that are able to oxidize organic sulfur compounds, such as
droplets (2 ppm) created with Macondo Oil in the presence of DesulfurococcaceaeandThermoproteaceae(Burggrafetal.,1997;
Corexit9500dispersant,Wangetal.(2016)reportedanincrease Zillig et al., 1982), were abundant, as well as methanogenic
in Alteromonadaceae (including Marinobacter species) and archaea, including Methanobacteriaceae, Methanocellaceae,
Alcanivoracaceaeduringthefirst9days.Furthermore,Colwellia, Methanoregulaceae, Methanosaetaceae, Methanosarcinaceae,
Cycloclasticus, and other Gammaproteobacteria increased and Methanotermaceae (Garcia et al., 2000; Sakai et al., 2008,
between days 27 and 36, and Flavobacteria at day 64 (Wang 2012). Methanogenic archaea were observed to co-occur with
et al., 2016). Hazen et al. (2010) found that Oceanospirillales Halobacteriaceae, known as an oil degrading archaea, which
dominated the oil plume samples from the DWH oil spill in is in accord with previous reports that methanogenesis is
GMandrepresentedonlyasmallfractioninthecontrolsample coupled to oil biodegradation in syntrophic communities
out of the oil plume (Hazen et al., 2010). In our experiments, (Zengler et al., 1999; Chang et al., 2006; Mayumi et al.,
Oceanospirillaleswasnotdominant.Thedifferencesbetweenthe 2016). Nevertheless, Halobacteriaceae, without methanogenic
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archaea only occurs with sulfur chemolithotrophic archaea oil droplets during the experiments resulted in changes in
in the CEWAF treatment (with dispersant). The presence of the functional profiles of both WAF/CEWAF treatments.
methanogenic archaea could imply that experiments reached The observed reduction in the carbohydrate metabolism and
anaerobic conditions. However, several methanogenic archaeal concomitant increase in metabolism of aromatic compounds,
species (e.g., Halobacteriaceae) are aerobic and facultative membranetransport,fattyacids,lipidsandisoprenoids,motility
anaerobic. The oxygen levels dropped to nearly 6 ml/L along and chemotaxis, nitrogen metabolism in WAF/CEWAF hints
both experiments (Supplementary Table 3). Although the at important changes in the microbial communities that were
oxygen levels were relatively high, it is possible that a suboxic driven by the presence of (dispersed) oil and dissolved oil
microenvironmentcouldbegeneratedaroundtheparticleswhere constituents in our incubation experiments. One of the first
thesearchaealgroupsgrows.Thisanaerobicmicroenvironment steps in oil degradation is particle colonization and protein
would be a result of intense respiration of the co-occurring secretion on the particle surface mediated by trans-membrane
groups observed with these archaea (oil degrading archaea or proteins;furthermore,motilityandchemotaxisallowprokaryotes
sulfur chemolithotrophic archaea). A remarkable difference torecognizeandcolonizeoildroplets.Weobservedadrasticdrop
between CEWAF treatments in the Foz and Barreirinhas inoildropletsthroughouttheexperiments.Thisdecreaseislikely
experiments is the presence of Nitrososphaera, an ammonia mediated by colonization (Wang et al., 2016). The increase in
oxidizingchemolithotrophicarchaea(Könnekeetal.,2005).The aromatic compound metabolism highlights the biodegradation
relationship between chemolithotrophy and oil degradation is of different types of hydrocarbon molecules, including the oil
unclear. components BTEX and PAH, whereas the increase in lipid
and isoprenoid metabolism indicates an increased potential for
Oil-Degrading Eukaryotes alkane biodegradation. Hydrocarbonoclastic bacteria synthesize
fatty acids, lipids and isoprenoids in the presence of n-alkanes
Our experiments demonstrated that in both treatments,
(Kalscheueretal.,2007).Theselipidsaccumulateontheinsideof
WAF and CEWAF, the growth of fungi (Microsporidia,
thecellsandareutilizedascarbonsourceforgrowth,particularly
Ascomycota, and Basidiomycota) was induced. Microsporidia,
in presence of a nitrogen source (Manilla-Pérez et al., 2010,
a PAH metabolizer, increased at T3, except for in the WAF
2011). However, we have no prove yet that the breakdown
treatment for Barreirinhas, where the PAH degrading bacteria,
of alkanes is linked to an increase in lipid and isoprenoid
Cycloclasticus, peaked. Ascomycota is extremely versatile
biosynthesis.
and metabolizes alkanes, BTEX, and PAHs (Harms et al.,
The use of dispersants in oil spill accidents has been
2011). The abundance of this group was high and stable
highly controversial (Kleindienst et al., 2015). In the present
throughout the experiment, suggesting that several different
study, the GC–MS- results showed that the half-life of
oil compounds were used by Ascomycota. Basidiomycota
BTEX dropped from 18.5 to 9.4 days in the dispersant-oil
abundance was lower than Microsporidia and Ascomycota
treatment (CEWAF) at Barreirinhas experiment, suggesting
abundance most of the time in both experiments. However,
a significant increase in the degradation of these aromatic
Basidiomycota abundance tended to increase toward the end
compounds in the presence of Corexit9500. However, the
of the experiment, having greater proportion than the Control.
bacterial community diversity results are not conclusive; while
Although Basidiomycota can metabolize PAHs (Cerniglia and
WAFT1,CEWAFT1andControlT0haveverysimilarprofiles
Sutherland, 2001, 2006, 2010; Sutherland, 2004; Verdin et al.,
in the Foz do Amazonas when compared to the Barreirinhas
2004), this fungi generally cannot use PAHs as the sole carbon
(Figures 3A,B). CEWAF had lower diversity compared to
andenergysource(CernigliaandSutherland,2001).Theabsence
WAF in both experiments, suggesting that the dispersant
of an additional nutrient source in our experiments may have
may reduce microbial community diversity, and thus have a
influenced the utilization of PAH by Basidiomycota. Fungi
negative effect in the water quality. The dispersant appears
produce multifunctional enzymes involved in the degradation
to induce the growth of certain types of microbes (e.g.,
of a large variety of organic molecules, including aromatic
Colwelliaceae, Flavobacteriaceae, and Oceanospirillaceae), that
hydrocarbons, chlorinated organics, polychlorinated biphenyls,
increased at CEWAF in the Barreirinhas experiment compared
nitrogen-containing aromatics, and many other pesticides,
to WAF. Furthermore, the BTEX, alkanes, and PAHs gene
dyes, and xenobiotics (Harms et al., 2011). Meanwhile, fungi
countswerehigherintheWAFthanintheCEWAFtreatments,
and bacteria may establish partnerships to more efficiently
particularly in the Barreirinhas experiments (Supplementary
degrade oil. Some positive correlations between the abundance
Figures 3, 4), suggesting that the dispersant may actually
of bacterial (e.g., Alcanivoracaceae and Alteromonadaceae) and
inhibitthegrowthofcertainimportantoildegradingmicrobes.
fungi (e.g., Ascomycota and Basidiomycota) metagenomics
This inhibition results on lower gene counts in the CEWAF
sequences were observed in our study (Supplementary
treatments.
Figures6,7).
Thisstudyhintsatpossiblerolesforbacteria,archaea,fungi,
andothermicroscopiceukaryotesintheAmazonas/Barreirinhas
WAF and CEWAF Treatments Influence
deep-sea water in oil biodegradation. We observed that
the Microbial Metabolic Profiles
chemolithotrophy, methanogenesis, nitrogen metabolism, and
The availability of dispersed oil as the sole carbon source utilizationofsulfurcompoundsarecoupledtooilbiodegradation
added for microbial growth from colonization of the buoyant (withandwithoutadditionofdispersants).Linksbetweenthese
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